The Folding, Assembly, and Intracellular Transport of Murine Class
I Major Histocompatibility Complex Heavy Chains
by
Robert Pulleyn Machold
B.S., Molecular Biophysics and Biochemistry (1991)
Yale University
SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
FEBRUARY 1996 (-11
© 1996 Massachusetts Institute of Technology
All rights reserved
Signature of Author
__
Department of Biology
September 20, 1996
Certified by
Hidde L. Ploegh
Professor of Biology
Thesis Supervisor
Accepted by
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LIBRARIES
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/Richard
A. Young
Professor of Biology
Chairman, Committee on Graduate Students
The Folding, Assembly, and Intracellular Transport
of Murine Class I Major Histocompatibility Complex Heavy Chains
by
Robert Pulleyn Machold
Submitted to the Department of Biology on September 20,
1996 in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
ABSTRACT
The folding and assembly of murine class I MHC molecules has been studied by
developing and characterizing novel antibodies directed specifically against denatured
class I MHC heavy chains. In Chapter 2, a rabbit polyclonal antiserum (RafHC) specific
for "free" (not I 2m-associated) murine class I MHC heavy chains was employed to
demonstrate that unassembled heavy chains, although normally retained in the ER, are
expressed at low levels at the cell surface even in the absence of P2m, and that both
TAP-dependent peptides and reduced temperature enhance the ability of free heavy
chains to attain a transport competent structure. In Chapter 3, three novel monoclonal
antibodies specific for unassembled H-2Kb heavy chains -- KU1, KU2, and KU4 -- were
characterized by epitope mapping and used to probe the structure(s) of unassembled Kb
heavy chains that appear transiently during the biosynthesis and disintegration of the
Kb class I MHC molecule. Unfolded Kb molecules reactive with the KU monoclonal
antibodies were detectable upon deposition of the former in the lumen of the ER, and
during the period of calnexin association, but reactivity with all three KU antibodies
was lost simultaneously after 5' -- a rate consistent with the assembly of Kb heavy chain
with P 2m. Class I MHC heavy chains that dissociated from P2m at the cell surface
rapidly unfolded into KU-reactive species, but remained folded at reduced temperature.
Degradation of cell surface-disposed free heavy chains was sensitive to inhibitors of
endosomal acidification. In Chapter 4, the mechanism by which class I MHC molecules
are downregulated by the human cytomegalovirus (HCMV) was analyzed by
comparing the abilities of two implicated viral gene products --US11 and US2-- to
degrade murine class I MHC allomorphs. The observed stability of the murine class I
MHC molecules H-2Kb and H-2Ld in US2-transfected cells, but not in US11+ cells,
demonstrates that the two viral proteins have distinct specificities for class I MHC
molecules.
Thesis Supervisor: Hidde L. Ploegh
Title: Professor of Biology
Acknowledgements
I would like to dedicate this thesis to my family, for being so supportive of me in
all of my pursuits, and for helping me through periods of indelible darkness.
I would like to thank Hidde for having me as a graduate student in his lab, and
for teaching me how to think critically and logically through experimental impasses.
And of course, a big thanks to all of the members of the Ploegh lab, past and
present, who shared many a humorous moment with me.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
CHAPTER 2
1
Peptide influences the folding and intracellular transport of free
Major Histocompatibility Complex class I heavy chains
Journal of Experimental Medicine (1995) 181:1111-1122
CHAPTER 3
Intermediates in the assembly and degradation of class I MHC
molecules probed with free heavy chain-specific monoclonal
antibodies
Submitted to the Journal of Experimental Medicine
CHAPTER 4
The HCMV gene products US11 and US2 differ in their ability
to attack allelic forms of murine MHC class I heavy chains
Submitted to the Journal of Experimental Medicine
CHAPTER 5: SUMMARY
104
Chapter 1
Introduction
Protein folding in the cell
Despite the wealth of structural data that has been amassed for a broad
spectrum of proteins, the mechanisms that determine the folding pathways of
newly synthesized polypeptide chains remain largely undefined. Studies on the in
vitro refolding of small globular proteins such as ribonuclease (Anfinsen et al., 1961)
and BPTI (Creighton, 1974) have demonstrated that, in principle, the amino acid
sequence of the protein contains all of the information necessary to guide the
denatured polypeptide chain to its proper conformation. A general paradigm that
has emerged from in vitro studies is that protein folding is initiated by a rapid
collapse of hydrophobic residues into the center of the molecule, and by the
localized folding of the polypeptide chain into secondary structures (eg. a-helices,
3-sheets) that guide the formation of disulfide bonds and other noncovalent
interactions (Kim and Baldwin, 1990). Intermediates in the protein folding pathway
have been described as 'molten globules' (Ptitsyn, 1995), a form of the protein which
is compact and contains substantial secondary structure, but whose hydrophobic
core may still be partially exposed, and whose overall tertiary structure remains
fluidlike. The rate limiting step in the folding process often entails the conversion of
these unstable molten globule states, which are in rapid equilibrium with the fully
unfolded state, into the final native conformation.
Although many proteins have been refolded sucessfully in vitro, there are
obvious limitations as to what conclusions can be applied towards the folding of
proteins within the cell. Protein folding in vitro is typically inefficient, and often
necessitates protein concentrations and physicochemical conditions that are vastly
different from those found within cellular folding compartments. A substantial
portion of proteins that attempt to refold in vitro misfold, and/or form aggregates
(Mitraki and King, 1989), whereas in the cell, folding of wild type nascent
polypeptide chains is usually >95% efficient (Copeland et al., 1986). In contrast to
the refolding pathways available to a denatured protein attempting to fold in vitro,
where the entire polypeptide chain may begin folding synchronously, proteins
synthesized in the cell may fold vectorially from N- to C- terminus as the nascent
polypeptide chain emerges from the ribosome. Proteins that are subunits in a
multimeric complex often require interactions with the other subunits to complete
the folding reaction, and thus are not easily studied in vitro. Likewise, proteins that
contain transmembrane domains must generally be inserted into a lipid bilayer
during folding, or their exposed hydrophobic regions will cause them to aggregate.
Proteins that reside at the plasma membrane, as well as soluble proteins
destined to be secreted, often possess a N-terminal signal sequence that directs their
cotranslational insertion into the lumen of the endoplasmic reticulum, a
compartment in the cell specialized for folding and assembly processes (Gething
and Sambrook, 1992). The redox potential in the lumen of the ER is maintained
such that the formation of disulfide bonds is promoted: the ratio of oxidized to
reduced glutathione is between 1:1 and 1:3 in the lumen of the ER, in contrast to the
1:30 to 1:100 ratio found in total cell extracts (Hwang et al., 1992). Disruption of the
proper redox potential with reducing agents such as dithiothreitol (DTT) results in
the misfolding and aggregation of proteins that normally form disulfide bonds
(Braakman et al., 1992). Another distinctive feature of the ER is that it contains
millimolar concentrations of calcium ions: although the role of calcium in ER
+2
processes such as folding and oligomerization is not yet clear, depletion of ER Ca
with membrane permeable calcium ionophores dramatically slows egress of
proteins from the ER to subsequent compartments in the secretory pathway (Lodish
and Kong, 1990). A number of ER-resident proteins have been identified that assist
the folding of nascent polypeptide chains; these include protein disulfide isomerase
(PDI), peptidyl prolyl cis-trans isomerase, and other 'chaperones' such as BiP and
calnexin (Gething and Sambrook, 1992). Unlike enzymes such as PDI, which
catalyzes the formation and isomerization of disulfide bonds, chaperones are
thought to assist in the folding process by transiently binding to and stabilizing
partially folded intermediates, thereby preventing nonspecific aggregation of
exposed hydrophobic regions.
In addition to providing a suitable chemical environment for folding and
assembly processes, the ER is also responsible for retaining and degrading proteins
that have misfolded, or have failed to assemble properly with the appropriate
partners (Hammond and Helenius, 1995). This sorting facility of the ER has been
termed "architectural editing" (Klausner, 1989), and has been well documented for a
number of oligomeric protein complexes, including immunoglobulins (Dul et al.,
1992)(Sitia et al., 1990), T cell receptors (Chen et al., 1988), and class I MHC
molecules (Salter and Cresswell, 1986), as well as for viral proteins such as the
influenza hemagglutinin (Copeland et al., 1986) and vesicular stomatitis virus
membrane glycoprotein G proteins (Hammond and Helenius, 1994b). The ERresident protein calnexin, a calcium-binding 64.5 kDa phosphorylated Type I
transmembrane protein (Wada et al., 1991), and its soluble homologue calreticulin,
have been shown to bind specifically to partially folded or assembled glycoproteins
in the course of their folding and oligomeric assembly (Ou et al., 1993)(Williams and
Watts, 1995). In the cases where the calnexin-associated protein is permanently
misfolded, or cannot oligomerize with other subunits, calnexin remains associated
with the polypeptide chain, thereby preventing disfunctional protein conformers
from being released throughout the cell (Hurtley and Helenius, 1989)(Klausner and
Sitia, 1990)(Bonifacino and Klausner, 1994). In Drosophila melanogaster cells, which
do not express detectable levels of any calnexin homologues, calnexin is both
necessary and sufficient to mediate the retention of unassembled subunits of
transfected class I MHC molecules (Jackson et al., 1994). Free CD3E chains expressed
in COS cells in the absence of other T cell receptor subunits were shown to
consistently colocalize throughout the cell with transfected calnexin molecules-either wild type, or lacking ER-retention sequences (Rajagopalan et al., 1994). These
and other studies implicate calnexin as the muscle behind the quality control
machinery.
Calnexin is a lectin: it binds specifically to monoglucosylated N-linked
oligosaccharides present on newly synthesized glycoproteins (Hammond et al.,
1994). This form of the glycan is reached initially by the sequential trimming of the
Glc 3Man 9GlcNAc 2 oligosaccharide by glucosidases I and II immediately upon
transfer of the oligosaccharide moiety from its dolichol-pyrophosphate precursor to
an asparagine residue contained within a glycosylation acceptor sequence on the
elongating polypeptide chain (Kornfeld and Kornfeld, 1985). Although the initial
stage of binding of calexnin to the newly synthesized glycoprotein appears to occur
via the carbohydrate side chain of the latter, calnexin may subsequently interact
directly with the polypeptide chain, as evidenced by the persistent association of
calnexin with class I MHC heavy chains even after removal of the glycan with
endoglycosidase H (Ware et al., 1995). For the class I MHC molecule, there is some
evidence that nonglycosylated mouse H-2Ld heavy chains interact with calnexin
(Carreno et al., 1995a). Inhibition of glucose trimming with castanospermine, 1deoxynojirimycin, or derivatives (Elbein, 1991), prevents the binding of calnexin to
newly synthesized glycoproteins (Hammond and Helenius, 1994a)(Hammond et al.,
1994), as does blocking the glycosylation of the polypeptide chain with tunicamycin
(Ou et al., 1993). Studies on murine class I MHC molecules (Vassilakos et al., 1996),
as well as the vesicular stomatitis virus G glycoprotein (Hammond and Helenius,
1994a), have shown that the maturation of these proteins is impaired in the
presence of castanospermidine, although the pleiotropic effects of the latter (eg. on
calreticulin) complicate the interpretation of these experiments. A recent report
characterizing a human cell line lacking calnexin found that the intracellular
transport and cell surface expression of endogenous class I MHC molecules in the
latter was similar to that observed in the parental cell line (Scott and Dawson, 1995).
In yeast, disruption of the calnexin gene is tolerated by Saccharomyces cerevisiae,
despite poorer retention of mutant proteins in the ER (Parlati et al., 1995), but is
lethal in Saccharomyces pombe (Jannatipour and Rokeach, 1995).
A fundamental issue that is largely unresolved in the current model of
quality control is: how do the molecules that participate in quality control judge
when folding and oligomerization has been completed for such a diverse range of
cellular proteins? More specifically, in these cases where calnexin is responsible for
the retention observed, what cues mediate the release of newly synthesized
glycoproteins from calnexin once they have completed their maturation? A study
on the folding and assembly of influenza hemagglutinin (HA) trimers translated in
a cell free system supplemented with microsomal membranes found that the
release of calnexin-bound HA molecules was impeded when the trimming of their
monoglucosylated glycans by glucosidase II was inhibited with castanospermine
(Hebert et al., 1995). This would suggest that the release of glycoproteins from
calnexin is determined primarily by the composition of the glycan on the
polypeptide chain, and not by the structure of the bound protein.
Once released from calnexin, misfolded HA subunits that contain high
mannose oligosaccharides are reglucosylated by the enzyme UDPglucose:glycoprotein glucosyltransferase, and thereby may reenter calnexinsupported folding and assembly pathways (Hebert et al., 1995). Interestingly, it has
been reported that UDP-glucose:glycoprotein glucosyltransferase has the ability to
discriminate between folded and unfolded glycoproteins, reglucosylating only the
latter (Sousa et al., 1992). Retention of glycoproteins in the ER may then
fundamentally depend on the action of the glucosyltransferase on the products
released by calnexin. The general mechanism of how the glucosyltransferase assays
the folded state of a protein is poorly understood, but there is recent evidence that
the enzyme may bind to exposed hydrophobic patches on the misfolded
polypeptide (Sousa and Parodi, 1995).
The structure and biosynthesis of the influenza hemagglutinin molecule has
been well characterized, and provides a model for how the folding and assembly of
a multidomain Type I membrane glycoprotein occurs in living cells (Wilson et al.,
1981)(Braakman et al., 1991)(Tatu et al., 1995)(Chen et al., 1995). Mature HA is
comprised of three large (84 kDa; 549 amino acids) transmembrane subunits, each
of which receive seven N-linked glycans, and acquire six intrachain disulfide
bonds, in the course of folding and trimerization. In addition to the fully oxidized
form of the unassembled HA polypeptide chain ("NT"), two partially oxidized fulllength intermediates have been characterized: IT1, which contains minimal
disulfide pairing, and IT2, in which one of the major loop-forming disulfide bonds
has formed. Translation and deposition in the lumen of the ER of the HA
polypeptide chain is completed within two minutes; formation of the fully oxidized
NT form is achieved about five minutes after chain termination (Braakman et al.,
1991). A recent study on nascent HA chains reveals that glycosylation of the
elongating polypeptide occurs immediately upon the emergence of the
glycosylation acceptor sites into the lumen of the ER, and that calnexin binding is
detectable prior to completion of the polypeptide chain (Chen et al., 1995). Using a
panel of conformation-specific antibodies raised against mature HA molecules, it
was shown that the formation of certain epitopes could occur in the IT1 and IT2
intermediate stages, and in the case of an epitope located in the N-terminal portion
of the molecule, on the emerging nascent chain (Chen et al., 1995).
The paradigm that has emerged from in vivo studies on the folding and
assembly of membrane glycoproteins such as the influenza hemagglutinin molecule
is that these processes can begin immediately upon entry of the nascent
polypeptide chain into the lumen of the ER. The folding of subdomains, and
disulfide bond formation within the polypeptide, appears to be rapid, and may
occur asynchronously, suggesting that large multidomain proteins are not
committed to a single folding pathway. Glycosylation of the nascent chain is a
prerequisite for calnexin association, and in addition, due to its bulkiness, the
glycan may serve to limit the conformational space available to the unfolded
polypeptide chain, and thereby may help prevent misfolding (Kern et al., 1993). The
binding of putative chaperones such as calnexin to the nascent chain may also
enhance the efficiency of folding and assembly by restricting improper
conformations or interactions of the latter.
In this thesis, I have sought to address some of the issues described above
concerning the folding and assembly of multisubunit glycoproteins in the ER, by
studying the biosynthesis and expression of the murine class I Major
Histocompatibility Complex (MHC) molecule. The murine class I MHC molecule
provides a highly appropriate model for studying membrane glycoprotein folding,
since it has already been extensively characterized immunochemically and
physically. The known three dimensional structure, and the availability of many
allelic variants, as well as mutant cell lines and mice defective for different stages of
class I biosynthesis, have greatly facilitated the experiments contained within this
thesis.
The overall approach of my thesis project has been to employ antibodies to
probe the structure of class I MHC molecules at different stages of their maturation,
and under conditions where folding and assembly cannot be completed.
Monoclonal antibodies are particularly useful as tools for monitoring the folding or
unfolding of regions of the polypeptide chain that surround the epitope recognized
by the antibody. However, because folding and assembly steps are often rapid in
comparison to the times required for cell lysis and immunoprecipitation, and can
occur asyncronously in a population of pulse-labelled nascent polypeptide chains,
it is often difficult to recover intermediates efficiently with this approach.
Generally, studies on the folding and assembly of class I MHC molecules have
employed antibodies specific for heavy chains that have assembled with 12m
(conformation- dependent), in combination with antibodies that recognize all forms
of the heavy chain (e.g., anti-cytoplasmic tail) to assess the extent of folding and
assembly.
Despite the wide array of conformation dependent antibodies that have been
prepared against murine class I MHC molecules, there were no immunochemical
reagents directed specifically against assembly intermediates of the mouse class I
MHC molecule at the onset of this thesis work. Much of the work contained within
this thesis is thus concerned with the characterization of novel antibodies raised
specifically against nonassembled ("free") class I MHC heavy chains, and utilization
of these reagents to characterize intermediates in the biogenesis and destruction of
the class I MHC molecule. In addition, we examine the mechanisms by which the
HCMV virus may exploit the vulnerability of newly synthesized class I MHC free
heavy chains prior to their assembly with f 2m and peptide.
Biosynthesis of class I MHC molecules
The immune system has evolved two major strategies for recognizing and
eliminating infectious agents from the body: the cellular response, which targets
antigens expressed within infected cells, and the humoral response, which is
directed against extracellular antigens. The molecules responsible for presentation
of antigens to the immune system in both the cellular and humoral responses are
the Major Histocompatibility Complex (MHC) class I and class II proteins
respectively. Class I MHC molecules are expressed at the plasma membrane in
virtually all cell types, and present short peptide fragments derived from proteins
synthesized within the cell to circulating CD8+ (cytotoxic) T lymphocytes (Yewdell
and Bennink, 1992). If the peptide presented is deemed by the T cell to be derived
from a foreign protein, the infected cell is then lysed.
Class I MHC molecules are comprised of three subunits: 1) a highly
polymorphic 44 kD type I transmembrane glycoprotein ("heavy chain"),
noncovalently associated with 2) beta-2-microglobulin (32m), a soluble 12 kD
polypeptide, and 3) a suitable peptide fragment of 8-11 amino acids (Yewdell and
Bennink, 1992). In humans, class I MHC heavy chains are encoded by the HLA-A,
-B, and -C genes, whereas in mice, these loci are referred to as H-2K, -D, and -L. At
least 50 alleles of the heavy chain are known for mouse MHC molecules (Klein,
1986). The specific allele is denoted with a superscripted letter, i.e., H-2Kb, and the
combination of alleles on one chromosome is termed the haplotype. Class I MHC
heavy chains are encoded by eight exons. Exon 1 encodes the signal peptide that
directs the cotranslational insertion of the heavy chain into the lumen of the ER.
Exons 2 and 3 encode the al and a2 domains (about 90 amino acids each)
respectively; most of the sequence polymorphisms among different class I heavy
chain alleles are clustered in these two domains. Exon 4 encodes a 90 amino acid Iglike domain that contains one of the two intrachain disulfide bonds present in the
fully assembled molecule. Exon 5 encodes the transmembrane domain, and exons 6,
7, and 8 encode the 25-35 amino acid cytoplasmic tail. The nascent class I MHC
polypeptide acquires one to three glycans, depending on the allotype.
The three-dimensional structures of a number of class I MHC molecules
have been determined to high resolution, and collectively provide a vivid image of
the basic mechanism of antigen presentation to T cells. The HLA-A2 molecule was
the first class I MHC allotype to be crystallized and characterized by X-ray
crystallography (Bjorkman et al., 1987b)(Bjorkman et al., 1987a). The initial 3.5 A
structure revealed that the al and oa2 domains folded together to create a peptide
binding groove consisting of two parallel a-helices 18 A apart supported by a
platform of eight anti-parallel P-sheets, creating a 25A long and 10A deep cleft. The
length of the cleft is constrained by residues at the ends of the al and a2 a-helices,
thus accounting for the strict length requirement of 8-10 residues for class I-binding
peptides. The a3 domain and P2m both fold into immunoglobulin-like domains.
Most of the contacts between k 2m and the heavy chain occur through the a3
domain of the latter, although some residues pointing down from the al-a2
peptide binding platform also participate.
The class II MHC molecule is essentially comprised of the same four
domains (al,P 2m, a2, and r3) as the class I MHC molecule, but is assembled from
two transmembrane subunits, a (contains class I MHC al and f 2m-like domains)
and p (a(2 and a3). Its structure has also been determined (Brown et al., 1993), and is
very similar to that of the class I MHC molecule; however, the peptide binding
groove of the class II MHC molecule is open at the ends, and therefore can
accomodate larger peptides (Wolf and Ploegh, 1995). The three dimensional
structures of the murine class I MHC molecules H-2Kb (Fremont et al., 1992) and Db
(Young et al., 1994) have also been solved by X-ray crystallography, and are
virtually superimposable upon the HLA-A2 structure.
Both the class I MHC heavy chain and 12m possess cleavable N-terminal
signal sequences that direct their cotranslational insertion into the lumen of the ER,
but peptides generated in the cytosol that are to be presented by class I MHC
molecules must also traverse the lipid bilayer of the ER membrane, and do so via a
heterodimeric peptide transporter comprised of two MHC-encoded membrane
spanning subunits, TAP1 and TAP2 (acronym for Transporter associated with
Antigen Presentation) (Heemels and Ploegh, 1995). Based on an analysis of their
primary sequences, the TAP1 and TAP2 proteins are predicted to span the ER
membrane six to nine times, with the bulk of each subunit consisting of a large
cytosolic domain that contains an ATP-binding cassette motif (Momburg et al.,
1994a)(Howard, 1995). Studies on microsomal vesicles enriched for ER membranes
(Androlewicz et al., 1993)(Shepherd et al., 1993)(Schumacher et al., 1994), and
permeabilized cells (Neefjes et al., 1993b), have demonstrated that the TAP complex
selectively translocates peptides of 8-15 amino acids, in an ATP-dependent manner.
In both rat (Heemels et al., 1993) and mouse microsomes (Schumacher et al., 1994 ),
TAP preferentially translocates peptides with a hydrophobic C-terminus (Momburg
et al., 1994b), which are known to be preferred binding substrates by most class I
MHC allomorphs ((Rammensee et al., 1993), see also below).
Cells in which TAP1 or TAP2 has been mutated or deleted express class I
MHC molecules at 5-10% of levels observed in wild type cells (Ljunggren et al.,
1989)(Van Kaer et al., 1992). The mutant mouse lymphoma cell line RMA-S (b
haplotype; expresses the mouse class I MHC molecules H-2Kb and Db), originally
selected for low cell surface expression of class I MHC molecules (Ljunggren and
Karre, 1985), and its nonselected parent RMA have been shown to translate class I
MHC subunits at similar rates; however, RMA-S cells are unable to present
endogenous antigens to class I-restricted cytotoxic T cells, and are defective in the
assembly of class I MHC molecules, due to a defect in the TAP2 gene (Attaya et al.,
1992). When RMA-S cells were incubated at lower temperature (Ljunggren et al.,
1990), or in medium containing suitable class I MHC binding peptides (Townsend
et al., 1989), class I MHC cell surface expression was partially rescued. These studies
demonstrate that in the absence of TAP-provided peptides, the class I MHC heavy
chain and
P 2m
can associate to form heterodimers, but that these "empty"
complexes remain in the ER, or if transported to the plasma membrane, rapidly
dissociate due to their thermolability in the absence of peptide. An interesting
exception to the dependence of class I MHC cell surface expression on TAP-
provided peptides is the human class I MHC molecule HLA-A2, which when
expressed in the human TAP-deficient cell line T2, can stably assemble with ERdisposed signal peptides (Henderson et al., 1992)(Wei and Cresswell, 1992).
In addition to translocating peptides into the lumen of the ER, the TAP
complex has been shown recently to interact directly with "empty" class I MHC
heavy chain-b 2m heterodimers, and may facilitate peptide loading of the latter (Suh
et al., 1994)(Ortmann et al., 1994). Like other membrane glycoproteins described
above, newly synthesized mouse class I MHC heavy chains associate with calnexin
immediately following their deposition in the ER, and remain bound while
assembling with P2m and peptide (Degen and Williams, 1991)(Degen et al., 1992). It
has been reported that for the mouse molecules H-2Kb and Db, calnexin remains
bound to the class I MHC molecule during the period of TAP-association and
peptide loading; release from both calnexin and TAP was observed to occur
simultaneously (Suh et al., 1996), concomitant with observed rates of egress to the
Golgi. Interestingly, in human cells, calnexin apparently dissociates from the class I
MHC heavy chains upon assembly of the latter with P2m (Ortmann et al.,
1994)(Sugita and Brenner, 1994)(Nobner and Parham, 1995), prior to the association
of the heterodimers with TAP, although there are conflicting reports on this issue
(Carreno et al., 1995b).
One basic question that has been addressed in studies on the assembly of
class I MHC molecules is: in what order do peptide and 12m normally associate
with the class I MHC heavy chain in the course of assembly? The results described
above for class I MHC expression in the absence of TAP-provided peptides
demonstrate that peptide is not required for the rapid assembly of class I MHC
heavy chains with P2 m per se. In addition, association of TAP with class I molecules
during assembly has only been observed for 32m-associated heavy chains, and not
free heavy chains (Ortmann et al., 1994)(Suh et al., 1994), suggesting that N2m
binding is a prerequisite for TAP-assisted peptide loading. Thus, in normal cells,
"empty" heavy chain-P2m heterodimers are likely to be the most prevalent assembly
intermediate. A study on the early events in the assembly of human class I MHC
molecules found that properly conformed but thermolabile (ie, lacking stably
bound peptide) heavy chain-1 2m complexes could be detected by
immunoprecipitation from lysates of briefly (<1 minute) pulse-labeled cells a few
minutes before stable complexes were observed (Neefjes et al., 1993a), lending
support to the above hypothesis.
Support for the alternative pathway, wherein the heavy chain binds peptide
first, followed by recruitment of 22m, has come primarily from in vitro experiments.
A study on the folding of free H-2Db heavy chains in detergent lysates of f 2mdeficient cells found that Db heavy chains could be induced to fold into a native-like
conformation, as assessed by immunoreactivity with a conformation-dependent
monoclonal antibody, upon addition of micromolar concentrations of Db-binding
peptides to the lysates (Elliott et al., 1991). In Chapter 2 of this thesis, we address
this issue in vivo by comparing the folding and export of class I MHC molecules in
cells from mice deficient in the expression of either 02m, TAP1, or both (Machold et
al., 1995).
For most of the allomorphs examined, class I MHC cell surface expression
was not detected in cells defective in the synthesis of P2m (Williams et al.,
1989)(Koller et al., 1990). Earlier studies have documented that the free class I MHC
heavy chains that are synthesized in P2m-deficient cells do not acquire
conformation dependent epitopes, and are retained in the ER until their
degradation, with the notable exception of two highly homologous molecules, H2Ld and H-2Db (Allen et al., 1986)(Smith et al., 1993). The low but detectable cell
surface expression of these two class I MHC allomorphs in the absence of I 2m was
accounted for by the presence of three glycans on these molecules, in contrast to
other apparently non-expressed allomorphs that only contained two glycans. We
have re-examined the issue of P2m-independent cell surface expression of mouse
class I MHC allomorphs in the experiments described in Chapter 2.
Under normal conditions, assembly and cell surface-deposition of class I
MHC molecules is completed after 25-60 minutes (Ploegh et al., 1981). For the
mouse class I molecules H-2Kb and Db, the relative rates of cell surface expression
are 25 and 40 minutes respectively. In an extreme case, the H-2Kk molecule is
transported to the cell surface at a 16 fold higher rate than the Dk molecule, even
though the two proteins are 80% homologous (Williams et al., 1985). When the al,
or al and a2 domains in the H-2Ld and Db molecules were exchanged, the resulting
chimeric molecules were transported at rates corresponding to their apparent rates
of association with f 2m (Beck et al., 1986). However, variability in the rate of stable
association with P2m among class I MHC allomorphs may also reflect the
differences in peptide binding requirements, or intrinsic folding rates of each class I
MHC heavy chain.
Downregulation of class I MHC expression by HCMV
The class I MHC antigen presentation pathway is the major route by which
virally infected cells are eliminated by the immune system. Many viruses have thus
evolved strategies for downregulating the cell surface expression of class I MHC
molecules (Maudsley and Pound, 1991)(Hill and Ploegh, 1995), at the
transcriptional, post-transcriptional, or post-translational stages of biosynthesis.
Selective attenuation of class I MHC expression at the transcriptional level has been
observed in cells infected with HIV-1 (Scheppler et al., 1989). The oncogenic
adenovirus type 12 has been reported to downregulate class I MHC expression by
post-transcriptional mechanisms (Vaessen et al., 1987). However, most of the
viruses that have been observed to interfere with host cell class I MHC expression
apparently do so at a post-translational stage of biosynthesis. For example, the
adenovirus type 2 virus encodes an ER-resident protein, E3/19K, that binds
specifically to newly synthesized human class I MHC molecules and retains them
in the ER (Del Val et al., 1992). An unusual mechanism of post-translational control
of class I MHC expression has been described for the herpes simplex viruses 1 and
2, which both express a small cytosolic protein, ICP47, that interferes with class I
MHC-mediated antigen presentation by inactivating the TAP1/TAP2 peptide
transporter (York et al., 1994)(Hill et al., 1995).
The human cytomegalovirus, a member of the herpesvirus family, also
downregulates the expression of class I MHC molecules, but does so by
destabilizing newly synthesized class I MHC heavy chains (Beersma et al., 1993).
The HCMV genes responsible for class I MHC downregulation were identified in a
systematic search of the HCMV genome for genes responsible for class I MHC
instability (Jones et al., 1995). One of these genes, US11, encodes an ER-resident type
I transmembrane glycoprotein that has been shown by transfection to be sufficient
to cause the destruction of newly synthesized class I MHC molecules (Jones et al.,
1995). Recently, it has been shown that the US11 gene product causes the
dislocation of newly synthesized class I MHC heavy chains into the cytosol upon
their deposition in the ER (Wiertz et al., 1996). When cells expressing the US11 gene
product were pulse labelled in the presence of inhibitors of the proteasome, the
major site of cytosolic proteolysis, a deglycosylated heavy chain species could be
detected that was cytosolic, as determined by subcellular fractionation (Wiertz et al.,
1996). In addition to the US11 gene product, the HCMV US2 gene product has also
been shown to cause the dislocation of newly sythesized class I MHC heavy chains
in transfected cells. In chapter 4, I have addressed the issue of why the HCMV virus
20
expresses two separate gene products that appear to downregulate class I MHC
molecules by similar mechanisms.
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Chapter 2
Peptide influences the folding and intracellular transport of free
Major Histocompatibility Complex class I heavy chains
Machold, R.P., Andree, S., Van Kaer, L., Ljunggren, H.-G., and Ploegh, H.L.
Journal of Experimental Medicine (1995) 181:1111-1122
Peptide Influences the Folding and Intracellular
Transport of Free Major Histocompatibility
Complex Class I Heavy Chains
By Robert P. Machold,* Sofia Andrbe,* Luc Van Kaer,t
Hans-Gustaf Ljunggren,* and Hidde L. Ploegh*
From the *Centerfor Cancer Research and Department of Biology, and the tHoward Hughes
Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Summary
Class I major histocompatibility complex molecules require both 12-microglobulin (32m) and
peptide for efficient intracellular transport. With the exception of H-2Db and L , class I heavy
chains have not been detectable at the surface of cells lacking 32m. We show that properly conformed class I heavy chains can be detected in a terminally glycosylated form indicative of cell
surface expression in H-2b, H-2d, and H-2 03 2m- - concanavalin A (Con A)-stimulated splenocytes incubated at reduced temperature. Furthermore, we demonstrate the presence of Kb molecules at the surface of 02m -'- cells cultured at 370 C. The mode of assembly of class I molecules encompasses two major pathways: binding of peptide to preformed "empty" hererodimers,
and binding of peptide to free heavy chains, followed by recruitment of 02m. In support of the
existence of the latter pathway, we provide evidence for a role of peptide in intracellular transport
of free class I heavy chains, through analysis of Con A-stimulated splenocytes from transporter
associated with antigen processing 1 (TAP1)-/- ,02m- -, and double-mutant TAP1/0/2m - mice.
M
HC class I molecules present short peptide fragments derived from proteins synthesized in the cytosol to specific CTL (1). The MHC class I molecule is a heterotrimer comprised of a polymorphic transmembrane glycoprotein (heavy chain), 32-microglobulin (02m),' and peptide
(2). Assembly of the MHC class I molecule takes place in
the lumen of the endoplasmic reticulum (ER) and requires
the presence of the transporter associated with antigen processing (TAP) 1/TAP2 heterodimer, a protein complex that
transports peptides produced in the cytosol into the ER (3,
4). In the absence of a functional TAP complex, heavy
chain-3 2m heterodimers are formed that are largely retained
in the ER, although their cell surface expression can be partially rescued by addition of peptide to the culture medium
(5) or incubation at low temperature (6).
In 32m-deficient cells, the vast majority of class I heavy
chains are also retained in the ER and are rapidly degraded
(7). However, limited cell surface expression of H-2Db molecules has been observed in lymphoid cells from mice lacking
3
2m (8). Cell surface-disposed non-f 2m-associated ("free")
rabbit aH-2b antiserum; aeHC,
anti-heavy chain serum; acp , Kb-specific antipeptide serum; ýzm, 32microglobulin; ID, one-dimensional; ER, endoplasmic reticulum; FTOC,
fetal thymic organ cultures; Staph. A, 10% fixed Staplhylococcus autres; TAP,
transporter associated with antigen processing.
aH-2,
1Abbreviations used in this paper:
8
1111
Db heavy chains were shown to exhibit conformationsensitive epitopes and sensitize target cells for killing by alloreactive CTL (9). Nevertheless, in studies with the 3 2mdeficient cell line R1E transfected with Db or Kb, only Db
molecules were detectable at the cell surface (7). The Ld molecule, which is quite similar to Db (94% identity), has also
been shown to be expressed at the surface of 1 2m-deficient
cells (10).
Are other class I molecules present at the surface of 32m-icells, but at levels below the threshold of available biochemical methods? H-2b cells deficient for 32m stimulate Kb-allo specific and -restricted CD8 T cells (11, 12). Fetal thymic
organ cultures (FTOC) prepared from 02m-/- mice support
positive selection of CDS - T cells expressing a Kb- restricted
TCR in the presence of added human 02m and peptide (13),
implying that free Kb heavy chains are present at the cell surface. By pulse-chase analysis of Con A-stimulated splenocytes from H-2b, H-2d, and H-2s mice bred onto a 02e-/background, we show here that intracellular transport of class
I heavy chains in 22m-"- cells can be demonstrated for most
class I alleles. Specifically, properly conformed Kb heavy
chains can be detected on the surface of H-2b 3_m -/ - cells.
Do class I heavy chains assemble with peptide in cells lacking
02m? Formally, the assembly of MHC class I molecules may
proceed either by the binding of peptide to an "empty" heavy
chain-0 2m intermediate, or by association of 32m with
J. Exp. Med. © The Rockefeller University Press - 0022-1007/95/03/1111/12 $2.00
Volume 181 March 1995 1111-1122
heavy chain-peptide complexes. Studies on the human cell
lines T1 and the TAP-deficient T2 have shown that the
HLA-B5 heavy chains readily assemble with 02m in the absence of TAP-dependent peptides (14), and have suggested
that, even in the presence of these peptides, empty complexes
may form before peptide binding (14, 15). Although these
data suggest that association of the heavy chain with 03 2m
usually precedes peptide binding, free Db heavy chains in detergent lysate of 2m - - cells can be induced to fold upon
addition of peptide alone (16). Thus, in the course of biosynthesis, a portion of class I molecules might assemble by forming
a complex with peptide first, followed by recruitment of
3:m. By comparing the conformation and cell surface expression of heavy chains in Con A-stimulated splenocytes from
H-2b 3 2m- - and H-2b TAPL,'/2m-'- mice, we demonstrate
that the folding and intracellular transport of free class I heavy
chains is enhanced by TAP-dependent peptides.
Materials and Methods
Antibodies. The following antisera and mAbs, prepared as tissue
culture supernatants, were used: rabbit aH-2b (aH-2; recognizes
conformed H-2K, D, and L locus products; a gift of Dr. S.
Nathenson, Albert Einstein College of Medicine, New York),
B22.249 (cal of D6; 17), 28-14-8s (ac3 of D-; 18), Y3 (al/a2 of
Kb; 19), K10.36.1 (specific for conformed Kb; a gift of Dr. G.
Waneck, Massachusetts General Hospital Cancer Center, Charlestown, MA), rabbit anti-p8 (recognizes the cytoplasmic tail of Kb;
prepared in our laboratory essentially as described; 20), and rabbit
aHC, a rabbit polyclonal antiserum directed against free class I
heavy chains. The latter was generated by immunizing New Zealand
white rabbits with inclusion bodies (kindly provided by Dr. S.
Nathenson) of soluble Kb or D6 heavy chains produced in bacteria by overexpression. Before immunoprecipitations, antibodies
were prebound to 50 AL 10% fixed Staphylococcus aureus (Staph.A)
for 60 min and then washed three times in ice-cold wash buffer
(0.5% NP-40, 50 mM Tris/HCI, pH 7.4, 150 mM NaCl, 5 mM
EDTA) to remove excess antibody and serum proteins. For FACS3
stainings, antibodies were purified via protein A-Sepharose (Repligen Corp., Cambridge, MA).
Mice. The generation of TAP1- - and 32m--' (H-2b haplotype) mice has been described (21, 22). l2m -"- mice of H-2b
haplotype (C57BL/6X129/Ola) were a gift from Dr. B. Koller
(University of North Carolina, Durham, NC). 0 2m- - mice of
H-2d haplotype were prepared by crossing H-2b / m - - mice with
BALB/c mice (H-2'), followed by a cross with B10.D2 mice and
five subsequent generations of brother-sister matings (mice were
a gift from Dr. T. Hansen, Washington University School of Medicine, St. Louis, MO). H-2' 3:m- - mice were generated by
crossing with SJL/J (H-2') mice, followed by 11 backcrosses (mice
were a gift from Dr. D. Roopenian, The Jackson Laboratory, Bar
Harbor, ME). To generate TAP1.3 2m double-mutant mice (designated TAP1/03m --),TAP1- - and 02m-/- mice (both H-2b) were
mated, and offspring were subsequently intercrossed and typed.
Control mice heterozygous for both the TAP1 and 32m genes
were generated from the same crosses or by mating TAP1/02m-/mice with B6 mice. All other control mice used were obtained from
The Jackson Laboratory. All mice used were at the age of 6-12
wk. Mice were maintained at the Division of Comparative Medicine, Center for Cancer Research, Massachusetts Institute of Technology (Cambridge, MA), in accordance with institutional
guidelines.
1112
Gel Electrophoresis. One-dimensional (1D) IEF was performed
as described (23, 24). Fluorography was accomplished by impregnating the gels with 2,5-diyhenyloxazol in DMSO before exposure
to film (XAR-5; Eastman Kodak Co., Rochester, NY).
Immunoprecipitations. 10' splenocytes were stimulated with
Con A (2.5 ig/ml) in DMEMv (GIBCO BRL, Gaithersburg, MD)
supplemented with 10% FCS, L-glutamine (2 mM), penicillin
(1:1,000 dilution U/ml), and streptomycin (100 Ag/ml) for 48 h
before labeling. After a 45-min starvation in methionine/cysteinefree medium, the cells were pulsed with 500 /Ci/ml ["S]methionine/cysteine (80/20) for 15 min, and then chased in complete
medium containing 1 mM nonradioactive methionine and cysteine,
at either 370 C or 260 C. Aliquots of cells (10:) were spun down
for each chase point and Ivsed in 0.5 ml ice-cold lysis buffer (0.5%
NP-40, 50 mM Tris/HCl. pH 7.4, 5 mM MgClz, 1 mM PMSF).
After 45 min on ice, the !vsates were centrifuged to remove the
nuclei and cell debris, and :hen precleared twice for 60 min (40 C)
with 2 jI normal rabbit se:r-:m prebound to 100 Al Staph. A suspension before specific immunoprecipitation. To minimize antibody
carryover between sequential immunoprecipitations, lysates were
incubated for 30 min with 50 4l Staph. A between immunoprecipitation steps. The Staph. A pellets were washed three times in icecold wash buffer before analysis by 1D-IEF.
Cell Surface lodinations. Con A-stimulated splenocytes from
normal and H-2b 3 2m-- mice were prepared as described above.
Before iodination, dead cells were removed by centrifugation with
Lympholyte-M (Cedarlane Laboratories Ltd., Hornby, Canada) according to the manufacturers protocol. Cells (10') were surface labeled with 1.0 mCi of Na"I (1 Ci = 37 GBq) in ice-cold PBS
by lactoperoxidase-catalyzed iodination. Class I molecules were subsequently immunoprecipitazed as described above.
Flow Cytometry. Splenocytes were stimulated in serum-free
medium (AIM-V; GIBCO BRL) supplemented with 250 ng/ml
Con A for 36 h at 370C, followed by incubation at either 370 or
260 C for an additional 16 h. Cells incubated at 260 C were buffered
by the addition of 20 mM He:es, pH 7.4. For cell surface staining,
106 cells were aliquoted per antibody. Cells were incubated on ice
for 30 min with 10-30 pg ml of purified Y3 mAb (aK"), B22.249
mAb (oaDb), or rabbit aHC serum (aeKb, Db), washed once with
cold PBS, and then incubated for 30 min with a 1:400 dilution
of goat anti-mouse or anti-rabbit FITC-coupled antibody (Southern
Biotechnology Associates, Inc., Birmingham, AL). Quantitation
of cell staining was performed on a FACScan® flow cytometer
(Becton Dickinson & Co., Mountain View, CA), with propidium
iodide used to gate out dead cells. Background staining values were
obtained by incubating cells with 10 Jg/ml purified IgG,, (for
B22.249), IgG2b (for Y3), or preimmune serum (for rabbit atHC)
before incubation with the FITC-conjugated secondary Ab.
Results
Folding and Intracellular Transport of Class I Heavy Chains
in H-2b H-2d, and H-2' ,3m-/- Cells. To generate an antiserum against class I MHC heavy chains that would recognize epitopes normally obscured by 2zm or present only on
unfolded heavy chains, we immunized rabbits with either
Kb or Db inclusion bodies produced by expression in bacteria. Initial characterization of the anti-Kb and -Db sera
showed that both reacted equally well against Kb and Db
heavy chains in immunoprecipitations, immunoblots, and
ELISA (data not shown). The antisera were combined for
the experiments described below, and this preparation is re-
Folding and Intracellular Transport of Free Heavy Chains
__~__
__I
aHC
B22.249
A
H-2b
S37
37*
acHC
aH-2
26 '
26"
1
37*
26"
1
26
37*
CISWANllI
p2m
S...
Db
Db,-
-
-
Kb -
Kb -
Sialylated
Slalylated
Kb, Db
-
-
Kb. b
Chase (hrs): 0 1 4
S1 -4
0 1 4
0
1
4
0 1 4
F
-
-
Chase (hrs): 0 1 4
0
1 4
0 1 4
28-14-8s
H-2d
aHC
aH-2
37'
'
37*
26
.37'
26
Ll
0
14
a3HC
26
37*
26
1 4
0 1 4
Bpmp
l-,
.
d
Kd D d
L L4,
fi3i
Slalylated
DChase
L
H-2s
0 1 4
aH-2
S37'
26
P2m [
0 1 4
I
37
--.•.
Chase (hrs): 0
0
HC
26'
-
.
[D
-
Chase (hrs): 0 1 4
0
1 4
1113
0 1 4
Machold et al.
1 4
014
0
1 4
K-
K'-S
Slalylated
Sialylated
Chase
(hrs):d,
Ld0 1 4
Chase (hrs): 0 1 4
K
_
CLI~
--
Salylated ,- -
lo
---0 1 4
'
Figure 1. Kinetics of class I MHC folding and transport in H-2b, H-2d,
and H-2' cells. Con A-stimulated splenocytes were labeled with [SS]mmethionine/cysteine for 15 min and chased for the times indicated at either
370 or 260C. (A and B) (b haplotype cells) Conformed MHC class I Kb
and Db molecules (c.H-2) and free heavy chains (aHC)were sequentially
immunoprecipitated from precleared cell lysates and analyzed on 1D-lEF
(A). Immature and sialylated forms of the Kb and Db heavy chains are
indicated. In parallel, conformed Db molecules were immunoprecipitated
with the mAb B22.249, followed by immunoprecipitation with the aHC
antiserum (B). (C and D) (d haplotype cells) Conformed MHC class I
Kd, Dd, and Ld (aH-2) and free heavy chains (aHC) were sequentially
immunoprecipitated (C). The positions of the immature and sialylated
S
D and Ld heavy chains are indicated. Ld molecules were immunoprecipitated in a separate experiment with the mAb 28-14-8s, followed by immunoprecipitation with the caHC antiserum (D). (E) (s haplotype cells), Conformed (aH-2) and free (o-HC) MHC class I K' and D' molecules were
sequentially immunoprecipitated as above. The positions of the immature
and sialylated K' and D' heavy chains are indicated.
A
H-2d
caH-2
26
37'
P2m-/-
!
! !
d
K -
,
•-
Dd
Ld
-4
Slalylated
d
-
Dd. L
-
,,,
01 4
Chase (hrs): 0 1 4
28-14-8s
37'
26'
I
I
I I
d
K -
D ed
qwqq
Slalylated
d
Dd, L
[
L
Chase (hrs): 0 1 4
0 1 4
ferred to as aHC. The aHC serum showed reactivity against
class I heavy chains from H-2K, D, and L locus products
aHC
of all haplotypes tested (b, d, s, k, 0, and also reacted with
," 1 ,--------I
human class I heavy chains in immunoblots, as do anti-free
class I heavy chain reagents produced against HLA-A, B,and
C heavy chains (25). This characterization revealed that the
acHC serum reacts broadly with non-23m-associated ("free")
class I heavy chains irrespective of conformation, peptide content, or glycan modification; no reactivity with heavy chainS 2m complexes was observed in immunoprecipitations on
' .-.
..•
Kb and Db molecules from biosynthetically labeled cell lines
(data not shown), including RMA and its TAP-2 deficient
counterpart RMA-S (26). Trace amounts of coprecipitating
02m were occasionally observed in immunoprecipitates from
H-2d cells (see Fig. 1 D).
To follow the fate of properly conformed and unfolded class
o 1 4 0 1 4
I heavy chains in the presence or absence of 32m, we performed a series of pulse-chase experiments on Con A-stimulated splenocytes prepared from H-2b, H-2d, and H-2' mice
ctHC
(Fig. 1) and their 32m- - counterparts (Fig. 2 and see Fig.
4, C and D). We included in our analysis cells chased at 26 0 C
37"
26' '
S
-- ' -~..-'
to determine the effect of low temperature on the stability
Sand conformation of free heavy chains (6). Aliquots of cells
were pulsed labeled for 15 min, chased at either 370 or 26*C,
and immunoprecipitated first with a conformation-sensitive
antibody (e.g., aH-2 serum), followed by a second round
of immunoprecipitation with the aHC serum. We analyzed
the immunoprecipitates by 1D-IEF to resolve the complex
mixture of biosynthetic intermediates. In the course of matuVF
ration, class I molecules acquire negatively charged sialic acid
- -f
residues, which results in a shift in isoelectric point toward
the anode. Sialylation is a marker for transport through the
S
tmns-Golgi network to the cell surface, and those heavy chains
S1 4
1 4
that are not sialylated reside in the ER or proximal to the
r
r
S1 4 0 ns-Golgi
14
network (27). The nonsialylated class I heavy chains
C
H-2-s
caH-2
02M-/-
37*
I'
a
26"
2HC
3, , 26'
II
K• -
Slalylated
[D.
-
are thus referred to as "immature:'
Fig. 1 A shows a pulse chase of H-2b Con A-stimulated
splenocytes analyzed with the acH-2 and aHC antisera. The
bulk of the assembled Kb and Db is rapidly sialylated at 26*
and 370 C, although more of the immature complexes are
detectable during the 26*C chase (compare 1-h time points).
The subsequent immunoprecipitation with aHC reveals a
transient pool of immature free Kb and Db at the onset of
the chase, most of which disappears by 1 h of chase. Some
sialylated free heavy chains appear after 1 h at 37*C, most
likely from dissociation of unstable complexes (see below),
but these are no longer detectable by 4 h. Because the aH-2
Slalylated
Chase (hrs): 0 1 4
0 1 4
0
1 4
0 1 4
Figure 2. Kinetics of class I MHC transport in H-2d and H-2*
s-2m/cells. Con A-stimulated splenocytes were pulse labeled as described above
and chased for the times indicated at 26* or 370 C. (A and B) Conformed
class I Kd, Dd, and Ld (2aH-2), and unfolded heavy chains (caHC) were
sequentially immunoprecipitated and resolved on ID-IEF (A). Sialylated
1114
forms of Ld and Dd that emerge at 26*C are boxed. In a separate experiment, Ld molecules alone (28-14-8s) were first immunoprecipitated, followed by aHC immunoprecipitations (B). The positions of the immature
and sialylated Kd, Dd, and Ld molecules are indicated. (C) Conformed
(aH-.2) and unfolded (aHC) class I KS and D' molecules were sequentially immunoprecipitated and analyzed on 1D-IEF. Immature and sialylated
K' and D' molecules (box) are indicated.
Folding and Intracellular Transport of Free Heavy Chains
·
antiserum recognizes both conformed Kb and Db (the latter
less efficiently), we performed a parallel immunoprecipitation
with the mAb B22.249, which recognizes a conformationdependent epitope on the al domain of Db (17). Consistent
with earlier reports (28), Db molecules were transported
with slower kinetics than Kb (compare 0-min time points,
370C, Fig. 1, A and B), and less completely (4-h time points,
370 C, arH-2 and B22.249 panels).
We performed a similar analysis on Con A-stimulated
splenocytes from H-2d and H-2' haplotype mice (Fig. 1,
C-E) to serve as a comparison for experiments performed
on 2m -- mice of these haplotypes (described below). Both
K a and Dd heavy chains were efficiently assembled and transported after 1 h of chase at 37*C, with somewhat slower
kinetics at 260 C (Fig. 1 C). In contrast, free Ld heavy
chains, as well as immature, conformed Ld molecules, were
detectable throughout the chase, even at 37*C (Fig. 1 C, aHC
and aH-2 4-h time points). To follow only Ld molecules,
we repeated the pulse-chase experiment and immunoprecipitated first with the Ld-specific mAb 28-14-8s, followed by
immunoprecipitation with aHC serum (Fig. 1 D). The rate
of transport of Ld is similar to that of the closely related Db
molecule (compare Fig. 1, B and D, B22.249 and 28-14-8s
immunoprecipitates), but in contrast to Db, immature free
Ld heavy chains persist throughout the chase, even at 370 C
(Fig. 1 D). Trace amounts of 32m can be seen in the aHC
immunoprecipitates (Fig. 1 D), suggesting that aHC may
also recognize a minor population of class I heavy chains that
are loosely associated with 3 2m (e.g., Ldalt; 10). Direct
aHC immunoprecipitation of class I heavy chains from lysates
of metabolically labeled Con A blasts (H-2d haplotype) also
coprecipitated minute amounts of 32m (data not shown).
Analysis of H-2' cells indicates that both K' and D' are
transported less completely than their H-2b counterparts
(Fig. 1 E, 4-h time points). Very little immature free D'is
detectable at either chase temperature, even at 0 min,althouih
small amounts of sialylated free D' heavy chains are evident
at 1 h (37*C chase). The apparent absence of free D'heavy
chains is not due to lack of reactivity with the aHC antiserum,
as demonstrated by pulse-chase analysis of H-2' 32m - - cells
(Fig. 2; see below). We conclude that rates of class I assembly
and intracellular transport show characteristic differences
among the alleles examined (29).
With the exception of Db (7) and Ld (10), transport of
class I heavy chains in the absence of 02m has not been observed. We performed pulse-chase experiments as described
above on Con A-stimulated splenocytes from H-2b, H-2d,
and H-2' mice devoid of 02m and analyzed the immunoprecipitated products by 1D-IEF to score for sialylation of class
I heavy chains. Unexpectedly, the aH-2 antiserum recognized
small amounts of class I material in all three 32m - - haplotypes (Fig. 2, A and C, and see Fig. 4, C), implying that
conformed free heavy chains are present in the absence of
02m. Does the polyclonal axH-2 antiserum contain some reactivity against unfolded free heavy chains, which could account for our results? This does not appear to be the case,
because reimmunoprecipitation of SDS-denatured Kb mole1115
Machold et al.
cules Wkith the aH-2 serum recovered only trace amounts of
material (data not shown). Similarly, the aH-2 serum does
not react with free K- heavy chains produced by in vitro
translation, even in the presence of oxidized glutathione (data
not shown; [301).
No sialylation of either conformed or unfolded H-2b class
K10.56.1
, ..
S37'
,,al
ap8
26
-mm1,
26'
37'
l4-
Kb
Slalylated
(hrs):
bChase
0
Chase (hrs): 0
14
1
0
B6
NANAse:
m
02
4
1
4
14
321m-/-
-+-
+
0
1
4
014
0
1
4
0
1
4
0zm-/-
-w
Kb 1C~i.
Sialylated
LIIC:
Kb
-n3k
Exposure:
overnight
1 week
Figure 3. Folding and cell surface expression of Kb molecules in H-2b
02m-I- cells. (A)Con A-stimulated splenocytes from H-2b 8zm-/- mice
were pulse labeled as described above and chased for the times indicated
at 26* or 370C. Conformed Pb molecules were immunoprecipitated with
the mAb K10.56.1, followed by immunoprecipitation of total Kb with
arp8, and resolved on 1D-IEF. (B)Con A-stimulated splenocytes prepared
from B6 and H-2b 82m-'- mice were surface iodinated, following by
immunoprecipitations of total Kb with ap8. The immunoprecipitates
were either digested with neuraminidase (+) or kept on ice (-) before
1D-IEF analysis. Labeled K' molecules from H-2b #2m-' - cells are apparent in the week-long exposure.
I heavy chains in H-2b 32m -- cells was observed after 4 h
of chase at 370 C (7); remarkably, transport of free heavy chains
was partially rescued by lowering the chase temperature to
260 C (see Fig. 4, C and D, 4-h time points). The sialylated
heavy chains immunoprecipitated with the czH-2 serum after
4 h of chase include both Kb and Db, as shown by a comparison with the parallel B22.249 (Db) immunoprecipitation
(see Fig. 4, D, 4- h time points, 260 C). Sialylated K5 molecules are also detectable in K10.56.1 (recognizes conformed
Kb; Fig. 3 A), p8 (recognizes all Kb; Fig. 3 A), and Y3
(recognizes conformed KS; data not shown) immunoprecipitates of A2m - - cells chased at 260C. Thus, by analogy to
earlier work on the TAP2-deficient cell line RMA-S, we find
that low temperature favors the cell surface accumulation of
K b heavy chains in the absence of 02m.
In light of the dramatic effect of temperature on the degradation of free class I heavy chains in 02m-'- cells (Fig. 4 C,
acHC, compare 370 and 260 C 4-h time points), we considered it likely that the heavy chains are transported to the cell
surface at 370 C, but are rapidly degraded such that the steady
levels are too low to be detected by metabolic labeling. To
assay more directly for cell surface-disposed K b molecules at
370 C, we performed iodinations of H-2b wild-type and
O3m-- cells and immunoprecipitated Kb heavy chains with
the anti-p8 serum (Fig. 3 B). Analysis by 1D-IEF showed
that Kb heavy chains are present at the surface of 32m- ! cells, albeit at levels well below that observed in nonmutant
H-2b cells, as expected (Fig. 3, B, compare overnight with
1-wk exposure). Digestion of the immunoprecipitated Kb
material with neuraminidase confirmed that the bands observed on 1D-IEF correspond to sialylated K b molecules
(Fig. 3 B, - and + lanes). We conclude that low levels of
free Kb heavy chains are expressed on the cell surface in the
absence of 32m at 370C.
Are free class I heavy chains transported to the cell surface
in other H-2 haplotypes? In H-2d 32m - ' - cells chased at
260 C (Fig. 2, A and B), degradation of heavy chains is reduced, and sialylated Ld and Dd are apparent by the end of
the chase (Fig. 2 A, 4-h time points). The induction of
sialylated L" at 260 C is visualized more clearly with the
mAb 28-14-8s (Fig. 2 B, 4-h time points). In H-2s' 2m- Icells, K, molecules remain largely as nonconformed immature heavy chains at both chase temperatures, whereas D',
like Ld, is partially sialylated after 4 h of chase at 260 C (Fig.
2 C). Thus, by incubating cells at 260 C, we can detect intracellular transport of free class I heavy chains other than Db
and Ld in H-2b, H-2d, and H-2s $2m-'- cells. In particular,
we observe transport of K b molecules in the absence of 02m,
which provides a biochemical basis for the Kb-restricted T
cell selection observed in 3m - / - FTOC (13).
Peptide Afects the Folding and Intracellular Transport of Free
Class I Heavy Chains. To assess the relative contributions
of the TAP1/TAP2 complex and 32m to the folding and intracellular transport of class I heavy chains, we performed
pulse-chase experiments as described above on H-2b Con
A-stimulated splenocytes lacking either TAP1 (TAP1-/-),
32m ( 3 2m- -), or both (TAP1/02m-/-). Immunoprecipita1116
tions of K b and D b class I molecules were performed with
either caH-2 or B22.249, followed by immunoprecipitation
with the oaHC serum, and then analyzed on 1D-IEF (Fig.
4). In cells lacking the TAP1 gene, class I transport was impaired at 370 C, but partially rescued at 260 C (Fig. 4 A),
consistent with previous reports (21). In contrast to normal
H-2b cells (Fig. 1), free heavy chains in the TAP1- - background persist throughout the chase and are present in amounts
approximately equal to that of f2m-associated material (Fig.
4 A). Sialylated free heavy chains, which appear only during
the 370 C chase, most likely arise from dissociation of thermolabile complexes in the course of intracellular transport.
We failed to detect sialylation of B22.249-immunoprecipitable Db molecules at either temperature in TAPI- - cells
(Fig. 4 B).
Cells lacking 32m are more deficient in intracellular transport (see above) and folding of class I heavy chains than TAP1deficient cells (compare Fig. 4, A and C). Like H-2d and
H-2s class I heavy chains (Fig. 2), K" and Db are present
predominantly as nonconformed free heavy chains in the
f 2 m - ' - background and are degraded by 4 h at 37°C (Fig.
4 C). The breakdown of free heavy chains is strongly inhibited at 260 C (aeHC, 4-h time points), and a greater proportion of both K b and Db appear to fold into a native-like conformation (Fig. 4 C, aH-2, and D, B22.249, 1-h time points).
Little, if any, folded Kb is detectable by the conformationsensitive mAb K10.56.1 during the chase at 370 C, but at
260 C, conformed free KS heavy chains are detectable by 1 h
(Fig. 3, 260 time points), in agreement with the results obtained with the aH-2 serum. The appearance of folded Kb
molecules at 260 C is not a consequence of impaired heavy
chain degradation, since the total amount of K` material
(op8) after 1 h of chase at 370 or 26 0 C is identical (Fig.
3 A). We conclude that, at low temperature, free K" heavy
chains can acquire a conformation similar to that observed
for properly assembled class I molecules.
Assembly of class I molecules in normal cells may proceed
via peptide binding to "empty" heavy chain-0 2m heterodimers, or alternatively, by association of i2m with heavy
chain-peptide complexes (31). To address the question of
whether TAP-dependent peptides affect the folding and cell
surface expression of free class I heavy chains, we crossed
H-2b TAP1-/- and 02m- - mice to obtain double-mutant
mice (TAP1/0 2m-'-). In Con A- stimulated splenocytes
lacking both f 2m and TAP1, the distribution of class I heavy
chains between free and conformed populations is similar to
that of 2m -/- cells, with the bulk of the Kb or D' heavy
chains in an unfolded conformation (aHC reactive). Loss of
TAP1 affects the folding of Db more profoundly than Kb
molecules, judging from the ratios of Db and K` immunoprecipitated with the mAb B22.249 and ceH-2 antiserum,
respectively, in the two cell types (compare Fig. 4, D and
F,for Db and Fig. 4, C and E for Kb, 1-h time points). The
most striking difference between the 02m - / - and doublemutant cells is that no sialylation of either Kb or D` is observed in the latter, even at 260C (Fig. 4, E and F). Extending
the chase of 8 h at 26 0 C still did not result in any detectable
Folding and Intracellular Transport of Free Heavy Chains
I_
_
____~_
·
_
alHC
B22.249
TAP 1-/-
aHC
aH-2
*
37'
-
02m -[
-
I
e5i
-U
S2m
I
26
I
J
26
I
Ii
37*
J
37'
26*
I
1
1'
I
'
37*
26 '
1
YwJ
i
i---
.Y~um.
"'
.
Db.
Kb
KbP
-
Slalylated I
F
Slalylated
Kb, Db
Kb, Db
Chase (hrs): 0 1 4
0 1 4
014
0 1 4
Chase (hrs): 0
B22.249
1
D3m-/-
0 1 4 0 14
0 14
1 4
37'
1
26-
I
37'
26
37'
26
1
I
-r
I-l"
I
I
I
26'
I I
~--
alHC
aH-2
aHIC
37'
V.'I-C
-
Db
ww
Db Kb-
-
~-
-mm~
Stalylated
Kb, Db
0
014
0
1 4
~~
Chase (hrs): 0 1 4
1 4
•-
H-2 26'
,
I
37
'
I
26 '
I
I
I
I
'0'F-C
Db -
.
m
V
Kb
"
g
Kb, Db
0 1 4
0 1 4
-B-
Kb -
Salylated
1
I
I
,'•
Chase (hrs): 0
0 1 4
1 37'
26
I
1 4
26
II
I
bJ
aIHC
B22.249
F
I-
37
0
014
37'
I
ii
Kb, Db
E.
TAP1-/m
~~~
T;T
Slalylated
13
Chase (hrs): 0 1 4
nun
Kb
4
0 1 4
"U itz
Slalylated
Kb. Db
Chase(hrs): 0
1 4
014
0
1 4
0
1 4
Figure 4. Kinetics of class I MHC transport in H-2b TAP1-'-, 2m-'-, and TAP1/f 2m-'- cells. Con A-stimulated splenocytes were pulse labeled
as described above and chased for the times indicated at 37* or 260C. Conformed class I Kb and Db (aH.1-; A, C, and E), and D b heavy chains alone
(B22.249; B, D, and F), were immunoprecipitated in parallel, followed by aHC immunoprecipitations. Immature and sialylated class I molecules were
resolved in 1D-IEF. (A and B) TAP1-/- cells. (C and D) 2zm- I- cells. The 0-min aH-2 time point (37*C) contains atypical background due to
cells,
an aspirator malfunction. (E and F) TAP1/02zm-1- cells. Note the appearance of sialylated Kb and Db in •2m-/-, but not in TAP.'2m-Iafter 4 h of chase at 260 C.
1117
Machold et al.
B22.249
Y3
O 37-C
L_.
B6
-f
S3L26*C
U26'C
-LL-
TAP1-/-
02m-/-
TAP1-/32m-/-
71
*.-2
r-1
* 26"C
B6
_r
TAP1-/-
I-
EI.
TAP1-/r2m-/-
Pzm-/-
aHC
l 37"C
U 26°C
L-
=-L
6.
B6
TAP1-/-
1118
I ri
02m-/-
TAP1-/p2m-/-
Figure 5. Flow cytometry of cells from B6, TAP1-/-, f2m-/-, and
TAP1/02zm-'- mice. Splenocytes were stimulated with Con A in AIM-V
medium for 36 h at 37*C, and then cultured an additional 16 h at either
37* or 26°C. Aliquots of cells were stained for conformed Kb (Y3; A),
conformed Db (B22.249; B), or free Kb and Db (CaHC; C) before
cytofluorimetry. Mean fluorescence values from normal (B6) Con A-stimulated splenocytes incubated with the appropriate control (see Materials and
Methods) and secondary antibodies alone were subtracted as background.
Folding and Intracellular Transport of Free Heavy Chains
sialylation of Kb or Db (data not shown). Within the limits
of detection of this experiment, we conclude that the presence of a functional TAP1/TAP2 complex is required for intracellular transport of free class I heavy chains.
Cytofluorimetry of Class I Heavy Chains at the Surface of
Normal and Mutant Lymphoid Cells. As an adjunct to our
pulse-chase analyses, we measured class I surface expression
at steady state by flow cytometric analysis of Con A-stimulated splenocytes from normal (B6), TAP1- -, 1:m- -,and
TAP1/0 2m-'- mutant mice (all H-2b haplotype) with the
Y3 (aKb) and B22.249 (caDb) mAbs, and the gamma
globulin fraction from the aHC serum (caKb, D6). Fig. 5
A shows the relative proportion of Y3-reactive cell surface
class I heavy chains in the four cell types. At 370 C, TAP1-/cells express conformed Kb molecules at 5% of the levels observed in normal cells, whereas at 260 C, the relative expression increases to 35%, consistent with previous reports (6).
Neither 0:m- - nor TAP1/f 2m-"- cells express detectable
amounts of conformed K S molecules at 37 0 C; however,
cells cultured at 260 C show a modest rescue of Kb
expression to 10% of wild-type levels. The absence of any
detectable Kb expression in the double-knockout cells, even
at 260C, is consistent with the pulse-chase experiments described earlier (Fig. 4). While quantitative data have not been
presented here, we have observed that the rates of synthesis
of class I heavy chains, as assessed in biosynthetic labeling
experiments, are indistinguishable for the four cell types.
The cell surface expression of conformed D b molecules in
the four cell types was assayed with the mAb B22.249 (Fig.
5 B). In contrast to Kb, the expression of properly folded
Db molecules in TAP1- - cells is only marginally rescued at
260 C (9-fold vs. 2-fold increase in staining), consistent with
the pulse-chase experiment shown in Fig. 4 B. Interestingly,
after incubation at 26 0C, 32m - -' cells exhibit greater Db
cells (1.7-fold), suggesting that the
staining than TAP1-iDb molecule relies more on TAP-dependent peptides than on
02m to be transported to the cell surface. Cells lacking both
02m and TAP1 show no increase in staining after incubation
at reduced temperature.
Free class I heavy chains (Kb and Db) were detected on the
cell surface by cytofluorimetry with the aHC serum (Fig.
5 C). While the loss of TAP1 results in a drastic decrease
in expression of conformed class I molecules at 370 C (Fig.
5, A and B), cells lacking TAP1 express twice the levels of
free heavy chains observed in normal cells. Culturing normal
or TAP1-deficient cells in the presence of 10% FCS results
in a two- to threefold decrease in free heavy chain staining
(data not shown), supporting earlier findings that exogenous
bovine 32m can associate with free class I heavy chains at
the cell surface (32, 33). In 32m - 1- cells, total heavy chain
expression is enhanced fivefold by overnight incubation at
260 C (Fig. 5 C). Similar to the results obtained with the
mAb B22.249, low levels of free heavy chain staining are observed in the double-knockout cells at both temperatures, suggesting that Db may be expressed at the cell surface, albeit
weakly, in the absence of both peptide and 32m.
S2m-`-
1119
Machold et al.
Discussion
We have demonstrated the presence of properly conformed
H2- K, -D, and -L locus products at the surface of cells lacking
32m, a finding that generalizes previous observations on Db
and Ld to other class I alleles. Furthermore, we have shown
here that the intracellular transport of free heavy chains in
32m-'- cells is dramatically enhanced by the presence of a
functional TAP1 gene.
Although many immunochemical reagents directed against
mouse class I molecules have been prepared, few recognize
non-3 2m-associated heavy chains exclusively. The Kb-specific
antipeptide 8 serum (ap8) has been used in combination with
conformation-sensitive reagents to document pools of free
Kb heavy chains (34, 35), but this antiserum is not specific
for free heavy chains. Lie et al. (36) have extensively characterized a partially folded form of the Ld molecule (Ldalt),
which is recognized by the 64-3-7 mAb. Ldalt heavy chains
are weakly associated with 32m and can be induced to fold
into a native conformation, as defined by the mAb 30-5-7,
upon incubation with pepride (10). Here, we introduce a rabbit
acHC that reacts primarily with class I molecules when not
associated with 02m. In this respect, the czHC serum is
analogous to the human class I reagents rabbit oaHC (37),
HC10 (37), HCA2 (25), and LA45 (38), all of which are
largely, if not exclusively, specific for free heavy chains. We
used the caHC serum to define a population of non-02massociated class I heavy chains, both in detergent extracts and
at the cell surface, that are immunochemically distinct from
heavy chains in a complex with 02m.
The conformed state of class I molecules is operationally
defined here by the presence of epitopes recognized by immunochemical reagents such as the aH-2 serum and the
mAb Y3 (19). How does the balance between conformed
and unfolded heavy chains depend on peptide and/or 02m?
To address this question, we compared the pools of conformed and unfolded class I heavy chains in H-2b Con
A-stimulated splenocytes deficient for TAP1, /2m, or both
(Fig. 4). We found that TAP1-/- cells express approximately
equal amounts of conformed and unfolded Kb and Db heavy
chains, whereas in cells lacking a2m or both TAPI and 02m,
the majority of the class I heavy chains are unfolded. Using
a panel of conformation-sensitive mAb and antisera, we detected low levels of properly conformed class I molecules in
H-2b (Figs. 3 and 4), H-2d, and H-2' (Fig. 2) 32m-/- cells
incubated at 370C. As expected, conformed free heavy chains
were unstable under physiological conditions; however, by
lowering the incubation temperature to 260 C, the proportion of folded to unfolded heavy chains in 32m- - cells was
increased (Figs. 3 and 4).
To demonstrate the presence of conformed free heavy chains
on the cell surface and to address the requirements for intracellular transport of the latter, we analyzed mutant mice
lacking either TAP1, I:m, or both for cell surface class I
molecules. The majority of class I complexes synthesized in
TAP1-!- cells are functionally "empty," based on their in-
stability in the absence of exogenous peptides (5) and thermolability (6). As anticipated, we found that free class I heavy
chains were expressed on the surface of TAP1-"- cells at
high levels relative to normal H-2b cells (Fig. 5), presumably as a consequence of increased dissociation of class I complexes in the absence of TAP-dependent peptides. In contrast
to K b , we found that the expression of Db molecules in
TAP-deficient cells was only rescued to a minor extent upon
incubation at 260 C (Figs. 4 and 5), suggesting that "empty"
Db molecules are less competent for intracellular transport
than their Kb counterparts.
Accumulation of free class I heavy chains at the surface
of.32m- - cells has been observed only for Db and Ld molecules (7, 9, 10). By cell surface iodination, we observed K b
heavy chains at the surface of 32m-;- cells incubated at
370 C (Fig. 3). We detected sialylation of class I heavy chains
in H-2b, H-2d, and H-2 032m -- cells (Figs. 2 and 4) chased
at reduced temperature (260 C). Low temperature partially
rescued the folding (Fig. 3), inhibited degradation (Figs. 3
'
and 4), and increased cell surface levels (Fig. 5) of free Kb
and Db heavy chains. The relationship between folding and
intracellular transport of class I molecules is well established
(39); thus, it is not surprising that we observed increased
intracellular transport of K b molecules under conditions that
favor the folding of the latter. We conclude that conformed
free class I heavy chains are expressed on the surface of
02m - - cells, thus demonstrating that i2m is not strictly
necessary for the transport of class I molecules.
Free Db heavy chains in detergent lysates have been shown
to fold into a nativelike conformation upon addition of peptide (16). In 02m -'- cells incubated at low temperature,
some proportion of free heavy chains may bind peptide in
the ER and thereby escape retention by calnexin (40). We
observed that Con A-stimulated splenocytes from the doublemutant (TAP1/0 2m-' - ) mice did not express detectable
levels of either sialylated Kb or Db, even at reduced temperature (Fig. 4). Likewise, we failed to detect either conformed
or unfolded class I heavy chains at the surface of double-mutant
cells by surface iodination (data not shown). When analyzed
by cytofluorimetry, double-mutant cells exhibited very low
levels of staining with Db-reactive antibodies (Fig. 5), although no increase in fluorescence was seen after incubation
at reduced temperature. Our results demonstrate that a functional TAP complex influences the cell surface expression of
free Kb and Db molecules, from which we infer that free
heavy chains can bind peptide in living cells.
Our results provide a biochemical basis for the ability of
Kb-restricted CD8 + T cells to interact functionally with
H-2b 02m-/- cells. One model system for studying positive
selection of immature thymocytes in vitro uses FTOC prepared from 3 2m- ' - mice (41). Recent work has demonstrated that functional CD8- T cells expressing a transgenic
Kb- or Db-restricted TCR can be generated in 22m - / FTOC upon addition of the appropriate peptide and 32m
(13, 42). Our data suggest that a proportion of Kb and Db
molecules at the surface of 3 2m- - cells may contain endogenously bound peptides, based on the requirement for
TAP-dependent peptides in the intracellular transport of free
Kb and Db heavy chains. In view of the widely different concentrations of peptide required to rescue emergence of a lymphocytic choriomeningitis virus peptide-specific Db-restricted
TCR in 2zm-1- (42) and in TAP1-/- FTOC (43), it is possible that the presence of TAP-dependent peptides bound to
Db in the former, but not the latter, situation may result in
different threshold concentrations of peptide required for key
selection events.
-/ We thank Dr. S. Tonegawa for making available the TAP1-/- mice, Dr. B. Koller for the H-2 $z2m
mice, Dr. T. Hansen for the H-2d $2m- - mice, and Dr. D. Roopenian for the H-2' 02m- - mice. In
addition, we thank Dr. S. Nathenson for providing us with purified soluble H-2Kb and Db and the aeH-2
serum, and Dr. G. Waneck for providing the mAb K10.56.1.
This work was supported by National Institutes of Health grants R01-AI33456-01 and R01-AI07463-17.
Dr. L. Van Kaer is supported by the Howard Hughes Medical Institute.
Address correspondence to Dr. H. L. Ploegh, E17-322, Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames St., Cambridge, MA 02139. S. Andr6e's and H.-G. Ljunggren's permanent
address is The Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden.
L. Van Kaer's present address is The Howard Hughes Medical Institute, Vanderbilt Medical School, Department of Microbiology and Immunology, Nashville, TN 37232.
Received for publication 25 July 1994 and in revised form 14 October 1994.
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1122
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Folding and Intracellular Transport of Free Heavy Chains
Chapter 3
Intermediates in the assembly and degradation of class I MHC
molecules probed with free heavy chain-specific monoclonal
antibodies
Machold, R.P. and Ploegh, H.L.
Submitted for publication to the Journal of Experimental Medicine
Abstract
Unassembled ('free') heavy chains appear during two stages of the class
I MHC molecule's existence: immediately after translation but prior to
assembly with peptide and 32-microglobulin, and later, upon disintegration of
the heterotrimeric complex. To characterize the structures of folding and
degradation intermediates of the class I heavy chain, three monoclonal
antibodies have been produced that recognize epitopes along the H-2Kb heavy
chain which are obscured upon proper folding and subsequent assembly with
32-microgloblin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193198). The Kb heavy chain is initially inserted into the lumen of the
endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and
KU4. Perturbation of the folding environment with the reducing agent
dithiothreitol (DTT) or the trimming glucosidase inhibitor N-7-oxadecyldeoxynojirimycin (7-O-dec) prolongs the presence of mAb-reactive Kb heavy
chains. At the cell surface, a pool of free Kb heavy chains appears following
60-120' of chase, whose subsequent degradation is impaired in the presence of
concanamycin B, an inhibitor of vacuolar acidification.
Introduction
Peptides derived from proteins degraded in the cytosol are presented to
the immune system by class I MHC molecules (1). Assembly of the class I
MHC heavy chain, a type I membrane glycoprotein, with 02- microglobulin
and peptide occurs rapidly after co-translational insertion of the subunits into
the endoplasmic reticulum (2). En route to heterotrimer formation, the
nascent heavy chain transiently associates with the ER - resident proteins
calnexin and calreticulin, lectins which bind to and retain incompletely
assembled or misfolded proteins in the ER (3). Inhibition of the binding of
calnexin/calreticulin to the nascent murine class I heavy chain decreases the
efficiency of assembly of the latter with 02-microglobulin(4), in agreement
with studies on influenza hemagglutinin maturation, in which newly
synthesized HA subunits were more likely to aggregate and/or form aberrant
disulfide bonds in the absence of calnexin/calreticulin association (5). After
the heavy chain and 1 2m associate, the heterodimer binds to the peptide
transporter complex TAP1/TAP2 (6)(7), an interaction that likely facilitates
peptide loading of the "empty" class I molecule. Properly assembled class I
complexes appear rapidly (<5') after initial completion of the polypeptide
chains (8), and are subsequently transported to the plasma membrane via the
secretory pathway. Once at the cell surface, class I molecules are largely stable
(9), although exchange of bound 32m for bovine t2m present in serum has
been observed for both murine and human class I molecules (10). Little if
anything is known about how class I molecules are destroyed after they have
exceeded their useful lifespan at the cell surface.
Antibody reagents that have been prepared against class I molecules
can be categorized as 1) folding -or assembly- independent, 2) folding -or
assembly- dependent, or 3) specific for the free heavy chain. For example, in
the case of the mouse class I molecules, the antiserum p 8 (11), raised against a
peptide derived from the cytoplasmic tail of H-2Kb molecules, recognizes all
forms of the H-2Kb heavy chain, whereas the mAb Y3 (12) binds only to
properly conformed states, and the rabbit anti free heavy chain serum binds to
non-assembled material exclusively (13). Unfolded forms of class I heavy
chain are likely to appear at two points during the lifetime of the molecule:
early in the course of folding and assembly in the ER, and later, immediately
prior to degradation. In order to characterize this population of class I heavy
chains in living cells, we have prepared three monoclonal antibodies against
denatured Kb molecules that recognize non - assembled heavy chains
exclusively. Folding and assembly studies on other glycoproteins such as the
mouse class I molecule H-2Ld (14)(15), or the influenza hemagglutinin
molecule (5), have utilized conformation-dependent antibodies to
characterize the structure of biosynthetic intermediates; this study
complements those by utilizing antibodies whose epitopes are exposed only
when the protein is unfolded and has not yet associated with t2microglobulin. We show that these antibodies can be used to
immunoprecipitate both assembly and degradation intermediates from pulse
labeled cells. Our results suggest that the folding of the extracellular region is
a rapid process, in which all of the epitopes detected by our antibodies
disappear synchronously.
Materials and Methods
Cell lines. The Rauscher virus-transformed mouse lymphoma cell line
RMA (16) was grown in RPMI 1640 supplemented with 10% fetal calf serum,
L-glutamine (2 mM), penicillin (1:1000 dilution U/ml), streptomycin (100
jgg/ml), sodium pyruvate (1 mM; Gibco BRL, Grand Island, NY), nonessential amino acids (0.1 mM; Gibco BRL), and P-mercaptoethanol (0.1 mM;
American Bioanalytical, Natick, MA). Splenocytes from C57BL/6 mice were
stimulated in the above medium supplemented with 2.5 [ig/ml concanavalin
A (ConA) for 48 hours prior to labelling.
Antibodies.
The following antisera and mAbs were used: Y3 (IgG2b;
recognizes the al/a2 domain of properly conformed Kb molecules; (12)),
rabbit anti-mouse free heavy chain (RafHC; recognizes non-02m associated
mouse class I heavy chains; (13)), rabbit anti-p8 (recognizes the cytoplasmic
tail of Kb; prepared in our lab essentially as described (11)) and rabbit anticalnexin (recognizes the C-terminus of calnexin; a gift of Dr. D. Williams).
The conformation sensitive rabbit anti-H2 serum was provided by Dr. S.
Nathenson. The mAbs introduced in this study (KU1, KU2, and KU4) were
generated by immunizing mice with inclusion bodies of recombinantly
expressed Kb heavy chains (a gift of Dr. S. Nathenson). Production of
hybridomas was performed as described (17). All three antibodies were
purified from cell culture supernatants with protein A-Sepharose (Repligen
Corp., Cambridge, MA), and used in immunoprecipitations at 10 gtg/ml lysate.
Western blotting. 107 freshly isolated splenocytes from C57BL/6 (b
haplotype), BALB/c (d haplotype), C3H (k haplotype), or PLJ (u haplotype)
mice were lysed directly in either 2x SDS sample buffer or 1x IEF sample
buffer and run on a 12.5% SDS-PAGE gel, or 1D-IEF gel (18), respectively. Prior
to blotting, the IEF gel was washed 5 times (10' each) with 300 ml of a solution
of 50% (v/v) methanol, 5 mm Tris/HC1, pH 8.0, and 1% SDS. The transfer to
nitrocellulose was performed in 25 mM Tris base, 200 mM glycine, and 20%
methanol for 2.5 hrs at 400 mA in a Biorad Trans Blot Cell (Biorad, Hercules,
CA). After a 2 hr incubation in blocking buffer (phosphate buffered saline
with 10% nonfat powdered milk and 0.05% TWEEN-20), blots were probed
with either RafHC (1/3000 dilution) or KU2 (5 ptg/ml) overnight, washed in
PBS/0.05% TWEEN-20, and probed for 1 hr with HRP-conjugated goat antirabbit or goat anti-mouse IgG (Southern Biotechnology Assoc., Birmingham,
AL) prior to visualization with ECL (Kirkegaard and Perry Laboratories,
Gaithersburg, MD).
Pulse chase analyses and immunoprecipitations. RMA cells or ConAstimulated splenocytes were starved in RPMI 1640 medium lacking
methionine and cysteine for 45' prior to being pulsed with 500 gCi/ml
[35S]methionine/cysteine (80:20) for the times indicated. When included, DTT
(5 mM) was added 5 minutes prior to labelling, and the inhibitors N-7oxadecyl-dNM (7-0-dec; 2 mM) and concanamycin B (ConB; 20 nM) were
added during the starvation period. Labelling was terminated by adding 1 mM
cold methionine/cysteine to the cell suspension. For the short (1-2') pulse
chase analyses (Figures 3 and 4), aliquots of cells (2-3x10 6) were removed at
each time point and directly lysed in ice cold digitonin lysis buffer (0.5%
digitonin (Sigma), 25 mM HEPES, pH 7.2, 10 mM CaC12, 1 mM PMSF, 10 mM
iodoacetamide) containing mAb or antiserum. Following centrifugation of
the cell lysates to remove nuclei and cellular debris, immune complexes were
isolated after a 2 hr incubation (with agitation) at 40C by incubation with 50 kl
10% fixed Staphylococcus aureus (Staph. A) for an additional 45'. The Staph.
A pellets were washed once in ice cold digitonin lysis buffer and then boiled
for 10' in denaturation/reduction buffer (2% SDS, 5 mM dithiothreitol, 50
mM Tris/HC1, pH 7.8, 1 mM EDTA). Following one preclearing step with
normal rabbit serum, the eluted Kb class I heavy chains were then reimmunoprecipitated in NP-40 lysis buffer (0.5% Nonidet P-40, 50 mM
Tris/HC1, pH 7.4, 5 mM MgCl 2, 1 mM PMSF, 10 mM iodoacetamide) with the
anti-p8 antiserum prior to gel analysis.
For the longer pulse (5') chase experiment shown in Figure 5, aliquots of
cells (ConA-stimulated splenocytes) were spun down at each timepoint, lysed
in NP-40 lysis buffer, and the post-nuclear supernatant precleared once with a
1:1 mixture of normal rabbit and normal mouse serum with 50 pl Staph. A
prior to immunoprecipitation of class I heavy chains. For the pulse chase in
Figure 6, due to the high background commonly observed in
immunoprecipitates from RMA cell lysates, we employed the direct
immunoprecipitation/re-immunoprecipitation methodology described
above. In vitro transcription and translation reactions were performed as
described (22).
Epitope mapping. The epitopes recognized by the mAb's KU1, KU2, and
KU4 were determined by immunoprecipitation from a library of M13 phage
displaying a random 10 amino acid insert (19). The library was constructed
from a vector encoding the fd-tet phage and the tetracycline gene. Prior to
immunoprecipitation, the phage suspension (6x10 10 phage units/0.8 ml
Nonidet P-40 lysis mix) was precleared with normal mouse serum (2 1l)
added with a 1:1 suspension of protein A - Sepharose beads for 1 hr at 40C.
Immunoprecipitations were performed by incubating the precleared phage
suspension with 10.tg/ml mAb for 2 hr at 40C, following which the
antibody/phage complexes were adsorbed onto protein A-Sepharose beads,
collected by centrifugation, and washed 5 times in NET buffer (0.5% NP-40, 50
mM Tris/HC1, pH 7.4, 150 mM NaC1, 5 mM EDTA). Phage were eluted from
the beads with 200 p1 of glycine HC1, pH 2.2 for 15' at 40 C, neutralized with
Tris/HC1 (pH 9) to pH 6-8, and then grown up on K91 (kanamycin resistant)
cells cultured in the presence of 20 gg/ml tetracycline. The mAb-selected
phage were further purified through two additional rounds of
immunoprecipitation, elution, and amplification, following which clones
were picked and amplified for sequencing.
RESULTS
Epitope mapping and specificity of free heavy chain monoclonal antibodies.
With the aim of characterizing partially folded or assembled forms of the Kb
class I heavy chain, we raised three monoclonal antibodies against denatured
Kb molecules: KU1, KU2, and KU4. The immunogen was injected as ureasolubilized inclusion bodies, and presumably contained little coherent
structure. We therefore expected that antibodies from this immunization
would recognize unstructured epitopes along the Kb polypeptide chain. Thus,
we employed an M13-based phage display library (19) encoding a random 10
amino acid insert to map the epitopes for each monoclonal antibody. Three
representative inserts are shown for each antibody (Fig. la), although we
sequenced at least 10 clones from each pool of selected phage to determine the
anchor residues for each epitope. Beneath each consensus sequence is shown
the corresponding Kb sequence and the sequences of other class I heavy chains
in the same region (20). The location of each epitope could be established
without ambiguity, and their projection onto the fully assembled Kb molecule
is shown in Fig. lb (21). The epitope recognized by KU1 (residues 49-54) is
normally folded into a 310 helix that immediately precedes the long ca helix in
the al domain that forms one side of the peptide binding groove. The
involvement of four out of five consecutive residues in generation of the
KU1 epitope argues in favor of recognition of this stretch in non-helical
configuration, and conversely, folding of this region should conceal the KU1
epitope. KU2 binds to an epitope that spans part of the second 03 strand and
connecting loop sequence in the cal domain (residues 23-30); this region
contains residues that directly contact P2m (Y27, E32). The KU4 epitope maps
to the first p strand in the ca3 domain (residues 193-198) and is adjacent to
residues that contact t2m (R202, W204) as well as one of the cysteines (C203)
that participate in the a3 disulfide bond. Note that the residues recognized by
each antibody, as identified by phage display, are buried or rearranged upon
folding of the heavy chain and assembly with 0 2m.
The results of the epitope mapping described above and sequence
comparisons for class I heavy chains in that region suggested that KU1 and
KU2 would recognize Kb exclusively, and that KU4 would recognize both Kb
and Kk molecules. To probe directly the specificity of the antibodies, Western
blots were performed on splenocyte extracts prepared from mice of b, d, k, and
u haplotype resolved on SDS-PAGE and 1D-IEF gels (Fig. 2). As a control, we
blotted with the rabbit anti-free heavy serum (RafHC), which recognizes most
murine class I heavy chains (13). As anticipated, KU2 recognized Kb heavy
chains exclusively (Fig. 2b), whereas RafHC decorated all of the class I material
present (Fig. 2a). Neither KU1 nor KU4 blotted as efficiently as KU2, although
the specificity for each was as expected based on the epitope mapping (data not
shown). Further confirmation of the predicted epitopes for the antibodies was
obtained from immunoprecipitations performed on Kb C-terminal truncation
fragments translated in vitro (KU1, KU2, and KU4 all react with translation
products containing Kb residues 1-204, only KU1 and KU2 react with
fragments containing residues 1-74; data not shown).
The monoclonal antibodies KUI, KU2, and KU4 recognize unfolded forms
of the Kb heavy chain.
Having mapped the binding sites for each
monoclonal antibody, we next sought to determine at which points these
epitopes would be exposed during the life of the Kb heavy chain. Little if any
detectable material could be immunoprecipitated with the antibodies from
RMA cells labeled for 60' (data not shown), suggesting that, while unfolded
forms of the heavy chain might appear transiently during both biogenesis and
degradation, they were too unstable to accumulate to appreciable levels at
steady state. We analyzed reactivity of KU1, KU2, and KU4 on Kb molecules
translated in vitro under conditions that support folding and assembly (22).
We observe that none of the monoclonal antibodies recognize any 02microglobulin containing forms of heavy chains, whereas the conformation
sensitive rabbit anti-H2 antiserum clearly does (Fig. 3a). The stronger
reactivity of KU2, compared to KU1 and KU4, is demonstrated in this
experiment, although the behavior of KU1, KU2, and KU4, in terms of loss of
reactivity with Kb heavy chains, appears indistinguishable (see also Figs 3b
and 4). Because of the stronger reactivity of KU2, much of the remainder of
the experiments was performed with this antibody.
We first chose to examine the early events in the assembly of the Kb
molecule, by performing a series of brief pulse chase experiments on RMA
cells with pulse times of one minute in order to resolve the rapid early stages
of Kb folding and calnexin association. To minimize the lag time between cell
lysis and exposure to antibody, aliquots of cells removed from the chase
mixture were added directly to lysis buffer containing the relevant antibody
("1o antibody") and immune complexes were recovered (8). The crude
immunoprecipitates were then denatured completely by exposure to 2% SDS
at 100 0 C, and re-immunoprecipitated with the p8 antiserum, which
recognizes all full length Kb heavy chains. The kinetics of assembly of the Kb
molecule are evident from the pulse chase experiment depicted in Fig. 3b:
properly conformed class I complexes (Y3 immunoprecipitates) form within 5
minutes, while conversely, free heavy chains (RafHC) are present
immediately following completion of the 1' pulse, but are largely assembled
beyond 5' of chase. KU2 also binds to nascent heavy chains extracted within
the first 5' of synthesis, suggesting that the zal domain P-strand recognized by
the antibody remains accessable until the heavy chain has assembled with
t2m. Although the recovery of calnexin-bound heavy chains was inefficient,
notwithstanding the use of digitonin lysis buffer, the peak of heavy chains coimmunoprecipitated with calnexin occurs around 2' - 5' after the 1' pulse,
slightly later than the appearance of the RafHC and KU2- reactive material.
This observed lag suggests that calnexin may not be stably bound to the
nascent heavy chains until 1 - 2' following completion of the Kb polypeptide
chain. While folding clearly is an early and rapid process, and is likely to start
on the nascent chain (5), it is completed post-translationally.
Both KU1 and KU4 could also immunoprecipitate nascent class I heavy
chains within the first few minutes following completion of the polypeptide
chain (Fig. 4a). To determine if disulfide bond formation is required for the
folding of the regions surrounding the epitopes recognized by the antibodies,
we performed a short pulse chase experiment in the presence of the reducing
agent dithiothreitol (DTT; 5 mM), which has been shown to prevent the
formation of disulfide bonds in living cells (Fig. 4b)(23). Under these
conditions, no properly conformed Kb molecules are detectable, even after 30'
of chase (Y3 panel). Unassembled Kb heavy chains persist throughout the
chase (RafHC panel), and all three monoclonal antibodies show prolonged
reactivity, suggesting that, in the absence of proper disulfide bond formation,
the folding of three distinct regions of the class I heavy chain is impeded.
To investigate the role of calnexin/calreticulin in the early folding
events of the class I heavy chain, we performed a short pulse chase
experiment in the presence of the glucosidase inhibitor 7-O-dec (24) (Fig. 4c).
By blocking the trimming of terminal glucose residues on the N-linked
oligosaccharides of the class I heavy chains, 7-O-dec effectively prevents the
association of calnexin/calreticulin with the latter (25). The formation of
properly conformed class I complexes (Y3 panel) still occurs in the absence of
calnexin/calreticulin binding, consistent with recent studies performed on
the folding of influenza hemagglutinin (5), although assembly of free heavy
chains with 02-microglobulin is clearly less complete over the chase period
(RafHC panel). All three monoclonal antibodies react with class I heavy
chains immediately after their synthesis, and by 15', much of the reactivity
has been lost. However, unfolded Kb heavy chains reappear by 30', most likely
from the disintegration of unstable class I complexes that were formed in the
absence of the quality control normally provided by calnexin/calreticulin.
Kb molecules unfold at the cell surface and are degraded in an acidic
compartment. To follow the fate of free Kb heavy chains at the cell surface,
we performed a pulse chase experiment on ConA-stimulated splenocytes
(prepared from C57BL6 mice) with a chase time of two hours, and resolved
the RafHC and KU2 - reactive class I material on SDS (Fig. 5a) and 1D-IEF (Fig.
5b) gels. The chase was performed at 26 0 C as well as 37 0 C since at the former
temperature, the stability of class I molecules has been shown to be markedly
enhanced. IEF allows the visualization of sialylated class I heavy chains.
Addition of sialic acids to the class I glycan occurs in the trans-Golgi network,
and immediately precedes surface deposition. For the description of the
results, we shall equate sialylation with cell surface expression. At the onset of
the chase, free heavy chains are observed exclusively in the ER (compare SDS
and 1D-IEF 0' timepoints), but appear at the cell surface by 30', most likely
from disintegration of unstable class I complexes. Fewer free heavy chains are
detectable during the 260 C chase, consistent with the increased stability of
class I complexes at this temperature.
KU2 - reactive heavy chains appear abundantly late in the chase,
following the loss of
P 2m
from unstable class I molecules at the cell surface
(compare KU2 immunoprecipitates, SDS and IEF panels, 120' timepoints). At
the lower temperature, free heavy chains that accumulate at the cell surface
(see RafHC, 260 chase, IEF panel) maintain a conformation that is not
recognized by the KU2 antibody (compare RafHC and KU2 panels, 120'
timepoints, 26 0 C chase). Thus, the free heavy chain is capable of retaining the
conformation of properly folded molecules, even after 02m dissociation,
consistent with studies on the H2-Ld heavy chain (26), as well as with our
previous observations (13). The KU2 antibody only recognizes a fraction of
the free heavy chains that have been retained in the ER at either temperature
(compare RafHC and KU2 panels, IEF gels); clearly, most of these free heavy
chains are maintained in a conformation or complex that obscures the
epitope. It is unlikely that calnexin is responsible for this effect, as the noncovalent complex between calnexin and the class I heavy chain does not
survive exposure to NP-40 (27). Because RafHC - reactive Kb heavy chains
persist at timepoints where little reactivity is observed with the monoclonal
antibody KU2, we infer that the latter is the more stringent tool for
monitoring folding, whereas RafHC reports on both folding and assembly:
association of Kb heavy chains with 0 2m obscures the RafHC reactivity. We
note that intact cells can be stained with the monoclonal antibodies as
revealed by cytofluorimetry, and that such staining is reduced for cells
maintained at 26 0 C (data not shown), a condition known to prevent
unfolding of otherwise labile Kb molecules (28).
By what means are class I complexes at the cell surface degraded? If
breakdown occurs in lysosomal compartments, pH neutralization of these
compartments might inhibit the dissociation of the heavy chain from the
class I molecule, or degradation of the latter, or both. To address this question,
we pulse-labeled RMA cells in the presence or absence of concanamycin B
(ConB), a fungal macrolide antibiotic that inhibits vacuolar H + ATPases (29),
and followed the pools of conformed (Y3), free (RafHC), or unfolded (KU2) Kb
molecules over the three hour chase period (Fig. 6). Due to the higher
background typically seen in immunoprecipitations from briefly pulselabeled RMA cells, we utilized the direct immunoprecipitation/reimmunoprecipitation methodology as described for the short pulse chase
experiments in Figures 3 and 4. This technique also results in improved
recovery of nascent heavy chains that are only reactive with KU2
immediately following their deposition in the ER. Heavy chains that
dissociate at the cell surface in untreated cells are largely degraded after three
hours of chase (Fig. 6a). In contrast, when cells are treated with ConB, the
destruction of both RafHC- and KU2- reactive heavy chains is inhibited.
Quantitation by phosphoimaging (6c) reveals that the decay of properly
formed complexes (Y3 panel) is similar in the presence and absence of the
inhibitor, suggesting that degradation, but not disintegration, of the class I
complex is dependent on lysosomal function. Neuraminidase digestion of
intact cells chased in the presence of ConB demonstrates that after three
hours, about half of the sialylated KU2-reactive heavy chains are cell surface
disposed, while the remainder resides within the cell, presumably within
lysosomes (data not shown).
Discussion
The pool of class I molecules within the cell is comprised of both
partial (i.e., monomeric and heterodimeric) and complete (i.e.,
heterotrimeric) complexes. Intermediate forms of the class I molecule are
most evident during the initial folding and assembly processes that occur in
the ER, as well as during the disintegration of the complex at the cell surface.
The three monoclonal antibodies introduced in this study have allowed us to
characterize in part the structure of non-p 2m associated free heavy chains as
they appear in the course of the class I molecule's lifetime. These reagents
recognize epitopes on the class I heavy chains that are buried upon folding
and assembly with P2-microglobulin, and thus can be used to monitor the
local conformation of the respective subdomains surrounding each epitope.
Nascent class I heavy chains interact with the ER-resident chaperones
calnexin and calreticulin rapidly after synthesis (30)(3). We show that three
distinct epitopes, recognized by KU1, KU2, and KU4, persist after synthesis for
at least 5 minutes, during which period, the heavy chains are associated with
calnexin (fig. 3). We have been unable to observe any differential loss of
reactivity over time with the three antibodies in the course of folding and
assembly of the class I heavy chain, in contrast to recent studies performed on
the much larger glycoprotein, influenza hemagglutinin, which have
delineated a number of discrete folding intermediates (5). If the
calnexin/calreticulin interaction is abrogated by incubation of cells with the
glucosidase inhibitor 7-O-dec, then a pool of mAb- reactive heavy chains
reappears later in the chase, demonstrating that, even though properly
conformed molecules are formed in the absence of calnexin/calreticulin
association, many of the heavy chains do not fold productively, in agreement
with results from the influenza hemagglutinin system (25) as well as with a
recent study on the murine class I molecules H-2Kb and Db (4). In the presence
of the reducing agent DTT, the folding of the heavy chains is blocked at an
early stage, prior to the packing of the epitopes recognized by each of the three
monoclonal antibodies. However, since calnexin contains disulfide bonds
that are essential for its function (31), addition of DTT most likely perturbs the
association between the class I heavy chain and calnexin as well, and thus
heavy chains synthesized under reducing conditions must attempt to fold
both without disulfide bonds and calnexin.
Once the class I molecule has been properly assembled, it proceeds to
the cell surface via the secretory pathway. Soon after deposition at the plasma
membrane however, a fraction of the class I complexes dissociates, as
evidenced by the appearance of sialylated free heavy chains 60' - 120' after
synthesis. In contrast to the pool of intracellular free heavy chains that persist
throughout the chase, the cell surface-disposed free heavy chains are also
reactive with the KU2 mAb, and thus are unfolded to a greater extent. One
explanation for this rapid disintegration of a portion of class I molecules is
that complexes that have bound low affinity peptides might attain a
transport-competent structure, but then lose peptide once they arrive at the
cell surface. This fraction may be stabilized by suitable peptide ligands added
extracellularly (32)(33), but should no peptides be available, the complex is
more likely to resolve into the separate subunits. Treatment of cells with
concanamycin B, a vacuolar proton pump inhibitor, prevents the subsequent
destruction of sialylated free heavy chains, implying that degradation occurs
intracellularly in an acidic compartment. While generally difficult to detect,
internalization of class I molecules is unlikely to be restricted to free heavy
chains, and thus, this pathway may provide an opportunity for "empty" class
I molecules which are not degraded to bind peptides derived from exogenous
antigens (34).
Acknowledgements
We would like to thank Dr. D. Williams for the rabbit anti-calnexin
antiserum, and Dr. S. Nathenson for the purified soluble H-2Kb and the rabbit
anti-H2 antiserum. This work was supported by National Institutes of Health
grants R01-AI33456-01 and R01-AI07463-17.
Figures
Fig. 1. Epitope mapping of the monoclonal antibodies KU1, KU2, and KU4. a)
Three representative phage insert sequences selected by each antibody are
shown, with translation, above the Kb sequence that is predicted to contain
the epitope. Residues in the phage sequences that match the actual Kb
sequence are shown in capital. For comparison, sequences of other class I
heavy chains are shown below each predicted epitope. b) Ribbon diagrams of
the properly conformed Kb heavy chain, highlighting in grey the locations of
the predicted epitopes.
Fig. 2. Western blot analysis of diverse class I material. Splenocyte extracts
from H-2 b, d, k, and u haplotype mice were resolved on SDS-PAGE or 1D-IEF
gels and blotted with either RafHC (2a) or KU2 (2b).
Fig. 3. The monoclonal antibodies KU1, KU2, and KU4 are specific for free
heavy chains. a) mRNAs for H-2Kb and mouse P2m were translated in vitro
for 60', following which class I molecules were immunoprecipitated with the
anti-H2 antiserum, or the monoclonal antibodies KU1, KU2, and KU4, prior
to analysis by SDS-PAGE. b) RMA cells were pulsed for 1' and chased for the
times indicated. Aliquots of cells were lysed directly in digitonin lysis mix
containing either Y3 (class I complexes), RafHC (free heavy chains), KU2, or Cacalnexin. Prior to SDS-PAGE analysis, the immunoprecipitated class I
material was re-immunoprecipitated with p8.
Fig. 4. Effects of redox potential and calnexin association on the folding and
assembly of the Kb molecule. RMA cells were pulsed for 1' and chased for the
times indicated. Nascent heavy chains were directly immunoprecipitated
from NP-40 detergent lysates as in Fig. 3 with the antibodies indicated in each
panel. The reducing agent DTT (4b) was added to cells at 5 mM 5' prior to
labelling, whereas the glucosidase inhibitor 7-O-dec (4c) was added to cells at 2
mM during starvation (45' prior to labelling).
Fig. 5. Appearance of unfolded class I heavy chains at the cell surface. ConA
stimulated splenocytes were pulse labeled for 5' and chased at either 370 or
26 0 C for the times indicated. Free (RafHC; recognizes both Kb and Db) or KU2reactive heavy chains (Kb alone) were immunoprecipitated from precleared
lysates and resolved on SDS-PAGE (5a) or 1D-IEF (5b) gels.
Fig. 6. Breakdown of unfolded Kb heavy chains at the cell surface. a) RMA
cells were starved for 45' in the absence or presence of the vacuolar H +ATPase inhibitor ConB, pulse labeled for 5', and then chased for the times
indicated. Class I heavy chains were directly immunoprecipitated from NP-40
detergent lysates (no initial preclear) with the antibodies indicated in each
panel, and re-immunoprecipitated with the p8 antiserum prior to SDS-PAGE.
b) Phosphoimager quantitation of the band densities in 6a. The data sets for
each pulse chase were normalized around the 60' chase point densities.
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Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold
Spring Harbor Laboratory.
18.
Ljunggren, H.G., M. Oudshoorn-Snoek, M.G. Masucci, and H.L.
PLoegh. 1990. High resolution one-dimensional isoelectric focusing of
murine MHC class I antigens. Immunogenetics 32:440-450.
19.
Smith, G.P., and J.K. Scott. 1993. Libraries of peptides and proteins
displayed on filamentous phage. Methods Enzymol. 217:228-57.
20.
Flaherty, L., E. Elliott, J.A. Tine, A.C. Walsh, and J.B. Waters. 1990.
Immunogenetics of the Q and TL regions in mouse. Crit. Rev Immunol. 10,
no. 2:131-171.
21.
Fremont, D.H., M. Matsumura, E.A. Stura, P.A. Peterson, and I.A.
Wilson. 1992. Crystal structures of two viral peptides in complex with murine
MHC class I H-2Kb. Science 257:919-926.
22.
Bijlmakers, M.J.E., J.J. Neefjes, E.H.M. Wojcik-Jacobs, and H.L. Ploegh.
1993. The assembly of H-2Kb class I molecules translated in vitro requires
oxidized glutathione and peptide. Eur. J. Immunol. 23:1305-1313.
23.
Braakman, I., J. Helenius, and A. Helenius. 1992. Manipulating
disulfide formation and protein folding in the endoplasmic reticulum. EMBO
J. 11:1717-1722.
24.
Tan, A., L. van den Broek, J. Bolscher, D.J. Vermaas, L. Pastoors, C. van
Boeckel, and H. Ploegh. 1994. Introduction of oxygen into the alkyl chain of
N-decyl-dNM decreases lipophilicity and results in increased retention of
glucose residues on N-linked oligosaccharides. Glycobiology 4:141-9.
25.
Hammond, C., I. Braakman, and A. Helenius. 1994. Role of N-linked
oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein
folding and quality control. Proc. Natl. Acad. Sci. USA 91:913-917.
26.
Solheim, J.C., N.A. Johnson, B.M. Carreno, W.R. Lie, and T.H. Hansen.
1995. Beta 2-microglobulin with an endoplasmic reticulum retention signal
increases the surface expression of folded class I MHC molecules. Eur. J.
Immunol. 25:3011-6.
27.
Galvin, K., S. Krishna, F. Ponchel, M. Frohlich, D.E. Cummings, R.
Carlson, J.R. Wands, K.J. Isselbacher, S. Pillai, and M. Ozturk. 1992. The major
histocompatibility complex class I antigen binding protein p88 is the product
of the calnexin gene. Proc. Natl. Acad. Sci. USA 89:8452-8456.
28.
Ljunggren, H.G., N.S. Stam, C. Ohlen, J.J. Neefjes, P. Hoglund, M.T.
Heemels, J. Bastin, T.N.M. Schumacher, A. Townsend, K. Karre, and H.L.
Ploegh. 1990. Empty MHC class I molecules come out in the cold. Nature
346:476-480.
29.
Yilla, M., A. Tan, K. Ito, K. Miwa, and H.L. Ploegh. 1993. Involvement
of the vacuolar H+-ATPases in the secretory pathway of HepG2 cells. J. Biol.
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30.
Degen, E., and D.B. Williams. 1991. Participation of a novel 88-kD
protein in the biogenesis of murine class I histocompatibility molecules. J.
Cell. Biol. 112:1099-1115.
31.
Tector, M., and R.D. Salter. 1995. Calnexin influences folding of human
class I histocompatibility proteins but not their assembly with P2microglobulin. J. Biol. Chem. 270:19638-42.
32.
Townsend, A., C. Ohlen, J. Bastin, H.G. Ljunggren, L. Foster, and K.
Karre. 1989. Association of class I major histocompatibility heavy and light
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33.
Rock, K.L., C. Gramm, and B. Benacerraf. 1991. Low temperatures and
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cell surface. Proc. Natl. Acad. Sci. USA 88:4200-4204.
34.
Rock, K.L. 1996. A new foreign policy: MHC class I molecules monitor
the outside world. Immunology Today 17:131-137.
Antibody
selected
sequences:
5' gcg
agg ctg atg gag aac 3'
Kd
Kk
Db
Dd
Dk
Ld
HLA-A2
HLA-B27
gag gtg tcg tac acg gac gac 3 '
5'tcg
aaa cca gag gcc aag
... EVsY t D D
Sk PEaK
gcg agg gtc atg gag gac
atg tgg gtg agc tac atg ttc gac
tcc ggg ccc gag tcg aaa
MwVsYm
f D
SgPE s K
ggg gag gtg ctc tac ttg tcg gac
tcc cgg cca gag gag aag
g EV L Y L S D
SRPEe K
gcc agg gag atg gag gac
Kb
5'...
AR iME n
AR vME d
Class I MHC
sequences:
KU4
KU2
KU1
AReMEd
R WM E
-- P
49A
Q 54
--P------
23M E V G Y V DD
IA ---IS ---IS ----
---._p
-- P -- I--- P -- I--
IS
-- --- -,
IS
-- ---
IA -----IT-----
30
193S R P ED K198
P -- SQV D
----N
----N
----N
----N
3'
P -- S KG E
R ------ G D
P -- S KV E
P -- S KG E
AVSD H E
P ISD HE
lb
RafHC
haplotype:
bd ku
bd ku
Kb.
SDS-PAGE
1D-IEF
Fig. 2a
I
KU2
I
bd ku
4.
bd ku
w*
-t·_
Kb-
SDS-PAGE
1D-IEF
Fig. 2b
aH2 RafHC
KIUi KU2
HC
p2m _
Fig. 3a
KU4
10 antibody:
Chase time:'
(min.)
0
1
2
5
15
30'0
1
2
5
15
a-calnexin
KU2
RafHC
Y3
301 0
Fig. 3b
1
2
5
15
30
0
1
2
5
15
301
ChaseTime:
o0
Y3
Tim 30
15
15 30
0 RafHC
1530
0
0
15
30 io0
RafHC
Y3
r0
15
0
15
30 1 0
15
KU
15 1 30
i15
+ 7-O-dec
RafHC
30
15
0
KU1
30 ' 0
15
+ DTT
vs
30
0
15
--· --·------
Fig. 4
15
s
30
0
30 o
KU2
30 ' 0
KUl
30
KU2
15
15
O
15
30i
KU4
30 ' 0
KU2-15
30
KU2
30
KU4
15
15
30
KU4
0
15
30'
RafHC
Chase temp. (OC):
370
Time (min.):
0
30
KU2
260
120 0
30
'
370
120 0
30
260
120 0
30
120
Class I HCactin -
RafHC
Chase temp. (oC):
370
I
Time (min.):
30 120
KU2
26
370
260
li
Kb
370
•
0 30 120'
Db
Kb-
Sialviated
-
.
-own-
000h
0 30 120'
M
-01
-
260
•
0 30 120
"No
F
260
370
.04ft
Figs. 5a (top) and 5b
.
- ConB
RafHC
Y3
KU2
3 120 150 180
(min.)
-
w
+ ConB
Y3
RafHC
KU2
120 150 180
-
--
-
Fig. 6a
Y3
85UUU
U
Or
-ConB
+
6000-
C)
-"
1-a
CZ
4000-
(D
N
E
2000-
0-
60
60
r·
-
9012
90
-I-
-r
120
108
150
chase time (min.)
-I-
180
C',
oII
B
RafHC
4000 -
- ConB
S+ ConB
M
3000 -
C
a)
-D
C
CZ
2000-
N
E
1000 -
0-
-r
60
-r
90
-r
120
-I
150
chase time (min.)
180
KU2
-ConB
5000
0 +ConB
4000
Cn
4
3000
Co
N
2000-
O
1000
0
60
60
90
90
120
120
150
150
chase time (min.)
180
180
Chapter 4
The HCMV gene products US11 and US2 differ in their ability to
attack allelic forms of murine MHC class I heavy chains
Machold, R.P., Wiertz, E.J.H.J., Jones, T.R., and Ploegh, H.L.
Submitted for publication to the Journal of Experimental Medicine
Abstract
Human cytomegalovirus downregulates the expression of human class I MHC
molecules by accelerating destruction of newly synthesized class I heavy chains. The
HCMV genome contains at least two genes, US11 and US2, each of which encode a
product sufficient for causing the dislocation of newly synthesized class I heavy chains
from the lumen of the endoplasmic reticulum to the cytosol. Based on a comparison of
their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the
US11 and US2 gene products have non-identical specificities for class I molecules. The
diversity in HCMV-encoded functions that interfere with class I- restricted presentation
likely evolved in response to the polymorphism of the MHC.
Introduction
MHC class I molecules present peptides derived from proteins degraded in the
cytosol to cytolytic T cells, and as such are targets for viruses seeking to evade immune
recognition (1). The ability to down-regulate the expression of class I molecules in host
cells has been documented for a number of viruses, including adenovirus (2), herpes
simplex virus (3), and both mouse (4) and human cytomegalovirus (5). In HCMVinfected cells, newly synthesized class I MHC heavy chains are rapidly degraded
following their deposition in the lumen of the ER (6)(7)(8). The HCMV gene product
US11 ("US11"), an ER-resident type I transmembrane glycoprotein, has been shown by
transfection to be sufficient to cause the selective degradation of endogenous class I
MHC molecules (9). In the presence of US11, class I MHC heavy chains are dislocated
from the lumen of the ER into the cytosol, where they are deglycosylated by host Nglycanase, and then degraded by the proteasome (10). A second HCMV gene product,
US2, has also been observed to cause the premature destruction of newly synthesized
class I MHC molecules, apparently by the same pathway as described above for US11
((9), Wiertz et al., submitted). Since both US11 and US2 are expressed at the same stage
of infection, we sought to determine why the virus might encode two separate proteins
that appear to function in the same manner.
One possibility is that US11 and US2 recognize overlapping but nonidentical
subsets of class I MHC molecules. In the initial comparison of the US11 and US2
transfectants, only the degradation of endogenous human class I MHC molecules was
studied, and no differences in allelic selectivity between US11 and US2 were apparent
((9)(10), Wiertz et al., submitted). In human fibroblasts infected with HCMV, all
endogenous class I MHC molecules appeared to be destabilized (6)(7)(8). In mouse L
cells (H-2k) transfected with HLA-B27, the human, but not murine class I MHC
molecules were degraded upon HCMV infection, but the reason for this difference was
not established (6). In virus- infected cells, both US11 and US2 are presumably
expressed, and these experiments therefore cannot resolve possible differences in
substrate specificity between the two viral proteins. To probe the specificities of US11
and US2, we infected cell lines transfected with either US11 or US2 with a panel of
recombinant vaccinia virus expressing different mouse class I heavy chains (H-2Kb, Kd,
Db, Dd, and Ld) to determine whether certain heavy chains would be resistant to US11-
or US2- mediated degradation. We observe that US11 and US2 differ in their abilities to
degrade murine class I heavy chains, and thus are not functionally redundant.
Specifically, we find that US11 degrades Kb, Db, Dd, and Ld efficiently, whereas US2 is
most effective in degrading Db and Dd. We suggest that MHC polymorphism drives
diversification of viral evasion strategies.
Materials and Methods
Cells and Vaccinia Virus Infections. U373-MG astrocytoma cells and the US11 and US2
transfectants prepared from this cell line have been described (9). Cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% (v/v)fetal calf serum,
penicillin (1:1000 dilution U/ml), streptomycin (100 pg/ml), and puromycin (Sigma, St.
Louis, MO) at a final concentration of 0.375 gg/ml. Recombinant vaccinia virus
expressing H-2Kb (lacking the cytoplasmic tail), Kd, Db, Dd, and Ld were obtained from
Dr. J. Yewdell. Between 1 and 5 x 106 cells per sample were detached by treatment with
trypsin, resuspended in phosphate buffered saline supplemented with 1%fetal calf
serum, penicillin, and streptomycin, and then infected for 45' with recombinant vaccinia
virus at a multiplicity of infection of 10, after which 10 ml of media was added. Five
hours later, cells were starved in methionine/cysteine-free medium for 45' with or
without the proteasome inhibitor Cbz-LLL (10 pm final) (10) prior to labelling with 250
gCi/ml [35S] methionine/cysteine (80:20). Labelling was terminated by addition of 1
mM cold methionine/cysteine to the labelling mix. Aliquots of cells were spun down at
each chase point, and the cell pellets frozen prior to immunoprecipitation of class heavy
chains.
Antibodies and Immunoprecipitations. For immunoprecipitation of mouse class I heavy
chains, a rabbit polyclonal antiserum (RafHC) which recognizes non-assembled or
unfolded heavy chains was used (11). Cell pellets were each lysed in 1 ml ice cold lysis
mix (0.5% NP-40, 50 mM Tris/HC1, pH 7.4, 5 mM MgCl, 1 mM PMSF, and 10 mM
iodoacetamide), and the postnuclear supernatant precleared twice with 10% fixed
Staph. A prior to specific immunoprecipitation of murine class I heavy chains with
RafHC. To enhance the visualization of degradation intermediates, the RafHC
immunoprecipitates were boiled in denaturation buffer (2% SDS, 50 mM Tris/HC1, pH
7.8, 1 mM EDTA, 5 mM DTT), and the murine class I material re-immunoprecipitated
with RafHC prior to gel analysis. The N-glycanase digestions in Fig. 2 were performed
according to the manufacturer instructions (Boehringer Mannheim, Germany). SDSPAGE and one dimensional isoelectric focusing were performed as described (12).
Results
The mouse class I molecule H-2Kb is degraded by US11 but not by US2.
To probe the
specificity of the HCMV gene products US11 and US2 for class I MHC molecules, we
infected parental U373-MG cells, and transfectants expressing either US11 or US2, with
a panel of recombinant vaccinia virus encoding different mouse class I heavy chains,
and then performed a pulse chase experiment (10' labelling, 0' and 20' chase points) in
the presence of the proteasome inhibitor Cbz-LLL (Fig. 1). At each chase point, aliquots
of cells were pelleted and frozen at -80 0C prior to lysis and immunoprecipitation with a
rabbit anti-free heavy chain antiserum (RafHC). Immunoprecipitates were denatured by
boiling in 2% SDS, and re-immunoprecipitated prior to analysis by SDS-PAGE. In the
case of H-2Kb (lacking cytoplasmic tail) vaccinia virus- infected cells (panel A), class I
heavy chains synthesized in the 10' pulse are stable throughout the 20' chase in control
cells and in US2 + cells. However, in US11 + cells, the fully intact Kb heavy chains
observed immediately upon completion of labelling (panel A, 0' timepoint) are rapidly
converted into a faster migrating species. If the pulse chase is performed in the absence
of Cbz-LLL, then the Kb heavy chains in US11 + cells are fully degraded by 20', and no
intermediates accumulate (data not shown).
US11 and US2 were equally capable of degrading the mouse class I heavy chains
H-2Db (panel C), and Dd (panel D). However, when challenged with the H-2Ld
molecule (panel E), US11 was more efficient than US2 at causing the breakdown of the
heavy chains, as evidenced by the nearly complete conversion in US11 + cells of full
length Ld heavy chains into the breakdown intermediate over the 20' chase. In the case
of H-2Kd (panel B), the heavy chains decay more rapidly in the presence of either US11
or US2 than in control cells, but little degradation intermediate accumulates in either
transfectant, despite the presence of the inhibitor Cbz-LLL. This is most likely due to the
comparatively lower rate of degradation for Kd, coupled with the earlier observation
that Cbz-LLL retards, but does not block, the subsequent degradation of the heavy
chain intermediate (10). Thus, while US2 can apparently destabilize the Kd and Ld
heavy chains to some extent, it does not cause their destruction at a rate sufficient for
the intermediate to accumulate.
The Kb degradationintermediateobserved is a deglycosylated heavy chain.
In the case
of human class I molecules, the US11-induced degradation intermediate that
accumulates in the presence of Cbz-LLL is a deglycosylated heavy chain (10). To
demonstrate that the Kb degradation intermediate observed in Fig. 1 is a deglycosylated
heavy chain, we performed a pulse chase experiment (10' label, 0' and 20' chase points)
on Kb vaccinia virus infected US11 cells, and immunoprecipitated with RafiHC the Kb
heavy chains from lysates of cells removed at each chase point. Each immunoprecipitate
was split in two, and either kept on ice (-) or treated with recombinant N-glycanase (+)
prior to re-immunoprecipitation with RafHC. The samples were then split again and
analyzed by SDS-PAGE (Fig. 2a) or 1D-IEF (Fig. 2b). As seen in Figure 2a, all of the Kb
molecules at the beginning of the chase are fully susceptible to N-glycanase treatment
(Fig. 2a, 0' timepoint), and no intermediate has yet accumulated. After 20', most of the
heavy chains have been converted into the US11-induced fragment, which migrates at
the same position as N-glycanase treated material (Fig. 2a, 20' timepoint). In
hydrolyzing the N-glycosidic bond of the Kb heavy chain's glycan, N-glycanase
converts the Asn sidechain to an Asp, and thus imparts an additional negative charge
upon the molecule. Isoelectric focusing of the samples in Fig. 2a reveals that the fully
intact Kb heavy chains present at the beginning of the chase have the same pI after Nglycanase treatment as the degradation intermediate observed after 20' of chase (Fig.
2b). We conclude that the mechanism of US11-mediated class I heavy chain degradation
is similar for mouse and human molecules, and that in both cases, a deglycosylated
heavy chain intermediate can be visualized when the proteasome inhibitor Cbz-LLL is
present during the chase.
HCMV downregulates the expression of MHC class I molecules by dislocating
newly synthesized class I heavy chains from the lumen of the ER back into the cytosol,
where they are rapidly degraded (10). The virus encodes two proteins, US11 and US2,
that are each sufficient for causing the premature destruction of class I heavy chains (9).
We show here that US11 and US2 have nonidentical specificities for a panel of murine
class I heavy chains. Over the 20' chase period examined, US11 degraded H-2Kb, Db, Dd,
and Ld with similar kinetics, while US2 only degraded Db and Dd efficiently. One
possible explanation for the observed differences in class I heavy chain degradation for
US11 and US2 positive cells is that each may interact with different regions of the class I
heavy chain in the process of dislocating the latter into the cytosol. Due to the sequence
variability between different class I alleles, this dual recognition strategy would allow
the virus to downregulate the expression of a broader range of class I molecules than
might be possible with either US11 or US2 alone.
References
1.
Yewdell, J.W., and J.R. Bennink. 1992. Cell biology of antigen processing and
presentation to MHC class I molecule-restricted T lymphocytes. Adv. Immunol. 52:1-123.
2.
Burgert, H.G., and S. Kvist. 1985. An adenovirus type 2 glycoprotein blocks cell
surface expression of human histocompatibility class I antigens. Cell 41:987-997.
3.
York, I.A., C. Roop, D.W. Andrews, S.R. Riddell, F.L. Graham, and D.C. Johnson.
1994. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8 + T
lymphocytes. Cell 77:525-535.
4.
Del Val, M., H. Hengel, H. Hacker, U. Hartlaub, T. Ruppert, P. Lucin, and U.H.
Koszinowski. 1992. Cytomegalovirus prevents antigen presentation by blocking
transport of peptide-loaded major histocompatibility complex class I molecules into the
medial golgi compartment. J. Exp. Med. 176:729.
5.
Browne, H.G., G. Smith, S. Beck, and T. Minson. 1990. A complex between the
MHC homologue encoded by human cytomegalovirus and 32-microglobulin. Nature
347:770-772.
6.
Beersma, M.F.C., M.J.E. Bijlmakers, and H.L. Ploegh. 1993. Human
cytomegalovirus down-regulates HLA class I expression by reducing the stability of
class I heavy chains. J. Immunol. 151:4455-4464.
7.
Warren, A.P., D.H. Ducroq, P.J. Lehner, and L.K. Borysiewicz. 1994. Human
cytomegalovirus- infected cells have unstable assembly of MHC class I complexes and
are resistant to lysis by cytotoxic T lymphocytes. J. Virol. 68:2822-2829.
8.
Yamashita, Y., K. Shimokata, S. Saga, S. Mizuno, T. Tsurumi, and Y. Nishiyama.
1994. Rapid degradation of the heavy chain of class I MHC antigens in the endoplasmic
reticulum of human cytomegalovirus- infected cells. J. Virol. 68:7933-7943.
9.
Jones, T.R., L.K. Hanson, L. Sun, J.S. Slater, R.M. Stenberg, and A.E. Campbell.
1995. Multiple independent loci within the human cytomegalovirus unique short region
down-regulate expression of MHC class I heavy chains. J. Virol. 69:4830-4841.
10.
Wiertz, E.J.H.J., T.R. Jones, L. Sun, M. Bogyo, H.J. Geuze, and H.L. Ploegh. 1996.
The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains
from the endoplasmic reticulum to the cytosol. Cell 84:769-779.
11.
Machold, R.P., S. Andree, L. Van Kaer, H. Ljunggren, and H.L. Ploegh. 1995.
Peptide influences the folding and intracellular transport of free major
histocompatibility complex heavy chains. J. Exp. Med. 181:1111-1122.
12.
Ploegh, H.L. 1995. One-dimensional isoelectric focusing of proteins in slab gels.
In Current Protocols in Protein Science. J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W.
Speicher and P.T. Wingfield, editors. John Wiley & Sons, New York. 10.2.1-10.2.8.
Acknowledgements
We thank Dr. J. Yewdell for the murine class I-vaccinia virus recombinants. This
work was supported by National Institutes of Health grants R01-AI33456 and R01AI07463-17, and by Boehringer-Ingelheim.
Figures
Figure 1. Fate of murine class I heavy chains in US11 + or US2 + cells. Control
(nontransfected), US11+, or US2+ cells were infected with vaccinia virus expressing H2Kb (lacking the cytoplasmic tail) (panel A), Kd (panel B), Db (panel C), Dd (panel D), or
Ld (panel E), labeled with [35S]methionine for 10' and chased as indicated.
Immunoprecipitated murine class I molecules were resolved on a 12.5% SDSpolyacrylamide gel and visualized by fluorography.
Figure 2. The Kb degradation intermediate is a deglycosylated heavy chain. US11+ cells
were infected with vaccinia virus expressing H-2Kb (lacking cytoplasmic tail), labeled
for 10' with [35S]methionine, and chased as indicated. Immunoprecipitated Kb heavy
chains from each chase point were either kept on ice (-) or treated with recombinant Nglycanase (+) prior to resolution by SDS-PAGE (a) or 1D-IEF (b).
Cells:
Chas e time:
Control US11+
i 0'
20'
0'
Kb -
Fig. la
20'
US2+
0'
20'
Cells:
Chase time:
Control US11+ US2+
i O'
20'
O'i
Kd-
Fig. lb
20'i
O'
20'
Cells:
Chase time:
Db-
Control US11+
I 0'
20' 110'
-W
Fig. ic
US2+
20' 110'
20'
Cells:
Chase time:
Dd-
Control US11+ US2+
....
__
I0 '
20' " 0'
L , --
20' " 0'
20' '
71
^IIY1ILI
Fig. ld
Cells:
Chase time:
Control US11+
I
O'
US2+
20' " O' 20' i" O'
20'
Ld-
Fig. le
1
Chase time:
N-glycanase: I
Kb
20'
+. II
*
Fig. 2a
I
N-glycanase:
20'
0'
Chase time:
I
+
Kb-
Fig. 2b
II
+
Chapter 5
Summary
104
In the experiments described within this thesis, I have developed novel
immunochemical reagents to characterize biosynthetic intermediates in the folding and
assembly of the class I MHC molecule with respect to the kinetics of their appearance,
and distribution within the cell. In addition, I have examined the mechanism by which
the human cytomegalovirus downregulates the expression of class I MHC molecules.
Outlined below are the critical findings from each chapter; a more detailed discussion of
each set of experiments has been written within each chapter.
In Chapter 2, the folding and cell surface expression of free murine class I heavy
chains was examined in different inbred mouse strains, including mutant mice lacking
TAP, 32m, or both. An antiserum directed against free class I MHC heavy chains
(RafHC) was characterized immunochemically, and was used to monitor the fate of free
class I MHC heavy chains in the ER and at the cell surface. The cell surface expression of
free class I MHC heavy chains in cells lacking p2m was found to be dependent on the
function of the TAP peptide transporter, and was enhanced at reduced temperature.
Definition of the requirements for cell surface expression of properly assembled
heterotrimeric class I MHC molecules, as well as partially assembled free heavy chains
may provide insights as to how the quality control machinery determines when folding
and assembly are complete for the class I MHC molecule.
In Chapter 3, three novel monoclonal antibodies (KU1, KU2, and KU4) directed
against free MHC class I heavy chains were introduced, and characterized by epitope
mapping. These antibodies were used to demonstrate that the folding and assembly of
three different epitopes located along the murine H-2Kb polypeptide chain is rapid and
synchronous, and that free heavy chains that appear at the cell surface are degraded in
an acidified intracellular compartment. Further studies on the folding and assembly of
class I MHC molecules using this approach would benefit from additional anti-free
heavy chain monoclonal antibodies mapped to the same resolution as the KU
105
antibodies, in order to follow the folding of as many regions of the polypeptide chain as
possible. However, our results suggest that the folding of the class I MHC heavy chain
may be too rapid a process in living cells for partially folded intermediates to be
isolated by pulse chase/immunoprecipitation techniques.
In Chapter 4, the specificities of the HCMV US11 and US2 gene products in
mediating the destruction of newly synthesized class I MHC molecules were compared
by introducing murine class I molecules into US11 or US2-transfected cells. It was
shown that US11 and US2 do not have identical specificities, and thus may interact with
different regions of the class I MHC molecule. Further defining the specificity of US11
and US2 by examining the stability of other class I MHC allomorphs, or chimeras, when
expressed in the respective US11+ or US2+ cell lines, should ultimately allow the sites of
interaction between the US proteins and the class I MHC molecule to be mapped.
Elucidating the pathway by which the US proteins dislocate newly synthesized class I
MHC heavy chains may also provide insights into the mechanism of protein turnover in
the ER.
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