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1986. 55:167-193
© 1986 by Annual Reviews Inc.
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LYSOSOMAL ENZYMES AND THEIR
RECEPTORS
Kurt von Figura and Andrej Hasilik
Physiologisch-Chemisches Institut, Westfalische Wilhelms-Universitiit, WaIdeyerstr.
IS,
D-4400
Munster, West Gennany
CONTENTS
PERSPECTIVES AND SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
167
BIOSYNTHESIS OF LYSOSOMAL ENZyMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168
168
170
171
173
Synthesis and Modifications in Endoplasmic Reticulum. . .. . . . . . . . . .. . . . . . . . . .... . . . . . . .. . .
Transport to the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Common Modifications in the Golgi . . . .. . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . .... . . . . . . . .
Formation of Mannose 6-Phosphate Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
MANNOSE 6-PHOSPHATE--DEPENDENT TRANSPORT IN VIVO. . . . . . . . . . . . . . . . . . . . ..
178
MANNOSE 6-PHOSPHATE--SPECIFIC RECEPTORS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Two Distinct Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
178
179
The Cation-Independent Receptor (MPRj .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . .
ITINERARIES OF LYSOSOMAL ENZYMES AND OF THE CATION-INDEPENDENT
RECEPTOR (MPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
182
MANNOSE 6-PHOSPHATE--INDEPENDENT TRANSPORT . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .
188
PERSPECfIVES AND SUMMARY
Interest in lysosomes and lysosomal enzymes was stimulated by the existence
of some 30 inherited lysosomal storage disorders in man. The enzyme defects
involved in most of these disorders were identified in the 1970s; see review by
Neufeld, Lim, & Shapiro in this series in 1975 (1). Presently , these mutations
are being characterized at the level of DNA and RNA.
Targeting of lysosomal enzymes is part of the more general question: how do
eukaryotic cells transport proteins synthesized in the rough endoplasmic reticu­
lum to diverse destinations? Hickman & Neufeld discovered, in 1972, that the
multiple deficiency of lysosomal enzymes in I-cell disease results from a
167
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1 68
VON FIGURA & HASILIK
deficiency in a recognition marker that is common to lysosomal enzymes and
required for targeting the enzymes to lysosomes (2). This observation provided
the basis for many subsequent studies that eventually led to the identification of
the recognition marker and its receptor. A 2 15-kd receptor, which recognizes
mannose 6-phosphate residues in lysosomal enzymes, has been identified as an
essential component of a system that in many cells allows for specific transport
of lysosomal enzymes to lysosomes. It was originally identified as a cell­
surface receptor binding exogenous lysosomal enzymes and mediating their
transfer to lysosomes along the pathway of receptor-mediated endocytosis. We
now know that this receptor functions also in transport of endogenous lysosom­
al enzymes and that its presence in organelles that constitute elements of the
secretory pathway is i mportant for that function. The combi ned application of
biochemical and cytological methods has significantly contributed to the
present knowledge of lysosomal enzyme transport. Further, the current applica­
tion of recombinant DNA methods to the study of lysosomal enzymes and their
receptors is expected to provide answers to many unresolved questions.
This review is limited to discussion of synthesis and transport of lysosomal
enzymes in mammalian tissues. We focus on the processing of the oligosac­
charides in lysosomal enzymes, on the mannose 6-phosphate-specific receptor
(MPR), and on its role in the transport of lysosomal enzymes. The transport of
lysosomal enzymes and the function of mannose 6-phosphate-specific recep­
tors have been the subject of recent reviews (3-5).
BIOSYNTHESIS OF LYSOSOMAL ENZYMES
Synthesis and Modifications in Endoplasmic Reticulum
Most of the proteins that are localized partially or fully to the extracytosolic side
of the endoplasmic reticulum, Golgi complex, lysosomes, and nuclear and
plasma membranes have in common a signal sequence that directs the ribo­
somes engaged in their synthesis to the rou gh endoplasmic reticulum [reviewed
in (6)]. This common signal sequence is a stretch of 1 5-30 mainly hydrophobic
amino acids that is localized to the N-terminus. When protruding from the
ribosome this stretch first forms a complex with a cytosolic ribonucleoprotein
called signal recognition particle (7). Subsequently, this complex binds to a
receptor at the surface of the rough endoplasmic reticulum that is called the SRP
receptor or the docking protein (8 , 9), and the nascent protein is transferred into
the lumen of the rough endoplasmic reticulum. Most of the soluble proteins that
are released into the endoplasmic reticulum lose the sign al peptide before the
synthesis of their polypeptide is completed.
A difference in size between polypeptides synthesized in vitro (nonglycosyl­
ated and possibly containing the signal peptide) and those synthesized in
cultured cells treated with tunicamycin, an inhibitor of glycosylation (nongly-
169
LYSOSOMAL ENZYMES AND RECEPTORS
cosylated products lacking cleavable signal peptide), is the commonly accepted
evidence for the existence of a signal peptide in a protein. Using this technique,
it has been shown that porcine cathepsin D (10), mouse cathepsin D, and rat
p-glucuronidase
( 1 1), as well as a and p chains of human p-hexosaminidase
(12), transiently contain signal peptides. Direct evidence for the presence of a
signal sequence in porcine cathepsin D has been obtained by Erickson et al ( 1 3),
who determined partial N-terminal sequences of porcine cathepsin D synthesized in
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vitro both in the presence and in the absence of membranes. The translation of
cathepsin D is also regulated by the signal recognition particle (14).
As has been demonstrated for several (nonlysosomal) glycoproteins, a glu­
cosylated oligosaccharide is transferred to certain asparagine residues in the
polypeptide intruding into the lumen of the rough endoplasmic reticulum
( 15- 17). Usually this transfer takes place prior to folding of the protein
backbone. The oligosaccharide transferred to asparagine residues is pre­
assembled in an "activated" form as a derivative of dolichol pyrophosphate
[( 18), see Figure 1]. The asparagin'1-linked oligosaccharides in lysosomal
(tronS-GOI91)
(CiS mid-GoI9i)
+
I
(0) (b) (c)
p, M M M
\
\ I
M M
\ I
M
I
G
2
G
3
G
3
M
M
M
2
,
,
M
M M
3.
M M
J'
M
..
Gn
..
Gn
I
p
I
P
I
Dolichol
2
M
I
M
\
o
M
1
M M
\ I
M M
\ I
M
1
Gn
1
•
Gn
I
Asn . X • Ser/Thr •
o
I
Gal
I
M
Gn
M M-P
\
\ I
M M
\ I
III
M
I
II
D
H
>
o
IT
SA
..3/6
Gal
114
2
M
\
Figure 1
I
SA
M
M
M
I
M
Gn
62
' I
Gol
114
Go
62
M M
\ I
M
1
M M
\ I
M
I
IV
Main stages in the processing of oligosaccharides in lysosomal enzymes. The organelles
to which the processing is localized are indicated at the top of the figure. Four typical structures that
have been found in lysosomal enzymes as formed in different parts of the Golgi complex are shown:
I
=
high-mannose, II
=
phosphorylated high-mannose, III
=
phosphorylated hybrid, IV
=
complex oligosaccharide. Hybrid oligosaccharides without phosphate groups have been found
also. The arrows indicate the approximate relative abundance of the four oligosaccharide types.
The symbols are: G
=
glucose, Gal
N-acetyl neuraminic acid, P
linked sugars.
=
=
galactose, Gn
=
N-acetylglucosamine. M
=
mannose, SA
=
phosphate. Single numbers indicate the positions of a.anomerically
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170
VON FIGURA & HASILIK
enzymes are subject to processing that follows the principles elucidated in the
past decade for secretory and membrane glycoproteins. These have been
reviewed in the previous issue of this series by Kornfeld & Kornfeld (18) and
will be only briefly mentioned. We will focus on the reactions that have been
studied in lysosomal enzymes and which in part are specific for them.
Processing of the oligosaccharide is initiated by "trimming" reactions. The
removal of the first glucose residue is effected by glucosidase I, takes place
within a few minutes of the transfer of the oligosaccharide, and may even
precede the completion of the polypeptide synthesis (19). Removal of the two
other glucoses by glucosidase II is a much slower process (20). Recently,
removal of outer glucose residues within 1 min of synthesis has been observed
in cathepsin D in human fibroblasts (2 1 ). The trimming in the rough
endoplasmic reticulum also involves a specific a mannosidase (22), and in
general seems to yield octarnannosyl chains. Specific removal of a mannose
residue from the terminus of the middle branch is likely to facilitate further
processing in the Golgi complex (23).
-
Transport to the Golgi
Several cytological observations suggest that the transport is mediated by
smooth vesicles formed in "transitional" elements of the endoplasmic reticulum
(24,25). The transit times from the reticulum to the Golgi complex vary among
different products (26-29), and it is not known whether this results from
characteristic interactions of the individual products with other components
remaining in or leaving the reticulum. In the case of membrane-associated
histocompatibility antigens, the transport depends on the availability of J3z­
microglobulin (30). Lodish et al (26, 3 1) postulated that a receptor protein in the
endoplasmic reticulum membrane regulates the selective transport of secretory
proteins into transport vesicles en route to the Golgi. l-Deoxynojirimycin, an
inhibitor of the trimming glucosidases (32, 33), has been shown to inhibit
secretion of aI-proteinase inhibitor in rat hepatocytes (34) and HepG2 cells
(31),and the transport of lysosomal enzymes into lysosomes in fibroblasts (2 1).
In the presence of I-deoxynojirimycin, these glycoproteins were retarded in the
endoplasmic reticulum. Upon subcellular fractionation of cells treated with the
drug, the retarded glycoproteins were found in the microsomal fraction and
their carbohydrates did not show the characteristics of processing in the Golgi
compl ex (21, 31).
In rat hepatocytes and HepG2 cells, the selectivity of the effect of 1deoxynojirimycin was indicated by the fact that it did not inhibit the secretion of
albumin (31, 34) or of the glycoprotein C3 and transferrin (31). In a mixed
popUlation of hybridoma cells, the drug inhibited the secretion of IgD and not
of IgM (35). This differential inhibition corresponded well to a differential
effect on the formation of complex oligosaccharides. In the case of the mem­
brane-associated glycoprotein v-erbB, the transport to the plasma membrane
171
LYSOSOMAL ENZYMES AND RECEPTORS
was not affected, although the processing was blocked (36). I t appears that the
inhibition of the processing in the presence of I-deoxynojirimycin interferes
with the transport of certain soluble glycoproteins, including lysosomal en­
zymes. The inhibition of transport of affected glycoproteins was incomplete
and molecules that eventually reached their normal extracellular or lysosomal
destinations contained at least some normally processed oligosaccharides (21,
34). Under normal conditions, the transport of proteins between organelles may
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be differentially influenced by interactions with other components of the
system. A well-known example is the retention of (3-glucuronidase in micro­
somal organelles containing a protein called egasyn (37).
Common Modifications in the Golgi
TRANSPORT
The Golgi complex is an elaborate membrane system, in which
a number of modification reactions, in particular the synthesis of the carbo­
hydrate portion of the various glycoconjugates, are accomplished [reviewed in
(38)]. The complex consists of a stack of flat or fenestrated cisternae with
associated tubules and vesicles with polar orientation. The so-called
cis part
receives the product of biosynthesis from the rough endoplasmic reticulum (24)
and the other pole, the
trans part, is marked in secretory cells by condensing
vacuoles and secretory vesicles (25).
Functionally, it is useful to consider three
regions within the stack: cis, mid, and trans, as suggested by Griffith et al (39)
and Rothman et al (40). Most studies on the intracellular transport deal with
membrane proteins. The various parts of the Golgi complex, though not rigidly
separated, are involved in different carbohydrate modification reactions (see
below). Lipids, membrane-associated proteins and soluble proteins that are
produced in the endoplasmic reticulum are subject to a vectorial
(cis to trans)
flow through the Golgi complex. The complex's own constituents
behave as a
stationary phase and their relative distribution through the cisternae may be
maintained through a counterflow resembling the distillation process
has been pointed out by Slot
(41).
It
& Geuze (42) that small vesicles with a large
membrane/volume ratio may efficiently accomplish the transport of membrane
components. Indeed, small vesicles in the vicinity of the Golgi complex
enriched in various receptors (42-44), and it should be of interest to test
are
the
possibility that some vesicles are enriched in the membrane constituents of
various parts of the Golgi complex and serve to reflux these constituents
between the neighboring organelles. As far as transport between the cis and mid
parts of the Golgi complex is concerned, it has been suggested by Rothman and
coworkers that it is the biosynthetic product that is passed over in small
vesicles. This suggestion is based on studies of the transport of the membrane
glycoprotein G of the vesicular stomatitis virus in an in vitro system, in which
the budding of vesicles from Golgi cisternae was observed
(45).
As judged from the modifications of the carbohydrates, lysosomal enzymes
172
VON AGURA & HASILIK
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can pass through all three parts of the complex. It is a matter of debate,
however, whether their passage through the trans-Goigi is obligatory. This
question is related to the localization of the sorting step and will be discussed
below .
COMMON MODIFICATIONS
Depending on the protein moiety, the outer­
chain mannose residues (see Figure 1) in glycoproteins entering cis-Goigi are
subjected to further trimming. This is accomplished by at least two (X­
mannosidases in an ordered sequence, and also involves a specific N­
acetylglucosaminyl transferase. The fIrst enzyme in this sequence, man­
nosidase I, is defIned as an enzyme hydrolyzing high-mannose oligosaccha­
rides to yield Man5 GleNAC2 (46), and is an accepted marker for cis-Golgi (47).
Its product may subsequently be processed by N-acetylglucosaminyl trans­
ferase I to yield GleNAc Man5 GleNAc2 (48, 49).
Recently, immunocytochemical evidence has been presented for localization
of this enzyme in the mid-cisternae of the Goigi complex (50). Mannosidase II
is highly specifIc for the product of N-acetylglucosaminyl transferase I and
converts it to GlcNAc Man3 GlcNAc2 . Mannosidase II seems to be rather
broadly distributed through the Goigi complex, with a maximum activity in the
middle portion of the complex (51). With the aid of specifIc antibodies directed
to mannosidase II, a rather uniform distribution of the enzyme in the elements
of the Golgi complex (52) has been fIrmly established. If mannosidase II does
not act on its substrate, addition of galactose and sialic acid results in formation
of hybrid oligosaccharides. The action of mannosidase II is prevented e.g. by
the presence of the so-called bisecting N-acetylglucosamine linked to 13mannose (53, 54). The product of mannosidase II is the preferred substrate of
N-acetylglucosaminyl transferase II. This reaction opens the pathway for the
synthesis of an array of complex oligosaccharides with two or more antennas
(18, 55). The fInal steps in this synthesis take place in trans-Golgi cisternae,
where galactosyl transferase is localized as has been demonstrated by Roth &
Berger (56).
In earlier studies, ample indirect evidence has been obtained of the presence
of hybrid or complex oligosaccharides in lysosomal enzymes. This evidence is
based on sensitivity to neuraminidase, binding to immobilized and cellular
lectins, and carbohydrate analyses, in which fucose, galactose, and sialic acid
were detected. Structural data on complex oligosaccharides in soluble lysosom­
al enzymes is available for l3-glucuronidase from human spleen (57). This
enzyme contains a small amount of complex oligosaccharides with two an­
tennas, of which one is incomplete. Bi- and triantennary oligosaccharides
comprising about 80% of the total oligosaccharides were found in human
J3-glucocerebrosidase (58). It should be pointed out that this hydrolase is an
example of a membrane-associated lysosomal enzyme (59). The observation,
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LYSOSOMAL ENZYMES AND RECEPTORS
173
in lysosomal enzymes,of short oligosaccharides containing only four (57,60)
or just a single sugar residue (61),points to intralysosomal degradation. This
may in part explain the low amounts of typical complex oligosaccharide
structures found in lysosomal enzymes. As determined by lectin binding and
resistance to endoglucosaminidase H, an enzyme cleaving most of the high­
mannose and hybrid oligosaccharides (62), complex oligo saccharides have
been found in metabolically labeled mouse �-glucuronidase (63), human fibro­
blasts cathepsin D, �-hexosaminidase and arylsulfatase B (64, 65), and in
Chang liver a-galactosidase (66),which contains some tri- or tetraantennary
oligosaccharides. In newly synthesized cathepsin D, oligosaccharides resistant
to endoglucosaminidase H and containing galactose comprise almost 30% of
the total oligosaccharide population (64, 67).
While the content of complex oligosaccharides in soluble lysosomal en­
zymes of normal fibroblasts is generally low, it is elevated in I-cell and
mucolipidosis III fibroblasts, predominantly in their secretions (64, 68-71).
This striking change results from a defect in these mutant cells in the phos­
phorylation of high-mannose oligosaccharides (see below).
Sialylated hybrid oligosaccharides have been found in cathepsin D and
�-hexosaminidase of human fibroblasts and contained 5-10% of the radioactiv­
ity in anionic oligosaccharides released by endoglucosaminidase H (72). A
group of hybrid oligosaccharides that contain phosphate in addition to sialic
acid was characterized by Varki & Kornfeld (73) and will be discussed below.
Formation of Mannose 6-Phosphate Residues
REACTIONS
Observations on the efficiency and saturability of the endocyto­
sis of lysosomal enzymes led Hickman & Neufeld to propose,in 1972 (2),that
lysosomal enzymes carry a specific recognition marker. By 1980, it became
apparent from a series of observations contributed from several laboratories that
the recognition marker in lysosomal enzymes is a carbohydrate (74),that it is
related to mannose (75),sensitive to alkaline phosphatase,and probably repre­
sented by mannose 6-phosphate residues (76-79). Mannose 6-phosphate was
identified in lysosomal enzymes as a component of oligosaccharides cleavable
by endoglucosaminidase H (80-83). Phosphate has been found in the carbo­
hydrate of all soluble lysosomal enzymes tested including �-glucuronidase
(82), �-hexosaminidase, a-glucosidase and cathepsin D (83), a-L iduronidase
(84), arylsulfatase A (85), arylsulfatase B (65), myeloperoxidase (86), acid
phosphatase (87), and cathepsin C (88). Subsequent to the identification of the
phosphorylated residue, it was observed that some of the phosphorylated
oligosaccharides contain N-acetyl-glucosaminyl-l -phospho-6-mannose diester
groups (72,89-91). The anomeric configuration of the N-acetylglucosaminyl
residue was found to be a (72, 89, 90, 92). Previously, phosphodiester
compounds of sugars or sugar alcohols had been found in cell walls of various
174
VON
FIGURA & HASILIK
microorganisms. In analogy to the synthesis of phosphomannan in yeast (93),
two carbohydrate modification reactions, which form the phosphorylated
recognition marker, were identified: transfer of N-acetylglucosaminyl 1phosphate from the UDP-N-acetylglucosamine to the C-6 hydroxyl of a man­
nose residue, and hydrolysis of the covering N-acetylglucosamine residue (see
Fig ure 2).
The enzyme catalyzing the first reaction, UDP-N-acetylglucosamine:lyso­
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somal enzyme N-acetylglucosaminyl-l-phosphotransferase (referred to as
transferase), was first demonstrated in membranes from rat liver, fibroblasts,
and Chinese hamster ovary cel ls (94-96), and was partially purified from rat
3
liver Goigi preparations (97). [[3- 2p]UDP-N-acetylglucosamine is a suitable
substrate for determination of transferase activity and can be prepared from
32
['Y- p]ATP using commercially available enzymes (98). N -ace tyl gl ucosamin e
I-phosphate can be transferred to the C-6 position of mannose residues in
precursor and mature forms of lysosomal enzymes, high-mannose oligosac­
charides, and a-methylmannoside (96, 97). How ev er lysosomal enzymes are
phosphorylated at least 100 times more efficiently than the oligosaccharides or
,
a-methylmannoside. Phosphorylation of high-mannose oligosaccharides in
nonlysosomal glycoproteins is not detectable (97), or only barely so (96). The
acceptor activity of lysosomal enzymes is destroyed by denaturation (96).
When lysosomal enzymes are deglucosylated with endoglucosaminidase H,
they become specific inhibitors of the N-acetylglucosaminyl-l-phosphate
transfer to lysosomal enzymes (99). Apparently, lysosomal enzymes contain a
unique, denaturable structure that is distinct from the acceptor oligosaccharide,
common to all lysosomal enzymes, and re cogni zed by the transferase. The dual
recognition of lysosomal enzymes by the transferase is strongly supported by
the failure of deglycosylated lysosomal enzymes to inhibit phosphorylation of
a-methyl mannoside (99), and the characterization of a mutant that phosphory­
lates high-mannose oligosacchrides and a-methyl mannoside but not lysosomal
enzymes
(100).
Man-Lysosomal enzyme precursor
UDP-GleNAe
GlcNAC
+
-+
UMP
1-0 -6-Man-Lysosomal
H,o
GleNAc
� -6-Man-Lysosomal
Figure 2
enzyme precursor
enzyme precursor
Two-step biosynthesis of mannose 6-phosphate residues in lysosomal enzymes.
LYSOSOMAL ENZYMES AND RECEPTORS
175
Primary structures of several lysosomal enzymes have been determined. No
homologies are found when the primary sequences in the vicinity of the two
glycosylation sites in porcine cathepsin D (101), one in rat cathepsin B (102),
and four potential glycosylation sites found in the established partial sequence
of human u-fucosidase (103) are compared. Therefore, the signal for the
phosphorylation is not represented (in different species) by a common sequence
adjacent to the glycosylation sites. Phosphorylation at different glycosylation
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sites within a single enzyme is random (63, 64). Thus, it is likely that the signal
is present in the lysosomal enzyme in a single copy. Because the signal is
sensitive to treatment with heat, sodium dodecylsulfate, or trypsin (99), it is
probably part of the protein and dependent on the tertiary rather than primary
structure.
In the cell, the precursor forms of the lysosomal enzymes serve as acceptors
for the transferase, which is localized to the Golgi complex (104, 105). Within
the Golgi complex, transferase activity decreases from cis to trans (51, 106,
107). At the time of phosphorylation, the bulk of oligosaccharides contain six to
nine mannose residues (63). Analyses of mutants showed that phosphorylation
of glucosylated high-mannose oJigosaccharides is possible in branches that do
not contain glucose (l08), and that truncated oligosaccharides with only five
mannose residues can be phosphorylated (108, 109). Mutants provide some
indication of whether a single transferase is responsible for phosphorylation. In
I-cell patients, in fibroblasts, and in all tissues examined, the transferase
activity is absent (94, 110-113), and lysosomal enzymes do not contain
phosphate (83,85,87,114), pointing to the existence of a single transferase. In
a milder form of the disease, mucolipidosis III, transferase activity (100, 111,
115) and phosphorylation of lysosomal enzymes (85, 116) are markedly re­
duced. The residual phosphorylation leads to the formation of oligosaccharides
with one as well as with two phosphate groups (1 15). This further supports the
concept of a single transferase.
The second enzyme in the specific pathway, N-acetylglucosamine 1phosphodiester u-N-acetylglucosaminidase, has been partially purified from
rat liver (117, 118) and human placenta (119). The enzyme is immunologically
and catalytically distinct from the lysosomal u-N-acetylglucosaminidase. It
hydrolyzes UDP-N-acetylglucosamine, but not the corresponding arylglyco­
side. The mechanism of hydrolysis is that of a glycosidase and not a phospho­
diesterase (120). It fractionates with Golgi membranes (104, 117, 118) and in
fractionated Golgi membranes, it is distributed at slightly higher densities than
the transferase (51, 105-107), which suggests a mid-Goigi localization.
STRUCTURE AND DISTRIBUTION OF PHOSPHOR YLATED OLIGOSACCHARIDES
IN LYSOSOMAL ENZYMES
In Figure 1, examples of the main types of
oligosaccharides found in lysosomal enzymes are shown. The structures of the
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176
VON FIGURA & HASILIK
phosphorylated oligosaccharides were studied in purified lysosomal enzymes
as well as in total cellular or secreted glycoproteins. Only a minor portion of the
oligosaccharides in lysosomal enzymes becomes phosphorylated. This portion
does not exceed 30% in l3-glucuronidase synthesized in mouse lymphoma cells
(63). Only about 25% of the radioactive mannose was released as phosphory­
lated oligosaccharides from cathepsin D and l3-hexosaminidase that were
isolated from NH4CI-induced secretions of human fibroblasts and therefore not
subjected to degradation in lysosomes (72). In lysosomal enzymes isolated
from tissues, the portion of phosphorylated oligosaccharides is even lower. In a
l3-glucuronidase preparation from human spleen containing forms enriched in
mannose 6-phosphate, phosphorylated oligosaccharides accounted for 10% of
total oligosaccharides (57, 92), in l3-glucuronidase from rat liver lysosomes
phosphorylated oligosaccharides were not detectable (91), and in cathepsin D
from porcine spleen they accounted for less than 4% of total oligosaccharides
(121). Pulse-chase labeling studies indicate the presence of secondary mod­
ifications to the oligosaccharides in lysosomal enzymes including removal of
blocking N-acetylglucosamine residues (63, 89) and removal of phosphate
(63). Therefore, the amount of phosphorylated oligosaccharides and the rela­
tive amounts of the different species depend greatly on the source and sub­
cellular location of a lysosomal enzyme.
In all studies, oligosaccharides with one phosphate group were found to be
two to five times more frequent than oligosaccharides with two phosphate
groups (63, 72, 73, 90, 92). Primarily, the phosphate is found in the diester
form. In the cathepsin D and l3-hexosaminidase preparations from human skin
fibroblasts mentioned above (72), more than 80% of the phosphorylated oligo­
saccharides contained one or two covered phosphates. A similar portion was
found in phosphorylated oligosaccharides in (3-g1ucuronidase and the total
cellular glycoproteins isolated from mouse lymphoma cells after a three-hour
labeling period (89, 90). In the lysosomal enzymes isolated from tissues,
phosphorylated oligosaccharides are found to contain either exclusively
monoesters [cathepsin D from porcine spleen, (121)] exclusively diesters
[microsomall3-g1ucuronidase from rat liver, (91 )], or predominantly diesters
[13-g1ucuronidase from human spleen, (92)]. Oligosaccharides that contain both
a phosphomonoester and phosphodiester group represent in all preparations a
minor species (73, 92). The underlying oligosaccharides contain four to nine
mannose residues, but species with six to eight mannose residues are the most
frequent forms (63, 72, 73, 89-92, 1 21).
Varki & Kornfeld (90) analyzed in detail the position of the phosphate groups
in cellular glycoproteins of mouse lymphoma cells. According to their analysis,
the mannose residues that can be phosphorylated are represented by the three
residues at the nonreducing termini of branches a through c and the penultimate
residues of branches a and c in the Mans GlcNAc2 oligosaccharide (shown in
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LYSOSOMAL ENZYMES AND RECEPTORS
177
Figure 1). This oligosaccharide is probably the predominant product of the
endoplasmic reticulum a-mannosidase (18,22). Phosphorylation of these five
mannose residues is not random and phosphorylated mannoses are most com­
monly found in branches c and a. During the processing covering N­
acetylglucosamine residues and outer nonphosphorylated mannose residues are
removed. As a result,the oligosaccharides underlying phosphomonoesters are
in general smaller than those carrying phosphodiesters,which suggests that the
cleavage of the covering N-acetylglucosamine residue is followed by removal
of mannoses (63,73,90). In oligosaccharides with two phosphate groups,the
phosphates always occur on two different branches.
Recently, phosphorylated oligosaccharides that contain one or two sialic acid
residues were identified by Varki & Kornfeld (73) in P388D1 macrophage-like
cells. These hybrid oligosaccharides contain only a single phosphate as mono­
or diester on branch c and lack a bisecting N-acetylglucosamine on the J3-linked
mannose. Hybrid oligosaccharides containing phosphodiester groups also were
found in thyroglobulin secreted by malignant thyroid tissue (122).
Lysosomal enzymes usually contain two or more oligosaccharides per sub­
unit. This holds true for human spleen l3-g1ucuronidase (63,57), cathepsin D,
the a and 13 chain of l3-hexosaminidase, and arylsulfatase B from human skin
fibroblasts (64, 65). Any of the oligosaccharides in mouse J3-g1ucuronidase
(63) and human cathepsin D (64) may become phosphorylated. From the extent
of phosphorylation,it appears that on the average one or less than one oligosac­
charide per subunit is phosphorylated. The percentage of phosphate groups that
becomes uncovered in the Golgi complex is unknown. The phosphate groups in
less than 20% of the phosphorylated oligosaccharides in cathepsin D and
J3-hexosaminidase become uncovered (72), provided that NH4CI does not
interfere with the action of phosphodiester a-N-acetylglucosaminidase. Under
these conditions,less than 10% of cathepsin D and J3-hexosaminidase subunits
leave the Golgi complex with high-mannose oligosaccharides containing
phosphomonoesters.
Analyses of the oligosaccharides in l3-g1ucuronidase from human spleen (57,
92),mouse lymphoma cells (89),rat liver microsomes and lysosomes (91),and
cathepsin D from porcine spleen (121,123) indicate that neutral high-mannose
structures are the prevailing oligosaccharides in these enzymes. The sizes of
these oligosaccharides depend on the enzyme sources. In l3-g1ucuronidase from
human spleen and from mouse lymphoma and P388D1 cells,the high-mannose
oligosaccharides with nine and eight mannose residues are most frequent (57,
63,89,92),whereas in J3-glucuronidase from rat liver lysosomes,forms with
five mannose residues prevail (91). The presence of neutral high-mannose
oligosaccharides depends largely on phosphorylation. This is indicated by the
paucity of high-mannose oligosaccharides in lysosomal enzymes from I-cell
fibroblasts (64, 68, 69).
178
VON FIGURA & HASILlK
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MANNOSE 6-PHOSPHATE-DEPENDENT TRANSPORT IN
VIVO
It appears that mannose 6-phosphate residues participate in the transport of
lysosomal enzymes in two physiologically significant ways. First, in certain
cells, they are indispensable for targeting endogenous lysosomal enzymes to
lysosomes. In 1972, Hickman & Neufeld (2) reported the important finding that
lysosomal enzymes in I-cell fibroblasts lack the recognition marker required for
receptor-mediated endocytosis. They proposed that the inability of I-cell fibro­
blasts to equip their enzymes with this recognition marker caused the in­
tracellular deficiency and extracellular accumulation of many lysosomal en­
zymes that is observed in I-cell fibroblasts. The subsequent demonstration that
the inability of I-cell fibroblasts to synthesize mannose 6-phosphate residues in
lysosomal enzymes (83, 114) was the result of a deficiency inN-acetylglucos­
amine I-phosphotransferase (94, 110) further identified mannose 6-phosphate
residues as indispensable for transport of lysosomal enzymes in fibroblasts. The
general importance of these residues is indicated by the excessive levels of
lysosomal enzymes in all body fluids of I-cell patients and the morphological
alterations in many tissues that result from deficiency in intracellular lysosomal
enzymes (124).
The second function of mannose 6-phosphate residues is to mediate in­
tercellular exchange of lysosomal enzymes. This function is exemplified in
female carriers of Hunter disease. Hunter disease is an X-linked lysosomal
disorder that is characterized by a deficiency in iduronate sulfatase. The female
carriers have two populations of cells, one of which is deficient in iduronate
sulfatase due to inactivation of the X-chromosome carrying the nonaffected
gene. Yet, the phenotype of carriers as well as the morphology of their tissues is
normal (125). This is explained by transfer of iduronate sulfatase from the
normal to the deficient cell population. This phenomenon of cross-correction
has been studied by Neufeld and coworkers (1) in fibroblasts and shown to
depend on the mannose 6-phosphate-containing recognition marker mentioned
above.
The functions of the phosphorylated recognition markers depend on their
interaction with specific mannose 6-phosphate receptors. Therefore, the two
functions also apply to at least one of the receptors, which will be discussed in
the following section.
MANNOSE 6-PHOSPHATE-SPECIFIC RECEPTORS
Two Distinct Receptors
Two mannose 6-phosphate-specific receptors are known. These receptors can
be differentiated by dependence on divalent cations. The cation-independent
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LYSOSOMAL ENZYMES AND RECEPTORS
179
receptor has been extensively characterized with regard to structure, biosynthe­
sis, turnover, subcellular location, and function and will be discussed in detail
below and be referred to as MPR. The existence of a second cation-dependent
receptor is suggested by a recent report by Hoflack & Kornfeld (126). These
authors found that membranes from mouse P388D1 macrophage-like cells bind
lysosomal enzymes in a saturable and mannose-6 phosphate-dependent man­
ner. More importantly, binding is strictly dependent on divalent cations.
P388D1 macrophages do not synthesize detectable amounts of the cation­
independent receptor (127). In these cells, the transport of lysosomal enzymes
to lysosomes is probably mediated by the cation-dependent receptors.
The Cation-Independent Receptor (MPR)
ISOLATION OF MPR
MPR is a membrane protein that can be solubilized in the
presence of nonionic detergents without loss of its binding properties. The first
purification of MPR was achieved by Sahagian et al (128). The authors isolated
the MPR from bovine liver using the lysosomal enzyme (3-galactosidase im­
mobilized to Sepharose 4B. Because of easier accessibility, yeast phosphoman­
nan and secretions from Dictyostelium discoideum became the preferred mate­
rials for preparation of affinity matrixes (73, 129, 130). The receptors have
been isolated from a variety of tissues and cells including bovine liver, human
fibroblasts, rat hepatocytes, Chinese hamster ovary cells, and rat chondrosarco­
ma (128, 131). In several cells of human origin, induding normal fibroblasts,
HepG2, U937, and HL-60 cells, the receptor constitutes 0.1--0.5% of total
membrane protein (our unpublished results).
STRUCTURAL FEATURES
In SDS-polyacrylamide gel electrophoresis, MPR
from different sources behaves as a glycoprotein with an Mr of about 215 ,000. It
contains fucose and terminal sialic acid residues (127, 131), phosphoserine
groups, and intrachain disulfide linkages (132). The latter are indicated by a
decrease in electrophoretic mobility after reduction. The receptors span the
membrane. A small portion of about 10 kd protrudes at the cytosolic side of
membrane and harbors the C-terminus of the receptor. The greater portion of
the receptor polypeptide is exposed at the luminal side of organelles and the
outer side of the plasma membrane. This portion contains the mannose 6phosphate binding site(s) (133, 134).
BIOSYNTHESIS AND TURNOVER OF MPR
The receptors undergo a series of
posttranslational modifications, some of which cause minor changes in the
mobility in SDS polyacrylamide electrophoresis (127, 132). The high-mannose
oligosaccharides in the receptor are converted predominantly to complex-type
structures. The processing of the oligosaccharides is remarkably slow and not
complete even two to three hours after synthesis (127, 132). Phosphorylation of
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180
VON FIGURA & HASILIK
the receptor on serine residues is restricted to the pool of mature receptors. The
phosphate has a half-life about seven times shorter than the protein in MPR
(132). Additional posttranslational modifications are indicated by sequential
acquistion of immunoreactivity and of binding activity in Chinese hamster
ovary cells (132). In fibroblasts and adherent Chinese hamster ovary cells, the
half-life of the protein moiety ranges from 10 to 29 hours (132, 135, 136). The
turnover is not affected if saturating amounts of exogenous ligands are added to
cells or if endogeneous ligands are lacking (135). Exposure to NH4Cl or
leupeptin, two inhibitors of lysosomal proteolysis, also has no significant effect
(135). Sahagian (137) found under specific conditions that the medium of
Chinese hamster ovary cells contained immunoreactive fragments, supposedly
representing degradation products of MPR. The author proposed a nonlyso­
somal mechanism for receptor degradation that depends on binding of secreted
lysosomal enzymes to MPR.
BINDING SPECIFICITY
Isolated receptors incorporated into liposomes (130,
131, 138) or immobilized on various matrixes (73, 130, 131, 139) bind
lysosomal enzymes in a saturable and mannose 6-phosphate-dependent man­
ner. The binding characteristics of the purified receptors (73, 128, 130, 139) are
comparable to receptors in isolated membranes (140-143) or at the cell surface
(144-146). The lysosomal enzymes bind with KD's in the nanomolar range.
The binding is competitively inhibited by mannose 6-phosphate (Ki 0.05-2
mM) and lysosomal enzymes (Ki
2-40 nM). It does not depend on divalent
cations and drops precipitously between pH 6 and pH 5.
By studying the binding of oligosaccharides to immobilized receptors, Fis­
cher et al (130) and Varki & Kornfeld (73) could show that phosphomonoester
groups in oligosaccharides are essential for binding. Oligosaccharides with one
phosphomonoester residue were retarded on receptor-substituted columns and
oligosaccharides with two phosphates in monoester linkage bound firmly to the
column. Neutral oligosaccharides or oligosaccharides with one or two phos­
phates in diester linkage to N-acetylglucosamine did not bind. The affinity to
immobilized receptors compared well with the ability of oligosaccharides to
become internalized (147, 148). The Kuptake of oligosaccharides with one or
two phosphates in monoester linkage were 3.2 x 10-7 and 3.9 X 10-8 M
(148). Neutral oligosaccharides and those with one phosphate in diester linkage
were not internalized. The 10-fold lower Kuptake of oligosaccharides with two
phosphates in monoester linkages argues for an extended binding site in the
receptor. Earlier observations of Sly and coworkers (149, 150), who compared
the uptake and inhibitor activity of phosphomannan fragments containing
multiple phosphomonoesters to that of pentamannosyl monophosphate, sug­
gested a cooperativity in binding. The polyvalent fragment was a potent
inhibitor and was internalized, whereas the monovalent pentasaccharide was
=
=
.
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LYSOSOMAL ENZYMES AND RECEPTORS
181
not internalized and was n o more inhibitory than mannose 6-phosphate.
Recognition of carbohydrates neighboring the mannose 6-phosphate residues
contributes to binding. This is evident from the following three observations.
First, the K uptake of monophosphorylated oligosaccharides is 1 00-fold lower
than the Kj of man nose 6-phosphate ( 1 48). Second, presence of outer mannQse
residues accessible to jack bean a-mannosidase can lower the affinity of
oligosaccharides with phosphomonoester groups for the receptor. Third, the
positions of phosphomonoester groups on the oligosaccharides· influence the
affinity for the receptors (73). Gabel et al (15 1 ) analyzed the oligosaccharides in
endogenous glycoproteins bound to the receptors in mouse lymphoma cells.
The receptor-bound material was highly enriched in oligosaccharides bearing
one or two phosphomonoesters, whereas phosphorylated oligosaccharides in
nonreceptor-bound material were enriched in phosphodiesters.
Lysosomal enzymes from Dictyostelium discoideum have been widely used
as tools to measure uptake via the MPR and to isolate the receptors. Their
binding properties ( 1 45, 1 46, 1 52) are indistinguishable from those of lysosom­
al enzymes. Yet, the recognition of the slime mold enzymes by the receptors is
resistant to exhaustive treatment with alkaline phosphatase ( 1 5 3). Gabel et al
( 1 54) recently identified these alkaline phosphatase-resistant groups as methyl­
phosphomannose residues. Thus, mannose 6-phosphate residues covered with
methyl groups are recognized by the MPR, whereas those covered with N­
acetylglucosamine, as occurs in lysosomal enzymes of mammalian origin, do
not bind to the MPR.
Early studies on binding
of lysosomal enzymes suggested that most of the receptor is localized to
intracellular membranes ( 143). In subcellular fractionation studies, the receptor
was found in the plasma membrane ( 1 28), in Goigi membranes ( 155), in coated
vesicles ( 1 34, 1 56) and in endosomes ( 157), but not in lysosomes (132, 1 36). In
various immunocytochemical investigations ( 1 58- 1 6 1 ), the consensus finding
is that MPR is present in the plasma membrane (including coated pits), in
coated vesicles present in the vicinity of the plasma membrane, in organelles
with tubular extensions that have been defined as CURL ( 1 62) [the vesicular
elements of CURL are equivalent to endosomes ( 163) or receptosomes ( 1 64)],
and in the Golgi area including the nearby coated vesicles, although in certain
tissues it may be difficult to detect MPR in the plasma membrane ( 1 6 1 ).
However, the findings with regard to the distribution of MPR in intracellular
membranes are strikingly divergent. Thus, in rat hepatocytes and some other
cells Brown & Farquhar ( 1 59) find MPR to be localized specifically to one or
two cisternae in cis-Golgi, whereas Geuze and coworkers ( 1 60) observe MPR
well distributed through all of the Golgi complex and the trans-Golgi reticulum
in rat hepatQcytes. In HepG2 cells, more MPR is present in the trans-Goigi
DISTRIBUTION OF MPR IN CELLULAR MEMBRANES
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182
VON FIGURA & HASILIK
reticulum than in the Golgi itself (161). The binding of the specific antibodies
used in these studies was visualized with an immunoperoxidase-conjugated
second antibody (159) or with protein A-coated gold particles (160, 161). In
these studies, little MPR was found in the endoplasmic reticulum. In contrast,
in Chinese hamster ovary cells endoplasmic reticulum was reported to be rich in
this receptor (158). Finally, extensive (44,159) or limited (158, 1 60) labeling
of MPR was observed in the membranes of lysosomal organelles. While it is
presently impossible to reconcile these conflicting findings, it is possible that
the use of different visualization techniques,the application of different criteria
for the identification of the organelles, and the study of different cell types all
may have contributed to the apparent differences in these observations.
ITINERARIES OF LYSOSOMAL ENZYMES AND OF THE
CATION-INDEPENDENT RECEPTOR (MPR)
Although the role ofMPR in targeting endogenous lysosomal enzyme is fmnly
established, no conclusive answers are available about where receptors and
ligands combine (binding site), where the receptor-ligand complexes are segre­
gated from the secretory pathway, where the ligands dissociate from the
receptors, where receptors and ligands are separated, and along which pathway
receptors recycle.
BINDING SITE
Since the binding property of a lysosomal enzyme depends on
formation of, at minimum, one uncovered mannose 6-phosphate residue, its
interaction with MPR would be possible at or trans to the location of the
uncovering enzyme. Localization of MPR to probably all membranes of the
Golgi complex and of the uncovering a-N-acetylglucosaminidase to mid-Goigi
suggests that mid-Golgi is the most proximal site where the binding of lysosom­
al enzymes can occur.
SEGREGATION SITE
The binding to receptors is thought to be followed by the
selective packaging into vesicles. The purpose of this packaging is to separate
newly synthesized lysosomal enzymes from other products that traverse the
Golgi complex and are destined for secretion and certain cellular membranes.
The most proximal site where segregation could occur is the binding site itself.
The restriction of MPR to the cis-Golgi cisternae and neighboring membranes
that Brown & Farquhar (159) observed led them to propose that the lysosomal
enzyme-receptor complexes are segregrated in the cis-Golgi into clathrin­
coated vesicles. This hypothesis is difficult to reconcile with the presence of
sialylated, phosphorylated hybrid and complex type oligosaccharides in lyso­
somal enzymes, which are bound to the receptors (151), en route to lysosomes
(67), or present in lysosomes (64, 165). Considering the trans-Golgi localiza­
tion of the terminal glycosyltransferases that add galactose and sialic acid
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LYSOSOMAL ENZYMES AND RECEPTORS
1 83
residues to hybrid and complex type oligosaccharides (4 1 , 56), lysosomal
enzymes are expected to pass the trans-Golgi cisternae. As processing appears
more likely to occur in nonreceptor than in receptor-bound form, a significant
fraction of lysosomal enzyme is likely not to bind before reaching trans-Golgi
cisternae.
The prevalence of neutral high-mannose oligosaccharides in lysosomal en­
zymes (see above) also has been utilized as an argument to propose that
segregation takes place predominantly in cis-Golgi. Lysosomal enzyme pre­
cursors secreted in the presence of NH4Cl retain their neutral high-mannose
oligosaccharides (72). These findings , together with the preferred occurrence
of complex oligosaccharides in lysosomal enzymes from I-cell fibroblasts (see
above) , indicate that the exemption of neutral high-mannose oligosaccharides
from processing to complex structures is due to the presence of phosphorylated
oligo saccharides rather than to binding to MPR or to a bypass of trans-Golgi
cisternae.
In double-labeling experiments with immunogold, Geuze and coworkers
( 1 60, 1 6 1 ) colocalized MPR, lysosomal enzymes, and albumin to the Golgi
cisternae and the trans-Golgi reticulum. In HepG2 cells, albumin was colocal­
ized with MPR and lysosomal enzymes even at the level of packaging in the
coated buds of the trans-Golgi reticulum ( 1 6 1 ) .
Theoretically, the most distal site at which segregation oflysosomal enzymes
could occur is the plasma membrane. A vailable data on lysosomal enzyme
transport neither contradict nor favor a pathway via the plasma membrane.
Such a pathway has been suggested to involve transport of MPR-ligand com­
plexes along the secretory route to the plasma membrane and internalization of
the complexes along the pathway of receptor-mediated endocytosis. The pool
of lysosomal enzymes bound to MPR detectable by immunofluorescence or
immunogold at the plasma membrane of fibroblasts ( 1 66) or HepG2 cells ( 1 6 1 )
may represent the newly synthesized endogenous lysosomal enzymes moving
via the plasma membrane. If transport via the plasma membrane constitutes a
major pathway, the enzymes at the plasma membrane must be protected from
displacement by mannose 6-phosphate ( 1 67) . However, the lysosomal en­
zymes at the plasma membrane also may represent enzymes that are reinternal­
ized following secretion. Although this secretion-recapture pathway ( 1 68) is of
importance in certain conditions such as the Hunter-carrier, it appears to
constitute only a minor pathway for delivery of lysosomal enzymes to lyso­
somes . If cells are grown in the presence of mannose 6-phosphate and related
compounds , which block MPR-dependent uptake of lysosomal enzymes , little
if any accumulation of lysosomal enzymes in the medium and no intracellular
reduction were found ( 1 67 , 1 69- 1 7 1 ) . A portion of the lysosomal enzymes
present at the cell surface may function in the degradation of the components of
the extracellular matrix ( 1 72) . Further studies may well show that the location
1 84
VON FIGURA & HASILIK
of the segregation site depends on the cell type and that within a single cell
segregation may be a smooth process occurring along an extended section of the
secretory pathway.
Ultrastructural studies localized MPR
and lysosomal enzymes to coated membranes and coated ve�icles neighboring
the Golgi complex or trans,Golgi reticulum and at the plasma membrane.
Willingham et al ( 1 73) identified the latter as being part of the endocytosis
pathway for exogenous lysosomal enzymes. Highly purified preparations of
coated vesicles from brain, liver, and human placenta contain lysosomal
enzymes. In such preparations, the enzymes are enriched in precursor forms
and at least in part bound to MPR ( 1 56, 174). Coated membranes im­
munoselected from fibroblasts, and analyzed for the lysosomal enzyme cathep­
sin D, contained exclusively the precursor forms of this enzyme ( 1 75). It was
estimated that during transport the precursors of cathepsin D are associated with
coated membranes only for a short period not exceeding a few minutes. This
agrees with the view that coated vesicles rapidly lose their coats. The observa­
tion that precursors of cathepsin D contain oligosaccharides of the complex type
(our unpublished results) supports the notion that coated membranes are in­
volved in lysosomal transport distal to trans-Golgi.
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COATED MEMBRANES AND VESICLES
DISSOCIATION OF MPR-LYSOSOMAL ENZYME COMPLEXES
Like many
other receptors involved in transport of ligands to lysosomal or prelysosomal
structures, MPR participates in many rounds of transport. The amount of
internalized ligand is far in excess of the number of MPR binding sites when the
protein synthesis is blocked by cycloheximide ( 14 1 , 144). Reutilization implies
dissociation and separation of receptors and ligands and transport of unoccu­
pied receptors back to the binding site. Receptor-ligand complexes dissociate
below pH 5 . 7 ( 1 28, 1 4 1 ). In vivo the same mechanism appears to be utilized for
dissociation of receptor-ligand complexes . This is illustrated by the effects of
conditions that prevent acidification of intracellular organelles . (Acidification
of organelles is reviewed by Helenius & Mellman in this volume. ) When
dissociation is inhibited, cells should rapidly become depleted in unoccupied
receptors. As a consequence, receptor-dependent functions (i.e. sorting of
endogenous and endocytosis of exogenous lysosomal enzymes) should be
blocked. This is exactly what is observed in cells exposed to weak bases (83 ,
84, 1 1 6, 141 , 1 69 , 1 76, 1 77) and the ionophore monensin ( 1 77- 1 79). Weak
bases and ionophores raise the pH of acidic organelles above 6 ( 1 80) Among
the acidic subcellular compartments ( 1 8 1- 1 84), lysosomes , CURL, and coated
vesicles are candidates for the dissociation site. In the laboratories of Robbins
( 1 85 , 1 86) and Sly ( 1 87, 1 88), several mutants of Chinese hamster ovary cells
were characterized that are defective in ATP-dependent acidification of endo.
LYSOSOMAL ENZYMES AND RECEPTORS
185
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somes but not of lysosomes. Such mutations result in an enhancement in the
secretion of lysosomal enzymes and a decrease in the endocytosis. These
observations define the dissociation of receptor-enzyme complexes clearly as a
prelysosomal event.
SEPARATION OF MPR AND LYSOSOMAL ENZYMES
Reutilization of receptors
implies that following dissociation. receptors and ligands are separated. Recent
morphologic studies by Geuze and coworkers (43 , 160, 16 1) shed some light on
where receptors might be separated from lysosomal enzymes. A membrane
reticulum containing tubular and vesicular structures extending in hepatocytes
from the peripheral cytoplasm down to the trans-Golgi area was described by
Geuze et al ( 162) as the compartment where, following endocytosis, the
asialoglycoprotein receptors are separated from their ligands. The receptors
concentrate in the tubular membranes. Free clathrin is frequently observed
adjacent to these tubules, suggesting that coated structures mediate retrieval of
receptors from the tubules. The ligands concentrate in the lumina of the smooth
vesicular structures, which may develop into or fuse with secondary lysosomes.
In rat hepatocytes , MPR colocalizes in CURL with the asialoglycoprotein
receptor (43). In HepG2 cells, MPR accumulates in the tubules of CURL , while
most of the lysosomal enzymes are found in the lumina of the smooth vesicular
structures ( 16 1). Thus, CURL is a likely candidate as the site where the
receptors are physically separated from lysosomal enzymes . The absence or
paucity of receptors in lysosome-enriched fractions from Chinese hamster
ovary cells ( 1 32) and fibroblasts ( 1 33) supports the view that separation of MPR
and its ligands occurs pre1ysosomally .
The pathway(s) along which receptors are transported for
reutilization are unknown. Models for transport of MPR have to take into
account that the receptors functioning in transport of exogenous and
endogenous lysosomal enzymes share at least one organelle, in which they mix .
This is indicated by the inhibition of segregation and of endocytosis of lysosom­
al enzymes ( 189, 190), and the rapid tagging of all cellular receptors with
antibodies (133, 137, 190) in cells exposed to antireceptor antibodies. CURL is
the most likely candidate for the organelle where receptors coming from the
Golgi and the plasma membrane mix. Separate pathways may exist for their
return to the Goigi and the plasma membrane. We favor a simple view, in which
all receptors are transported from the CURL-tubules to the Golgi complex,
where they mix with newly synthesized receptors. Unoccupied MPR following
the secretory route would replenish the pool of plasma membrane receptors. As
discussed above , MPR occupied with endogneous ligands may follow the same
pathway or diverge somewhere before reaching the plasma membrane. In the
latter case, MPR ferrying endogenous ligands would gain access to CURL
CYCLING OF MPR
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1 86
VON FIGURA & HASILIK
along a route different from the pathway of receptor-mediated endocytosis, and
mix in CURL with MPR coming from the cell surface.
Conditions perturbing the transport of MPR to or from CURL, as well as
those inhibiting the dissociation of ligands, may be expected to change the
subcellular distribution of MPR. Gonzalez-Noriega et al ( 1 4 1 ) observed a
depletion of MPR binding sites at the ceIl surface with use of chloroquine­
treated fibroblasts, and proposed this depletion resulted from inhibition of
receptor recycling to the cell surface. When measured as antigenic sites, the
number of cell surface receptors is also decreased. However, the uptake of
iodinated anti-MPR antibodies is largely resistant to chloroquine and related
weak bases (our unpublished results). This indicates that chloroquine alters the
steady-state concentration in membranes , but allows for recycling of receptors.
Since the ligands do not dissociate in cells exposed to weak bases, ligand­
occupied and hence functionally inactive receptors may recycle in such cells. A
significant portion of lysosomal enzymes internalized by Chinese hamster
ovary cells is returned to the cell surface, indicating that ligand occupation per
se does not prevent the backflow of receptors (4).
Farquhar and coworkers (44, 1 9 1 , 1 92) proposed that the movements of
MPR are induced upon its occupation with or dissociation from ligand. In cells
that do not synthesize ligands for MPR (I-ceIl fibroblasts or hepatocytes
exposed to tunicamycin), receptors accumulated in the cis-Golgi cisternae and
in adjacent coated vesicles, whereas they were steadily removed from endo­
somes/lysosomes. An opposite distribution was observed in chloroquine­
treated hepatocytes , where ligands cannot be separated from receptors . The
authors proposed that MPR shuttles nonconstitutively with movement from the
cis-Golgi to endosomes/lysosomes being triggered by occupation with ligand,
and movement from endosomes/lysosomes to the cis-Golgi area by dissociation
of ligands. At present, this attractive model is difficult to accommodate with the
observation that cycloheximide (which inhibits the synthesis of endogenous
ligands) is without apparent effect on receptor distribution in hepatocytes ( 1 93),
and on uptake of antireceptor antibodies ( 1 90). Furthermore, cycloheximide
does not affect the exchange of receptors between ceIl surfaces and intracelluar
membranes, and the kinetics of receptor exchange between intracellular mem­
branes and cell surfaces are similar in I-cell and control fibroblasts and are not
significantly affected by weak bases and monensin (our unpublished results).
Thus, the ultrastructural and biochemical analyses indicate that dissipation of
pH gradients within cells (e.g. by chloroquine or monensin) may alter the
relative rates of the movement of the receptor to and from a compartment such
that the overall cycling rate is not appreciably changed.
The route
of the transport of lysosomal enzymes from CURL to lysosomes is not known.
DELIVERY OF LYSOSOMAL ENZYMES INTO DENSE LYSOSOMES
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LYSOSOMAL ENZYMES AND RECEPTORS
1 87
It may involve packaging into specific transport vesicles, which fuse with
existing lysosomes. A more likely possibility is a gradual transition of (light)
CURL elements to (dense) lysosomes ( 163, 194). Vesicular elements of the
CURL may lose their tubular, receptor-dense connections and detach from the
continuous network of CURL. These vesicles, which may be considered as new
(light) lysosomes, gradually acquire the high density characteristic of lyso­
somes . The traditional view that secondary lysosomes are the organelles in
which lysosomal enzymes first encounter their substrates and initiate degrada­
tion has to be modified. Ligands that are internalized by receptor-mediated
endocytosis are separated from their receptors in CURL ( 1 62 , 193) and mix in
the lumen of vesicular CURL elements with lysosomal enzymes . Since CURL
is an acidic organelle ( 1 82), degradation may well be initiated therein. Some
recent experiments designed to trace the compartments in which endocytosed
ligands are subject to proteolysis indicate that the degradation is initiated in
light organelles that cofractionate with endosomal markers [( 195) , and personal
communication from S. Diment and P. Stahl] . At present, these organelles
cannot unequivocally be identified with CURL (vesicles still connected with
tubules and subject to membrane recycling) or with light organelles defined
above as new lysosomes. New Iysosomes should share many properties with
CURL, such as composition of the fluid content, but the former should be
distinguished by the lack of membrane components, which are subject to
recycling. Since internalized lysosomal enzyme can induce degradation of
material stored in secondary lysosomes ( 1 96) , the possibility has to be consid­
ered that new Iysosomes can fuse with existing secondary Iysosomes.
Lysosomal enzymes are converted by a series of proteolytic steps into mature
forms, which are typically represented by the forms isolated from tissues. For
cathepsin D, the conversion is initiated in light organelles and completed in
dense lysosomes (67). In the light organelles the precursor is converted into an
intermediate independent of ATP-driven acidification (our unpublished re­
sults). Generation of mature forms from the intermediate depends on ATP­
driven acidification and thiolproteinases and is localized in lysosomes ( 197) .
The processing is likely to be initiated in light organelles discontinuous with
CURL, such as new (light) lysosomes according to the definition given above.
This view is based on the absence of proteolytically processed forms of
cathepsin D in the medium of cultured fibroblasts ( 1 69) , in spite of the
exocytosis of fluid from CURL ( 1 98).
For details of proteolytic maturation of lysosomal enzymes and their fate in
lysosomes the reader is referred to recent reviews (37, 1 99) .
In
this section we propose a speculative model (Figure 3), which attempts to
reduce the number of signals required for transport to a minimum. Nascent
MODEL FOR MPR-DEPENDENT TRANSPORT OF LYSOSOMAL ENZYMES
1 88
VON FIGURA & HASILIK
lysosomal enzymes are sorted into the endoplasmic reticulum with the aid of
amino-terminal signal peptides. A yet undefined structure common to all
lysosomal enzymes serves as a signal for the phosphorylation by N­
acetylglucosaminyl- l -phosphotransferase. This process, in turn, initiates the
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generation of mannose 6-phosphate residues, which serve as signals for the
MPR. Binding to MPR somewhere in Golgi complex removes lysosomal
enzymes from the fluid.content that is destined for secretion. By utilizing the
secretory route, the complexes can reach the plasma membrane, where a signal
residing in MPR allows for collection in coated pits . Along the pathway of
receptor-mediated endocytosis, the complexes are ferried to CURL, where
receptors and ligands are separated. The same signal allowing for concentration
in coated pits at the plasma membrane may be utilized in CURL for collecting
the receptors into vesicles that move to the Golgi complex.
The proposed model might reflect a primitive pathway, from which more
sophisticated pathways evolved. The latter may allow for a shorter path be­
tween the binding site and the segregation site and avoid transport via the
plasma membrane . Necessarily, the evolution of these pathways would require
the formation of additional signals. Functionally analogous signals have been
postulated to direct transport of Golgi-derived products to distinct destinations,
such as different domains of the plasma membrane (200, 20 1 ) or elements of
either a constitutive or a regulated secretory pathway (202). Such signals may
trigger packaging of receptor-ligand complexes into specific vesicles that
mature into CURL or guide the return of receptors from CURL to either Golgi
or plasma membrane. In Figure 3 such shorter pathways are indicated by dashed
lines .
MANNOSE 6-PHOSPHATE-INDEPENDENT TRANSPORT
Several lines of evidence indicate that lysosomal enzymes can be ferried in a
manner that is independent of mannose 6-phosphate-specific receptors . In
I-cell fibroblasts , variable residual amounts of lysosomal enzymes are found
within the lysosomes (69). The fraction of newly synthesized enzyme targeted
to lysosomes may be as high as 20-50% for a-glucosidase and cathepsin D (87 ,
e.>:ogerlOVS
Figure 3
A model of the transport of lyso­
somal enzymes. The thin lines refer to path­
ways of the enzymes and the bold lines to
those of MPR. For further explanation see
text.
� /':�":;::
'I��;::�
'o;,
GOl��
PLASMA
:)M/MBRANE
enzyme. F,om
ER
"
I
I
I
I
I
I
I
'\\ \
\\
\\
\'
"
CURL
t
LYSOSOME:'.>
Annu. Rev. Biochem. 1986.55:167-193. Downloaded from arjournals.annualreviews.org
by WIB6151 - Deutsche Forschungsgemeinschaft on 04/20/09. For personal use only.
LYSOSOMAL ENZYMES AND RECEPTORS
1 89
1 69), whereas for other enzymes, such as �-hexosaminidase, a-L-iduronidase ,
and ary1su1fatase A it is below the limit of detection (84 , 87 , 1 69 , 203). The
activity of acid phosphatase is normal in I-cell fibroblasts. This has to be
attributed to secondary effects, since only one third of the newly synthesized
acid phosphatase polypeptides are targeted to lysosomes (87). The normal
activities in I-cell fibroblasts of �-glucocerebrosidase and acetyl CoA:a­
glucosaminide N-acetyltransferase, two integral membrane enzymes, suggest
that membrane proteins find their way independent of mannose 6-phosphate
residues. This agrees with the absence of mannose 6-phosphate residues in
membrane proteins of the lysosomal membrane (our unpublished results),
including �-glucocerebrosidase (204).
In liver, spleen, kidney, and brain from I-cell patients, the activities of many
lysosomal enzymes are normal, although these tissues are deficient in N­
acetylglucosaminyl l -phosphotransferase 0 1 2, 1 1 3). As the residual lysosom­
al enzymes in I-cell fibroblasts, these enzymes must use mannose 6-phosphate­
independent mechanisms for transport into lysosomes. Therefore, we must
consider that mannose 6-phosphate-independent mechanisms also contribute
to targeting of lysosomal enzymes in normal tissues.
ACKNOWLEDGMENTS
Thanks are due to Dr. J. Conary for critical reading of the manuscript and Mrs.
R. Rumpff for typing the manuscript. Our research on lysosomal enzymes is
sponsored by the Deutsche Forschungsgemeinschaft and Fonds der Chemi­
schen Industrie.
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ANNUAL
REVIEWS
Further
Quick links to online content
Annual Review of Biochemistry
Volume 55, 1986
CONTENTS
Annu. Rev. Biochem. 1986.55:167-193. Downloaded from arjournals.annualreviews.org
by WIB6151 - Deutsche Forschungsgemeinschaft on 04/20/09. For personal use only.
A CUPFUL OF LUCK, A PINCH OF SAGACITY, Martin D. Kamen
1
LECTINS AS MOLECULES AND AS TOOLS, Halina Lis and Nathan
35
Sharon
ARACHIDONIC ACID METABOLISM, Philip Needleman, John Turk,
Barbara A. Jakschik, Aubrey R. Morrison, and James B.
69
Lefkowith
SINGLE-STRANDED DNA BINDING PROTEINS REQUIRED FOR DNA
REPLICATION, John W. Chase and Kenneth R. Williams
103
REACTIVE OXYGEN INTERMEDIATES IN BIOCHEMISTRY, Ali Naqui,
Britton Chance, and Enrique Cadenas
137
LYSOSOMAL ENZYMES AND THEIR RECEPTORS, Kurt von Figura and
167
Andrej Hasilik
TRANSMEMBRANE TRANSPORT OF DIPHTHERIA TOXIN, RELATED
TOXINS, AND COLICINS, David M. Neville, Jr. and Thomas H.
195
Hudson
MOLECULAR ASPECTS OF SUGAR: ION COTRANSPORT, J. Keith Wright,
Robert Seckler, and Peter Overath
225
GENETICS OF MITOCHONDRIAL BIOGENESIS, Alexander TzagoloJf and
249
Alan M. Myers
CARBOHYDRATE-BINDING PROTEINS: TERTIARY STRUCTURES AND
PROTEIN-SUGAR INTERACTIONS, Florante A. Quiocho
287
MUTANTS IN GLUCOSE METABOLISM, Dan G. Fraenkel
317
TRANSCRIPTION TERMINATION AND THE REGULATION OF GENE
EXPRESSION, Terry Platt
339
DOUBLE-STRANDED RNA REPLICATION IN YEAST: THE KILLER
SYSTEM, Reed B. Wickner
373
BACTERIAL PERIPLASMIC TRANSPORT SYSTEMS: STRUCTURE,
MECHANISM, AND EVOLUTION, Giovanna Ferro-Luzzi Ames
397
TAURINE: BIOLOGICAL UPDATE, Charles E. Wright, Harris H.
427
Tallan, Yong Y. Lin, and Gerald E. Gaull
EXTRALYSOSOMAL PROTEIN DEGRADATION, S. Pontremoli and E.
455
Melloni
(continued)
v
vi
CONTENTS
(continued)
PLATELET ACTIVATING FACTOR: A BIOLOGICALLY ACTIVE
PHOSPHOGLYCERIDE, Donald J. Hanahan
483
STRUcruRAL ASPECTS OF THE RED CELL ANION EXCHANGE PROTEIN,
Daniel Jay and Lewis Cantley
511
PROTEOGLYCAN CORE PROTEIN FAMILIES, John R. Hassell, James H.
Kimura, and Vincent C. Hascall
539
THE ROLE OF ANTISENSE RNA IN GENE REGULATION, Pamela J.
Annu. Rev. Biochem. 1986.55:167-193. Downloaded from arjournals.annualreviews.org
by WIB6151 - Deutsche Forschungsgemeinschaft on 04/20/09. For personal use only.
Green, Ophry Pines, and Masayori Inouye
•
569
BIOLOGICAL CATALYSIS BY RNA, Thomas R. Cech and Brenda L.
Bass
599
NONVIRAL RETROPOSONS: GENES, PSEUDOGENES, AND TRANSPOSABLE
ELEMENTS GENERATED BY THE REVERSE FLOW OF GENETIC
INFORMATION, Alan M. Weiner, Prescott L. Deininger, and
Argiris Efstratiadis
631
ACIDIFICATION OF THE ENDOCYTIC AND ExocYTIC PATHWAYS, Ira
Mellman, Renate Fuchs, and Ari Helenius
663
DISCONTINUOUS TRANSCRIPTION AND ANTIGENIC VARIATION IN
TRYPANOSOMES, Piet Borst
701
733
EUKARYOTIC DNA REPLICATION, Judith L. Campbell
NEUROPEPTIDES: MULTIPLE MOLECULAR FORMS, METABOLIC
PATHWAYS, AND RECEPTORS, David R. Lynch and Solomon H.
Snyder
773
TRANSCRIPTION OF CLONED EUKARYOTIC RIBOSOMAL RNA GENES,
801
Barbara Sollner-Webb and John Tower
831
DNA POLYMORPHISM AND HUMAN DISEASE, J. F. Gusella
�-AMINO
ACIDS: MAMMALIAN METABOLISM AND UTILITY AS
a-AMINO ACID ANALOGUES, Owen W. Griffit h
855
THE TRANSPORT OF PROTEINS INTO CHLOROPLASTS, Gregory W.
Schmidt and Michael L. Mishkind
879
METALLOTHIONEIN, Dean H. Hamer
913
MOLECULAR PROPERTIES OF VOLTAGE-SENSITIVE SODIUM CHANNELS,
William A. Catterall
953
ACTIN AND ACTIN-BINDING PROTEINS. A CRITICAL EVALUATION OF
MECHANISMS AND FUNCTIONS, Thomas D. Pollard and John A.
Cooper
987
BIOCHEMICAL INTERACTIONS OF TUMOR CELLS WITH THE BASEMENT
MEMBRANE, Lance A. Liotta, C. Nageswara Rao, and Ulla M.
Wewer
1037
HORMONAL REGULATION OF MAMMALIAN GLUCOSE TRANSPORT, Ian
A. Simpson and Samuel W. Cushman
1059
(continued)
CONTENTS
(continued)
vii
COMPLEX TRANSCRIPTIONAL UNITS: DIVERSITY IN GENE EXPRESSION
BY ALTERNATIVE RNA PROCESSING. Stuart E. Left. Michael G.
1091
Rosenfeld. and Ronald M. Evans
, SPLICING OF MESSENGER RNA PRECURSORS, Richard A. Padgett.
Paula 1. Grabowski, Maria M. Konarska, Sharon Seiler, and
Phillip A. Sharp
1119
THE HEAT-SHOCK RESPONSE, Susan Lindquist
1151
Annu. Rev. Biochem. 1986.55:167-193. Downloaded from arjournals.annualreviews.org
by WIB6151 - Deutsche Forschungsgemeinschaft on 04/20/09. For personal use only.
INDEXES
Author Index
1193
Subject Index
1263
Cumulative Index of Contributing Authors, Volumes
Cumulative Index of Chapter Titles, Volumes
51-55
51-55
1284
1287