AN ABSTRACT OF THE DISSERTATION OF
Mei-Chuan Wang for the degree of Doctor of Philosophy in Animal Science presented
on October 29, 2003.
Title: Purification and Characterization of Recombinant Calpain-5
Abstract approved:
Redacted for privacy
Neil E. Forsberg
Recombinant human calpain-5 was expressed in insect cells using a baculovirus
system. The expressed calpain-5 was purified by both traditional chromatography and
by affinity-column chromatography. Both methods yielded active protease. Calpain-5
displayed very limited hydrophobicity. This indicated that calpain-5 is not a membrane
binding protein. Calpain-5 had apT of 8.3. The recombinant calpain-5 also exhibited
calcium-dependent proteolytic activity. The calculated calcium requirement for halfmaximal activity was 9.6 mM when incubated at 37 °C and 26.5 mM when incubated at
30 °C. Compared to traditional calpains, which require less than 1 mIM calcium for half-
maximal activity, calpain-5 exhibited weaker proteolytic activity. This is an unusual
observation because calpain-5 lacks the typical calcium-binding domain of the calpains
and implied that other calcium-binding region of the protein account for calciumbinding and sensitivity. Our results also showed that calpain-5 was different from
traditional calpains because its activity was higher at 37 °C compared to 30 °C and
remained active at 37 °C for more than 2 hours. This differs from traditional calpains
which display better proteolytic activity at lower temperatures and become inactive
within 30 minutes of incubation in 37 °C. Calpain-specific inhibitors, calpastatin and
E64, did not inhibit calpain-5. Only one calcium-binding inhibitor, PD 150606, inhibited
calpain-5 proteolytic activity. These results confirmed that calpain' s calcium-binding
domain is important in calpastatin binding and calpain-5 possesses other calcium-
binding regions. Calpain-5 was able to degrade spectrin, a ubiquitous cytoskeletal
protein. This indicates that calpain-5 might have a role in cell remodeling. Finally,
calpain-5 has the ability to degrade itself. It is not clear if this is the result of inter- or
intra-molecular proteolysis and whether this leads to activation of the protein or is,
instead, the first step in its degradation. Calpain-5 is expressed at highest concentrations
in testis, brain, liver and gastrointestinal tract. It is not clear why these tissues require a
unique calpain. Calpain-5 may provide these tissues with an additional calciumdependent proteolytic activity which is not regulated by calpastatin and which could
participate in cytoskeletal protein turnover.
©Copyright by Mei-Chuan Wang
October 29, 2003
All Rights Reserved
Purification and Characterization of Recombinant Calpain-5
by
Mei-Chuan Wang
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented October 29, 2003
Commencement June 2004
Doctor of Philosophy dissertation of Mei-Chuan Wang presented on October 29, 2003.
APPROVED:
Redacted for privacy
Major Professor, representing Animal Science
Redacted for privacy
Head of the Department of Animal Sciences
Redacted for privacy
Dean of tl
Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.
Redacted for privacy
Mei-Chuan W,anj, Author
ACKNOWLEDGMENTS
I sincerely appreciate my major professor, Dr. Neil Forsberg, for his guidance,
encouragement and financial support throughout my program. He provided me a great
opportunity to expand my knowledge and inspired my enthusiasm for research.
I greatly appreciate my other committee members, Dr. Mark Leid for guidance
and suggestions; Dr. Philip Whanger, for allowing me to use his lab equipment; Dr. Jeff
Widrick for friendly talk and sharing of antibodies; and Dr. Mina McDaniel who
substituted at a critical moment on my committee. I am also grateful for my colleagues,
Dr. Bor-rung Ou, Dr. Juntipa Purintrapiban, Dr. Yoji Ueda, Dr. Ying-yi Xiao, Mr. Mike
Beilstein, Mr. Kevin Marley, and Mr. Yong-Quiang Wang, for their wonderful
friendship.
Finally, 11 give my special thanks to all the staff in the Department of Animal
Sciences and Oregon State University, for providing me an excellent learning and
working environment during my studies.
TABLE OF CONTENTS
1.
INTRODUCTION ......................................................................
1
2.
LITERATURE REVIEW ..............................................................
4
2.1
Calpain structure and properties ................................................... 4
2.2
Calpain substrates and inhibitors ................................................. 10
2.3
Calpain function ..................................................................... 13
2.3.1
2.3.2
2.3.3
2.3.4
Calpaininceilcycle ...................................................... 14
Calpain in cell differentiation .......................................... 15
Calpaininapoptosis ..................................................... 16
Calpain and human diseases............................................ 17
2.4
Calpain superfamily ..............................................................
18
2.5
Calpain-5 ...........................................................................
25
MATERIALS AND METHODS ....................................................
30
3.1
Calpain-5 expression vector .....................................................
30
3.2
Insect cell culture ..................................................................
30
3.3
Baculovirus expression system ................................................... 32
3.4
Expression of calpain-5 ............................................................
34
3.5
Cell lysis ...........................................................................
35
3.6
Chromatography column test ...................................................... 36
3.
TABLE OF CONTENTS (Continued)
3.7
Chromatography ..................................................................
37
3.8
SDS-PAGE and Western Blot ..................................................
38
3.9
2D electrophoresis ..............................................................
39
3.10
His-tag protein purification......................................................
39
3.11
cMyc tagged protein purification ..............................................
40
3.12
Calpain-5 activity assays ........................................................
41
RESULTS ...........................................................................
42
4.
4.1
Calpain-5 purification by chromatography .................................... 44
4.2
Calpain-5 2D electrophoresis ..................................................... 58
4.3
Calpain-5 purification by polyhistidine affinity column .................. 58
4.3.1
4.3.2
4.3.3
4.4
5.
5.1
Calpain-5 activity assay .............................................. 61
Caseinolysis ............................................................ 67
Spectrin degradation ................................................. 70
Calpain-5 purification and activity by double affinity columns ......... 70
DISCUSSION ........................................................................
75
Calpain-5 proteolytic activity .................................................
77
5.1.1
5.1.2
5.1.3
Calcium-dependency of calpain-5 autolysis ........................ 77
Effects of inhibitors on calpain-5 activity ............................ 80
Domain T and its suspected function ................................ 81
TABLE OF CONTENTS (Continued)
5.2
Calpain-5 proteolytic activity ...................................................
5.3
Limitations
85
5.4
Future work ........................................................................
86
BIBLIOGRAPHy
83
90
LIST OF FIGURES
Fjgure
1.
Domain structure of calpain .............................................................
2.
Crystal structure of calpain-2 large subunit ........................................... 8
3.
Some typical members of the calpain superfamily ................................. 20
4.
Calpain-5 alignment ....................................................................
5.
Map and cloning site of pSEC4O and human calpain-5 ............................ 31
6.
Diagram of the Bac-to-Bac Baculovirus Expression System ..................... 33
7.
PCR and restriction digestion ofpFastBacl-Capn5 ................................ 43
8.
Western blot of calpain-5 in insect SF-9 cells ...................................... 45
9.
Western blot of chromatography column test..s .................................... 47
10.
Partial purification of calpian-5 from SF-9 cells by DEAE Sepharose
chromatography ............................................................................ 48
11.
Purification of calpain-5 from SF-9 cells by Q-Sepharose, Superdex-200
and Source-Q chromatography ......................................................... 49
12.
Western blot analysis of calpain-5 fractions obtatined from
chromatography .........................................................................51
13.
Calpain-5 calcium-sensitivity assay .................................................. 53
14.
Detection of calpain-1 in calpain-5 samples ........................................ 55
15.
Calpain-5 degradation over time ...................................................... 56
16.
Effects of calpain-specific inhibitors on calpain-5 activity ........................ 57
5
27
LIST OF FIGURES (Continued)
Figure
17.
2D-electrophoresis of calpain-5 ........................................................
59
18.
Talon elution of calpain-5 ...............................................................
62
19.
Calpain-5 (from Talon column) calcium-sensitivity assay .......................... 63
20.
Reletive immunoblot density of calpain-5 calcium sensitivity in 37 °C and
30°C .......................................................................................... 64
21.
Calpain-5 (from Talon column) degradation over time ............................. 65
22.
Inhibition of calpain-5 (from Talon column) by different calpain
inhibitors ...................................................................................
66
Immunoblotting of calpain-1 and calpain-2 in the Talon elution
sample ......................................................................................
68
24.
Caseinolytic activity of calpain-5 ......................................................
69
25.
CBB staining of spectrin breakdown by calpain-5 ................................... 71
26.
Double affinity purification of calpain-5 .............................................. 73
27.
Silver staining of calpain-5 autolysis in various calcium
concentrations .............................................................................
23.
74
LIST OF TABLES
Table
1.
Calpain superfamily
.19
2. Purification of recombinant human calpain-5 from SF-9 cells .......... 52
3. Isoelectric points of calpains .................................................
60
4. Differences of house-keeping calpains and calpain-5 .....................
89
DEDICATION
This thesis is dedicated to my family:
My parents, Ti-Chiou, and Yi-Mei
My husband, Jan-Shiang Taur
My daughter, Sophia, and the oncoming baby
Purification and Characterization of Recombinant
Calpain- 5
1. INTRODUCTION
Calpain was first reported in 1964 as a calcium-sensitive enzyme (Guroff,
1964). Until recently, the calpains were categorized as a family of calcium-dependent
cytosolic cysteine proteases. Although the exact physiological roles of calpains remain
to be elucidated, calpains respond to many environmental, pathological or physiological
changes and their wide distribution suggests that they have important cellular roles (for
reviews, see Sorimachi et al., 1997; Carafoli and Molinari, 1998; Huang and Wang,
2001).
The best understood calpains are calpain- 1 and calpain-2 which are also known
as "conventional calpains" (Sorimachi
et
al., 1994). These two calpains are expressed in
all human tissues, at all age stages, and exert essential roles in life maintenance.
Calpains were shown to be involved in cell proliferation, differentiation, cell death
(apoptosis), cell motility and many human diseases. These proteases do not digest
proteins completely. Instead, calpains process certain proteins (such as those at a check
point of cell cycle control, at key points of cell events, or those which are unusually
stable for maintain cell function) for further degradation. Several experimental models
like stroke-type excitotoxicity, hypoxialischemia, vasospasm, epilepsy, toxin exposure,
brain injury, kidney malfunction, and genetic defects have established that calpain
activation is an early and causal event in the degeneration that ensues from acute,
definable insults (for review, see Vanderklish and Bahr, 2000). Thus, calpains are
2
critical to the development of pathology and therefore represent distinct and potential
therapeutic targets.
Beside these two conventional calpains, many calpain-like cysteine proteases
have been discovered. Fourteen large subunit and two small subunit isoforms have been
reported and are characterized as the calpain superfamily. Members in this superfamily
share a unique homology in the cysteine protease domain, but have little divergence in
structure and expression pattern (for reviews, see Sorimachi and Suzuki, 2001; Huang
and Wang, 2001). Calpains-5, -6, -7, -10, and -15 have no EF-hand calcium binding
structure. Calpains-3, -6, -8, -9, -11, and -13 are tissue specific. Most of these members'
genes were found by EST
(Expressed Sequence Tags)
and genomic sequence database
search. Therefore, their physiological functions remain unclear. However, some
members of the calpain superfamily exert critical roles in pathological conditions. For
example, defects in calpain-3 have been identified as the cause of Limb-Girdle
Muscular Dystrophy 2A. Defects in calpain-lO were shown to be responsible for type 2
diabetes, whereas calpain-9 appears to be a gastric cancer suppressor. Additional
knowledge of calpains will surely assist the understanding and prevention of human
diseases.
Calpain-5 was first reported in 1997 as a human homolog of C. elegans sex
determination gene tra-3 by PCR-based screening of EST sequences (Mugita et
al.,
1997). From its deduced amino acid sequence, calpain-5 shares the cysteine-protease
domain which is noted in other calpains but replaces the calcium-binding domain with a
TRA-3 C-terminal (called "Domain T"). It is predicted that calpain-5 retains proteolytic
activity but it was not clear whether the proteoytic activity is calcium-dependent.
Calpain-5 protein has not been detected in in vivo samples. Interestingly, human
calpain-5 mRNA expressed in all fetal tissues tested, including brain, heart, kidney,
liver, spleen, lung and thymus, but only in selected adult tissues. In adult tissues, high
levels are expressed in testis, brain, colon, and intestine. This indicates calpain-5 might
have a role in fetal development. The high expression of calpain-5 in testis also suggests
a possible relation with sex determination in human as function of its homologous
TRA-3. However, its physiological function remains unknown.
The goal of this research was to examine calpain-5 protelytic activity and extend
the current knowledge of calpain-5. Calpain-5 has a cysteine protease domain but lacks
a calcium-binding domain. How it exerts its proteolytic function, its selective
expression in development, and controls sex determination is still unknown.
Establishment of calpain-5's proteolytic properties and identification of its relevant
physiological substrates seems to hold the key to understand its physiological function.
In this study, we expressed human calpain-5 in baculovirus-infected insect cells to yield
an active protein. The protein was then collected and purified for biological assays.
Calpain-5 was assayed for its calcium-sensitivity, time and temperature effects,
inhibitor interaction and substrates to gain insight in its physiological function. Results
of these studies can be applied into the understanding ofother atypical calpains (which
also lacks a calcium-binding domain) and help us predict roles of other vertebrate
calpain-5 homologs.
2. LITERATURE REVIEW
2.1
Calpain structure and properties
Calpain [EC3.4.22.17, clan CA family C2 (Sorimachi
et al.,
1997) was first
found as a calcium-activated cysteine protease in the rat brain soluble fraction (Guroff
and Guroff, 1964). Since then, calpain-related research has expanded rapidly. More than
14 calpains have been found in animals. These are categorized as the calpain
superfamily (for reviews, see Sorimachi and Susuki, 2001). It has been shown from the
accumulated studies that calpains are multi-functional proteases playing very important
roles in life maintenance in different cell stages, including proliferation, differentiation,
apoptosis and cell migration. Therefore, calpain has long been targeted in therapeutic
drug design.
The best characterized calpains are
and mcalpains. )i-Calpain (calpain-1)
and mcalpain (calpain-2) were named because they require a micromolar and a
millimolar concentration of Ca
for activation, respectively (Sorimachi
et al.,
1997).
These two isoforms exist ubiquitously in mammalian tissues and consist of a unique
catalytic (80 kDa) subunit and an identical regulatory (30 kDa) subunit (also called
calpain-4) which form a heterodimeric structure (for review, see Sorimachi and Suzuki,
2001). Domains in the large subunit include the amino terminal Domain-I, the protease
Domain-Il, Domain III, and the calcium biding Domain-IY (Figure 1). Small subunit
comprises two domains, the N-terminal Domain V and calcium-binding Domain VI.
The domain structures are highly conserved in all calpains, especially, the protease
Domain-Il.
5
"Pre-autolysis Calpain"
/Ca
80k
II
I
III
IV
[V
30k
VI
autolysis
autolysis
Ca
Autolytic
Activation
"Post-autolysis Calpain"
II
76k
III
IV
18k
1
new N-terminus
t
new N-terminus
Substrate proteolysis
Figure 1. Domain structure of calpain. Native enzyme contains four domains in large
subunit and 2 domains in small subunit. When Ca is present, calpain is activated by
autolysis. Substrate proteolysis follows autolysis.
It is very important to elucidate domain structure in regard to calpain's function
and activity. Domain-I shows less similarity in the corresponding domain in the typical
thiol protease (for review, see Jia et al., 2002). The sequence and structural differences
may contribute to proteolytic specificity, stability or activation in calpains. For example,
in calpain-1 and calpain-2, the N-terminal region of Domain-I could function in
inhibiting the enzyme or preventing access of substrate to the active site. It is removed
during autocatalytic activation (Saido et al., 1993).
Domain-Il is the cysteine protease domain. Its active site residues are similar to
other cysteine proteases such as cathepsins B, L, or S (Groves et
al.,
1998), and highly
conserved in the calpain super-family (Jakely and Friedrich, 1999). Domain II can be
further divided into two clusters from its structure; Ila and lib (Hosfield et al., 1999).
The active site cleft is located at the interface of JIa and lIb. In calpain-2, the protease
domain contains the catalytic triad residues, Cys 105, His 262 and Asn286 at the
interface of Domain-IIa and Domain-Jib where the active site resides (for review, see
Jia et al., 2002).
Domain-Ill shares similar characteristics with C2-domain found in several Ca
regulated proteins such as protein kinase (Rizo and Sudhof, 1998). Its function was long
unclear. However, many C2-domains posses roles in Ca
binding (Sutton et
al.,
-dependent phospholipid
1995) and calpain activation in vivo involves an interaction with
membrane (Meligren, 1987). Therefore, Domain-Ill has been considered to participate
in calpain membrane-association processes.
Domains-IV and -VI are highly similar and both contain 5 sets of EF-hand
sequences that are potential calcium-binding sites (Sorimachi et al., 1997). The
5th
EF-
7
hand motif of Domain-TV and -VI interact with one another to form a heterodimer.
When calcium binds, the heterodimer contact is blocked and results in dimer
dissociation (Strobi et al., 2000). Besides this, the small subunit makes several contacts
with the large subunit (Elce
et
al., 1997) including one at the N-terminal of Domain I
(refered to as an anchor region; Suzuki and Sorimachi, 1998). Domain V of the 30K
small subunit is hydrophobic and rich in Gly residues. It is suggested that Domain-V
interacts with membrane phospholipids to localize or stablize the enzyme complex
(Sorimachi
et
al., 1997).
In vitro studies revealed that the Ca concentration required for calpain halfmaximal activity ranges from 3-50 tM for calpain-1 and 400-800 iM for calpain-2
(Cong et al., 1989; Kapprell and Goll, 1989). This Ca concentration is significantly
higher than physiological Ca
concentration, which are generally less than 1 tM
(Guyton, 1991). However, an increase in intracellular free Ca concentration up to 1-2
jtM results in increasing calpain activity (Hong et aL, 1994; Turner et al., 1991). This
indicates that calpain activity in living cell is controlled not only by
but also by
some other mechanisms. In fact, calpain activity is tightly controlled in vivo.
The crystal structure of calpain-2 has been determined and has provided
considerable insight relating to calpain's Ca -dependent activity. The molecule is an
elongated, multidomain assembly with dimensions of approx 1 00x60x50 A (Figure 2).
Jia et al. (2002) proposed a model to address the aspects of calpain activation,
regulation and cellular localization. According to this model, when Ca
is absent, the
small subunit of calpain facilitates the proper folding of the large subunit. The
regulatory subunit also makes contact with the N-terminal anchor of Domain I and
Domain IV. This heterodimer forms a structure in which three active residues are held
apart. When calcium is present, the binding of Ca
to the EF-hand motif in Domains IV
and VI induces a conformational change in the two subunits and interfere with the
interaction between the two subunits. Following the conformational change, Domains I
and II are able to move closer to form an active site and calpain autolysis occurs.
Autolysis removes the N-terminal 27 or 19 (calpain- 1 and calpain-2) amino acids from
the 80 kDa large subunit and 91 amino acids from the 30 kDa small subunit (Suzuki,
1990). Calpain autolysis is important for expression of its catalytic activity by
permitting activity at lower Ca concentrations. Subunit dissociation, if it occurs,
releases the larger subunit which retains full catalytic activity (Yoshizawa et al., 1995;
Elce
et
al., 1997). However, if the calpain remains as a heterodimer during catalysis, the
small subunit functions in stabilizing the conformation of the large subunit and in
preventing rapid degradation (Zhang and Mellgren, 1996).
Finally, translocation of calpain to membrane has been observed following
autolysis. The C2-like Domain III may be responsible for promoting binding of active
calpain to membrane in response to Ca
in the cell membrane lowers the Ca
(Saido
et
al., 1993). Binding to phospholipids
concentration requirement for activity (Goll et al.,
1992). Some studies have revealed that calpain binds to membrane proteins as well as to
phospholipids. A membrane-associated activator, specific for calpain- 1 and calpain-2
was detected in rat brain and skeletal muscle (Melloni et aL, 1998). This activator
competes with endogenous calpain inhibitor, calpastatin, for the binding of the catalytic
subunit, increasing its Ca
heterodimeric structure.
sensitivity and promoting the dissociation of the calpain
10
2.2
Calpain substrates and inhibitors
Calpain cleaves a variety of substrates. These include cytoskeletal proteins
(spectrin, integrin, fibronectin, desmin, vimentin, cadherin, talin, ankyrin, filamin);
myofibrillar proteins (troponin, tropomyosin, myo sin light chain kinase A, nebulin, Cprotein, M-protein); soluble or membrane-associated proteins (protein kinease A,
protein kinase C, a-subunit of G-proteins, protein tyrosine phosphatase, phospholipase
C); receptor proteins (EGF receptors, rydodine rceptor, retinoid X receptor), cytokines
(interleukin 1 a); transcription factors (Fos, Jun) and lens proteins (crystallins; Potter et
al.,
1998; Belcastro
et al.,
1998; Shoshan-Barmatz
et al.,
1994; Kawasaki and
Kawashima, 1996). The wild range of calpain substrates displays calpain's wide spread
influence in life maintenance of different cell stages. Calpains exert roles in cell
function by altering gene function through the cleavage of specific transcription factors,
affect cell viability by controlling cell apoptosis andlor cell signal transduction
pathways, and respond to extracellular stimuli and modify physiological functions by
degrading specific proteins, enzymes, or receptors (reviewd by Carafoli and Molinari,
1998; Tidball and Spencer, 2000).
Calpain does not degrade proteins to small peptides or amino acids. Rather,
cleavage occurs at specific sites, leaving large polypeptide fragments with altered
physical properties or for further degradation. Proteins containing PEST primary
sequences (segments rich in proline (P), glutamate (E), serine (S) and threonine (T)) are
possible calpain substrates (Barnes and Gomes, 1995). New studies have shown that
secondary and tertiary conformational features surrounding the cleavage site of target
protein are as important as well as the PEST sequence (Stab ach et
al.,
11
1997).
It is interesting that calpain isoforms have distinct substrate preferences. In
vitro
studies demonstrated that quantitative parameters, such as Kcat and Km, may differ
depending on substrates (Stabach
et al.,
1997). Two autolysised active calpain-1
isoforms (with differences in N-terminal autolysis, 78K and 76K) showed distinct
substrate specificities (Schoenwaelder
et al.,
1997). This indicates that multiple active
forms of calpain with unique substrate specificities contribute differently to cellular
function and may underlie plasticity of cell signaling (Ono
et al.,
1998).
The search for calpain inhibitors developed following the discovery of calpains.
Current knowledge about calpain substrate specificity and the nature of the active site of
enzyme have accelerated the search for calpain inhibitors, which benefits in biological
studies in the area of calpain mediated event and calpain-related disease treatment.
Generally, Ca
chelators, such as EDTA and EGTA, and cysteine protease inhibitors,
such as leupeptin were first applied as calpain inhibitors. Most of these inhibitors are
neither selective nor potent compared to calpain-specific inhibitors such as calpastatin
and PD 150606 (Donkor 2000).
Calpastatin is an endogenous calpain inhibitor expressed ubiquitously in
mammalian cells. Current research has shown that calpastatin is an inhibitor of calpain1 and calpain-2 but not calpain-3 (p94). Four isoforms of calpastatin (Types Ito IV)
exist as a result of alternative splicing or post-translational proteolysis of a single
calpastatin transcript (Geesink
et al.,
1998; Takano et
al., 2000; Li
and Goldberg,
2000). Under physiological conditions in which Ca concentration is lower than that
required to initiate proteolytic activity, calpastatin binds to calpain and completely
12
inhibits calpain activity in a substrate-competitive manner (Kawasaki and Kawashima,
1996). One molecule of calpastatin can interact with four calpain molecules (Uemori et
al., 1990). An imbalance between the activities of calpain and calpastatin is believed to
be responsible for the pathological role of calpain in many diseases and has been
considered as a therapeutic point of treatment (Spencer and Mellgren, 2002).
A variety of peptidic, synthetic peptide mimetic and non-peptide inhibitors have
been recently developed (Donka 2000). Calpain inhibitor I (Ac-Leu-Leu-Nle-H) and II
(Ac-Leu-Leu-Met-H), calpeptin, and leupeptin are widely used (Sorimachi-Y et al.,
1997; Tao et a!, 1998). Although these inhibitors efficiently inhibit both calpain-1 and 2, they are not very specific as they also inhibit other cysteine proteases (Figueiredo-
Pereira etal., 1994; Tsubuki et al., 1996). For example, calpain inhibitor I is a better
inhibitor of cathepsin L and calpain inhibitor II inhibits cathepsin B as well (Sasaki et
al., 1990).
E64 (L-trans-Epoxysuccinyl-leucylamido [4-guanidino]butane) is a naturally-
occurring expoxysuccinate first isolated from fungi as a papain inhibitor (Hanada et al.,
1978). E-64 and its derivates (E-64c and B-64d) inactivate cysteine proteases by
forming a covalent bond with the active site thiolate (Tamai et al., 1981). The inhibition
of calpain by E-64 is highly-selective but not specific to calpain (Barrett etal., 1982).
Recently, several peptidic and peptide mimetic inhibitors were tested in attempts
to enhance potency, selectivity and cellular permeability of the inhibitors. Peptidyl
calpain inhibitors consist of a region for enzyme recognition and an "active site" for
covalent modification of the catalytic site thiol (Donka, 2000). These inhibitors usually
contain a functional group such as an aldehyde or ketone. A dileucyladehyde, ZLLa1
13
(benzyloxycarbonyl-leu-leucinal), was very effective in inhibiting calapin activity
(Tsubuki et al.,
1996). Even more effective, a synthesized oxoamide, AK295, was
reported to show 1000-times inhibition of calpain comparing to inhibition of cathepasin
B (Bartus
et al.,
1994).
PD 150606, a a-mercaptoacrylyic acid derivative, is a relatively potent,
selective, cell-permeable and reversible calpain inhibitor developed by Wang and
colleagues (Wang
etal.,
1996). PD-150606 inhibits calpain by binding to the Ca++
binding domain of calpain, which is different from the active-site-specific inhibitors
described above (Lin
et al.,
1997). Therefore, when PD-150606 is used with other site
specific protease inhibitors such as E-64, the combination produces a very specific
inhibition of calpain.
2,3
Calpain function
The physiological importance of the calpains has long been discussed from
many aspects. Ubiquitously expressed calpains- 1 and -2 appear to be essential in life
maintenance while functions of most other calpain family member remain unclear.
Here, we summarize roles of calpains- 1 and -2, with emphasize on manmials, to
establish the significance of calpains.
14
2.3.1
Ca/pain in cell cycle
Calpain involvement in the eucaryotic cell cycle has been established by
numerous studies, mainly based on work with calpain-specific inhibitors (for review,
see Santella etal., 1998). Studies have shown that calpain inhibitors block the G1- to Sphase transition in serum-stimulated fibrob lasts (Zhang et al., 1997). This effect was
reversible upon withdrawal of the calpain inhibitor. A calpain substrate, p53 tumor
suppressor protein, is suspected to play a key role in the G1 check point. p53 was
identified as a calpain substrate in vitro (Gonen et al., 1997). Blockage of calpain
activity by calpain inhibitor or chelator caused p53 accumulation in serum-stimulated
fibroblasts (Zhang et al., 1997). The result was confirmed by immunofluorescence
localization studies which showed accumulation of calpain- 1 correlated with the critical
time period for p53 proteolysis in serum-stimulated fibroblasts. Another cell cycle
controlling protein, cyclin-dependent kinase inhibitor (p27), is also a calpain substrate.
Calpain-mediated degradation of p27 is required for growth-arrested 3T3 cells to pass
through the G1 to S checkpoint (Patel and Lane, 2000).
Calpain participates in cell cycle during both mitosis and meiosis. Changes in
calpain location during mitosis and meiosis have been reported in several cell lines, rat
eggs and starfish oocytes (Schollmeyer, 1988; Lane et al., 1992; Malcov et al., 1997;
and Santella et al., 1998). Calpain relocation from the plasma membrane to the nucleus
has also been reported. Direct evidence was given by direct injection of calpain in the
vicinity of the nucleus (Schollmeyer, 1988). When the injection was made at interphase,
it quickly promoted transition to metaphase, whereas injection in late metaphase
15
accelerated the transition to anaphase. These effects were inhibited when calpains
were co-injected with calpastatin.
2.3.2 Ca/pain in cell differentiation
Calpain regulates numerous aspects of cell differentiation by limited proteolysis
in myoblasts, osteoblasts, chondrocytes, and adipocytes (for review, see Yajima and
Kawashima, 2002). Calpains- 1 and 2 respond differently in the differentiation system.
In HaCaT keratinocytes, cartilage chondrocytes and rodent osteoblasts, calpain-2 is upregulated 3- to 10-fold compared to calpain-1 (Sato and Kawashima, 2001). Studies also
show that the calpain-calpastatin balance can be an important factor in controlling the
amounts of cellular components that participate in cell differentiation. For example,
Barnoy et
al.
(1996) found that a transient reduction of calpastatin occurred following
the initiation of myoblast fusion. This change allows calpain activation and degradation
of selective proteins. This promotes the destabilization of membrane and creation of
membrane fusion-potent regions (Barnoy et
al.,
1998).
Calpain participates in differentiation in many ways (for review, see Yajima and
Kawashima, 2002). Directly, calpain functions in the turnover of cytoskeletal proteins,
and membranes in the cell remodeling. In response to environmental differentiation
simuli, calpain also has roles in differentiation signaling by digesting specific signal
transduction proteins, such as p107 in restinoblastoma (Jang et
adipocytes (Pate! and Lane, 2000).
al.,
1999) and p27 in
16
2.3.3 Ca/pain in apoptosis
Apoptosis, also called programmed cell death, can be induced in several ways,
some clearly involving Ca (McConkey and Orrenius, 1996). Calpain's critical role in
apoptosis was reported in a number of findings (for review, see Johnson, 2000). For
example, calpain cleaves cyclin-depedent kinase 5 (CDK5) activator protein p35 to p25
leading to neuronal cell death (Lee et
al.,
2000). Furthermore, calpain substrate p53 is
activated in apoptosis (Shen and White, 2001) and the cleavage of fodrin, a calpain
substrate, is considered as a hallmark of apoptosis (Nath et
al., 1996).
Although apoptotic signals can be blocked by calpain inhibitors in several cell
systems, calpain is not the only enzyme involved. In addition, caspases play key roles in
apoptotic pathways (Solary et
al., 1998).
Several lines of evidence indicate calpain is an
upstream factor in apoptosis signaling (Waterhouse et
al., 1998).
Calpain substrate p53
mediates the Bax/caspase-9 pathway in apoptosis (Shen and White, 2001). Calpain
activation of caspase-3 was reported in radiation-induced lymphoma apoptosis, drug
induced t-cell apoptosis (Varghese et
(McCollum et
al.,
al.,
2001) and UV-induced neuronal death
2002). Calpain-mediated caspase-12 activation was also observed in
oxygen- and glucose-deprived cells (Nakagawa and Yuan, 2000). This "crosstalk"
between calpain and caspase proteolytic systems has been emphasized in apoptosis
research recently (Neumar et
al.,
2003).
17
2.34 Caipain and human diseases
Calpain has long been targeted in therapeutic drug design because of its
multifunction and specific and limited proteolysis. The most promising calpain research
in this regard relates to neuro-muscular disorders. Tn addition, calpain inhibitors have
been tested in several disorders including cataract development (David and Shearer,
1993), autoimmune diseases (Hayashi
et al.,
2000), vasospam, toxin exposure, kidney
malfunction and genetic defects (for review, see Vanderklish and Bahr, 2000).
Generally, calpains-1 and -2 both contribute to apoptosis and necrosis in injured
neurons (Wang and Yuan, 1994). In or near the core of cerebral ischemia (during stroke
and cardiac arrest) and traumatic brain injury, neurons experience a large and rapid rise
in intracellular calpain activity. This might resulte from decreased blood flow,
membrane depolarization, and opening of voltage-gated neuronal Cachannels. In
Alzheimer' s disease, intracellular Ca
(Hartmann
et al.,
is also elevated in susceptible central neurons
1993). Over-activated calpain could lead to uncontrolled degradation
of cytosolic proteins and ultimately, to neuronal death.
In neuromuscular degeneration conditions, such as muscular dystrophy or
denervation atrophy, calpain activity increases several-fold (Stracher, 1997). In muscle
wasting diseases, such as Duchenne muscular dystrophy (DMD), an intracellular Ca
imbalance causes muscle damage by activating calpain. These lines of evidence suggest
calpain participates in multiple forms of muscle pathogenesis.
In vitro
studies have
shown calpain can degrade myofibrillar proteins very specifically and in a rate-limiting
maimer (Nelson and Traub, 1983; Goll
et al.,
1992). Therefore, several calpain
inhibitors have been tested as therapeutic agents in nerve and muscle degeneration.
2.4
Calpain superfamily
In addition to "conventional", "house-keeping" or "classical" calpains (calpains-
1 and -2), tissue-specific calpains, ubiquitous calpain and calpain-like proteins have
been reported in mammals, vertebrates, nematodes, Drosophila, budding yeast, and
plants (Sorimachi and Suzuki, 2001, Ono
et al.,
1998). In mammals, more than fouteen
large calpain subunit homologous and two small subunit isoforms have been studied
(Table 1). These proteins form the calpain superfamily. Members of the calpain family
have divergent structure, but the primary sequences of the protease domains show
significant similarities to each other (Figure 3). Most of the members of this
superfamily have Ca-binding andlor probable Catbinding domains, and C2 or C2like domains. Interestingly, some calpain superfamily members, such as calpain-6, lack
the conserved active site Cys. This is probably because their physiological functions
have been adapted and specialized to the cells in which they function.
The first tissue-specific calpain to be discovered was p94 (now called calpain-3).
This is expressed exclusively in skeletal and cardiac muscle (Sorimachi et
al.,
1989). It
possesses three unique insertion segments (NS at the N-terminal, IS 1 in Domain II and
1S2 between Domains III and IV) not found in other calpains. Calpain-3 does not
associate with the conventional calpain small subunit (Kinbara et
al.,
1998). The
19
Calpain protein #
Calpain
Other Names
EF-hand
Tissue Type
+
ubiquitous
lJ-Calpain,
Calpain 1
capnl
CAPN1
m-Calpain,
Calpain 2
Calpain 3
capn2
capn3
CAPN2
nCL-1, p94
Ubiquitous
Skeletal muscle
(Lp82, Lp85, Rt88)
lens, retina
htra3
caIn5
nCL-3
CAPNX,
-
Ubiquitous (High in
colon, small intestine
and testis)
Calpain 6
CaDn6
Calpamodulin
-
Placenta?
Calpain 7
capn7
palBH
-
Ubiquitous
Calpain 8
Capfl8
nCL-2
+
Stomach mucosa
Calpain 9
can9
nCL-4
CAPN1O,
+
Digestive track
Calpain 10
caIDnlO
CaPn8
Calpain 11
cainll
CAPN11
Calpain 5
Ubiquitous
+
Testis
Ubiquitous
Calpain 12
Calpain 13
Calpain 14
capnl2
capnl3
capnl4
Calpain 15
capnl5
Calpain Small Subunit capnsl or
1
Cpnsl
CAPN12
CAPN13
CAPN14
+
(High in hair follicle)
+
Testis/Lung
Ubiquitous
Sol H
CAPN4
Ubiquitous
+
Ubiquitous
+
NA
Calpain Small Subunit capns2 or
2
cpns2
Table 1. Calpain superfamily. Fourteen calpains and two small isoform have been
discovered so far. See text for details. (Note: NA = not available)
21
insertion segment-2 (1S2) contains a nuclear localization signal-like basic sequence.
Hence p94 has been localized in the nucleus as well as the cytosol (Sorimachi et al.,
1993). In skeletal muscle, p94 binds to a gigantic muscle protein, connectin/titin, via its
insertion segment-2 (1S2). Mutation of p94 gene is responsible for Limb-Gridle
Muscular Dystrophy Type 2A (Sorimachi et al., 2000; Broux et aL, 1995). Four splice
variants of calpain-3 have been detected in rat lens (Lp85, Lp82), retina (Rt88) and
cornea-specific Cn94 (Fukiage et al., 2002; Fukiage et al., 2000). These eye-specific
calpains were classified as AX1 subclass (altenative eon fl. Lp85 and Lp82 were
reported to have roles in lens development and maturation (Ma et al., 2000) while
functions of Rt88 and Cn94 are unknown.
Calpain-5 and -6 are highly homologous (Matena et al., 1998) to each other but
are divergent from other calpains identified. They both possess most of the amino acid
sequence conserved with calpain family members but lack the C-terminal Ca-binding
domain (Dear et al., 1997). Both calpains-5 and -6 were initially identified as a
homologous to the Caenorhabditis elegans sex determination gene tra-3 (Mugita et al.,
1997). Interestingly, calpain-6 is not a functional protease because it lacks the critical
active site Cys residue. Therefore, calpain-6 was suspected to participate in regulatory
processes by competing with alpain-binding substrates or cofactors (Dear etal., 1997).
The mRNA expression pattern is also different for both proteins. Calpain-5 mRNA is
expressed ubiquitiously in human tissues but calpain-6 was found only in placenta.
Calpain-6 seems to be a sex-related protein because its gene (capn6) is located on the
X-chromosome. The physiological functions of both proteins remain unclear.
22
Calpain-7 (PaIBH) is the most divergent calpain family member identified so
far (Franz et al., 1999). PalB was first found in Aspergillus nidulans as a protease
involved in alkaline ambient pH adaption (Denison et al., 1995). This protein has a
cysteine-protease domain showing significant similarity to mammalian conventional
calpain but lacks an EF-hand Ca binding cluster. Calpain-7 is a mammalian
homologous form of Aspergillus PalB and its mRNA was expressed nearly ubiquitously
throughout mammalian tissues (Futai et al., 2001). It shares only 26-35% identity with
the rest of the calpain members. Calpain-7 contains the protease Domain II, but has
little homology in Domain I (a long N-terminal region) and III (Pal homologous
domain; PBH) (Sorimachi et al., 1997). When expressed in COS cells, calpain-7 was
enriched in the nucleus, suggesting its role is distinct from conventional calpains (Futai
etal., 2001).
A stomach smooth muscle-specific nCL-2 (calpain-8) and it's variant nCL' were
first reported in 1993 (Sorimachi et al., 1993). Like other typical calpains , nCL-2 has
calpain distinct domains, a protease, a C2-like, and a 5 EF-hand Ca-binding domain.
Therefore, it is suggested that nCL-2 is a cytosolic Ca-active protease regulated by
calpastatin and the calpain small subunit. nCL-2' is an alternative-splicing product of
the nCl-2 gene, capn8. nCL-2' lacks the C2-like and EF-hand domains but retains the
N-region of nCL-2. Unlike nCL-2, nCl-2' is also found in the anterior pituitary
following estrogen treatment (Duan et al., 2002).
It is most intriguing that human capn2 and capn8 genes overlap in a head to
head orientation on chromosome 1 (Hata et al., 2001). Here, the expression of both
calpain-2 and calpain-8 in stomach may influence each other.
23
Calpain-9 (nCl-4) was found to be a digestive tract-specific calpain (Lee et
al.,
1998). it possesses a highly-conserved sequence with calpain family members with
significant similarity of protease and Ca-binding domains and interacts with the 30K
calpain small subunit for it's full activity (Lee et
aL,
1999). Recently, nCL-4 was shown
to be involved in tumorigenesis as a tumor suppressor in fibroblast (Liu
et al.,
2000).
This finding agrees with a study which indicated that nCL-4 mRNA decreases in gastric
cancer tissue in vivo(Yoshikawa et
al.,
2000). Thus, identification of nCL-4 substrates
may reveal the mechanism of gastric cancer.
Calpain- 10 is an atypical calpain in that it lacks the EF Catbinding domain
and, instead, has a T-domain homologous to calpain-5 (Horikawa et
al., 2000).
The
calpain- 10 gene is expressed ubiquitously and encodes at least eight alternative splicing
variants. Some of these variants lack an intact protease domain, which suggests that
calpain-lO may have diverse non-proteolytic cellular functions. h a genome-wide
screen for Type 2 diabetes (non-insulin-dependent diabetes mellitus, NIDDM), the
human calpain- 10 gene was identified as a type 2 diabetes susceptibility gene
(Horikawa
capnl 0
et al.,
2000; Baier et
al., 2000).
The polymorphisms in noncoding regions of
are believed to alter risk by affecting transcriptional regulation of calpain-1 0.
Dear and colleagues reported four novel calpain large subunit genes by
searching the public EST and NCBI genomic sequence database (Dear et
1999b;
2000;
genes,
capnll,
Dear and Boehm,
2001).
al,
1999a;
As reported today, protein products of these four
12, 13 and 14, are unclear. Calpain-1 1 is predicted to be an intracellular,
Ca-dependent cysteine protease. Calpain- 11 mRNA exhibits a highly-restricted tissue
distribution with highest levels present in testis. Calpain- 12 was first described in the
24
mouse skin. In situ hybridization and Northern blot analysis showed that calpain-12
is expressed mainly in the cortex of the hair follicle during the anagen (proliferating)
phase of the hair cycle. Three variants of calpain-12 mRNA were found as a result of
alternative splicing. The isoforms differ in their C-terminal ends, ending with aberrant
domain-Ill and lacking Ca-binding domains. Human calpain-13 mRNA was detected
only in testis and lung while calpain-14 mRNA could not be detected in any of 76
tissues examined (Dear and Boehm, 2001). The predicted calpains- 13 and -14 proteins
were more homologous with each other (37.9%) than with other calpain large subunits.
Calpain-15 (Sol H) is a human homologue of the Drosophila melanogaster
small optic lobes gene (sol) and its cDNA was first isolated from human brain (Kamei
et al., 1998). The Sol H protein is a 117 kD protein, which is much longer than other
calpain family members. In fact, the catalytic domain contains the only similarity
between So! H and the classic calpains, while the N-terminal contains a C2-C2 zincfinger-like repeat and C-terminal function unknown. It is suspected that So! H
mightplay a role in development of sensory system neurons in Drosophila and
mammals (Kamei et al., 2000).
As described before, capn4 gene encodes calpain small subunit (named small
subunit-i). The small subunit is indispensable for calpain activity and function in vivo
because the knockout of capn4 is lethal in mouse (Elce et al., 2000). The two
housekeeping ca!pains (calpains- 1 and -2) share a common small subunit. Recently, a
novel human small subunit of calpain, small subunit-2, was reported and shown to be a
functional equivalent of the small subunit-i in vitro (Schad et al., 2002). This protein
helps the large subunit to fold properly as well and its Ca
sensitivities are almost
25
identical as the small subunit-i. However, the small subunit-2 binds to the large
subunit weakly. Another notable difference between the small subunit-2 and regular
small subunit-i is the different autolysis pattern. The conventional small subunit is
thought to undergo autolytic cleavage upon activation by Ca, resulting in an 1 8kDa
fragment (Tompa etal., 1996). The novel subunit shows no sign of such a specific
cleavage. It is also suggested that the expression of small subunit-2 is tissue specific.
2.5
Calpain-5
In C.
elegans,
sex determination is controlled by a complex cascade of negative
regulation. TRA-3 is involved in this pathway as a potentiator of TRA-2 which
promotes female development in XX hermaphrodites (Sokol and Kuwabara, 2000).
TRA-3 is a cytosolic protein with Mr of 73.6 kDa. TRA-3 has similarity to the calpain
family throughout the Domains 1-111 (Barnes and Hodgkin, 1996). In particular, 63% of
residues in the TRA-3 Domain II are similar to those of rat p94 and with three catalytic
residues identical to other calpains. This indicates that TRA-3 has proteolytic activity.
In fact, TRA-2 was established as a TRA-3 substrate (Sokol and Kuwabara, 2000).
However, TRA-3 lacks the C-terminal calcium-binding domain, Domain IV, which is
highly-conserved in the calpain family. Instead, TRA-3 has a Domain T. The absence
of calpain Domain IV indicates that TRA-3 does not associate with small subunit for its
activity and is not regulated by the small subunit. Domain T's function is not clear but
does not cappear to be critical for protelytic function of TRA-3 (Barnes and Hodgin,
1996).
26
As stated previously, calpain-5 was first identified as a homologous of the C.
elegans sex determination gene tra-3 (Mugita et al., 1997; Dear et al., 1997). Originally
capn5 was cloned from a human cDNA library and a mouse embro cDNA library. The
encoded amino acid sequences are highly conserved across species. For example,
human capn5 shares 92.2% homology to mouse capn5 (Figure 4). Human capn5 has a
calculated M 73KD, predicted p1 of 8.3 and capn5 gene was located on chromosome
1 1q14. The overall protein sequence shows a high similarity with C. elegans and
highly-conserved Domains Ito III with other calpains as well. The catalytic residues in
Domain II are particularly highly conserved. As with the TRA-3 protein in C. elegans,
Domain IV is exceptional and distinct from other calpains: it is replaced with Domain T
that has no calcium-binding EF structure. Because the Domain IV is thought to be
essential to be the interaction with the small subunit or calpastatin, which play
regulatary roles in calpain' s activity, this indicates that calpain-5 could be monomeric
and potentially constitutively-active. Interestingly, an EF-hand structure was predicted
in Domain III. Thus not all calcium-induced activity may be lost in calpain-5. Human
calpain-5 seems to be expressed ubiquitously with highest level in testis. The high
expression of calpain-5 in testis supports a sex-related function as in TRA-3. Calpain-5
mIRNA can also be found in other tissues including brain, colon, small intestine and, at
low levels in other tissues. During mouse embryogenesis, the major expression of
calpain-5 occurs in the developing thymus, and in sympathetic and dorsal root ganglia
(Dear and Boehm, 1999). However, no calpain-5 protein was detected in vivo to date.
Hence, its physical function remains unclear.
27
House Capn5
Rat Capn5
Human Capns
Tra-3
Domain I]
MFSCAKA--YEtXIYSALKRACLRKKVLFEDPLFPATDDSLYYKGTPGPWRbWRPKDIC 58
MFSCTKA--YEYYSALKRACLRIU(VLFEDPHFPASDDSLYYKGTPGP1VRbJKPPFDIC 58
HF5CVKP--yEDc'jY5ALRQDCpRRKVLFEDpLFpAT])D5LYYKGTPGpAVRJKpPKGIC 58
HTRSEKTPEFGNJYEKLR(IC IKKKQPFVDTLFPPTNLFLEQRQ5SDIVTJKRPGELH 60
*
*:: * :.* * *. **:::**:
..
:
House Capn5
Rat Capn5
Human Capn5
Tra-3
[ Dcinainll
:
:
DDPRLFVDGISSIilThHQGQVGHCbJFVAACSSLASRE5LWQKVIPDUKEQEWNPEID5YA 118
DDPRLFVDGISSi-WLHQGVGNC1JFVAACS5LASRE5LWQKVIPDWKEQETJNPEKPDSYA 118
EDFRLFVDGI5S1U)LHQQVGNC1JFVAAC5SLASRESL1JQgVIPDREQETJDPRKAQAYA 118
PDPHLFVEGASPNDVTQGILGNCWFVSACSALTHrJFKLLAQVIPDADDQHW5TK--HAYA 118
:******:*:*:
. :k. .
::
.
House Capn5
Rat Capn5
Human Capn5
Tra-3
Human Capn5
Tra-3
:
GIFHFNFWRFGEWDVIVDDRLPTVNNQLIYCHSNS0JEFbJCALVEKAYIXLAGCYQALD 178
GIFHFNFWRFGEWDVVVDDRLPTVNNQLIYCHSNSI'IEFIJCAPVEKAYAXLAGCYQALD 178
GIFHFHFNRLG-HVIDERLPTc1NNQLIYCH5N5PNEFtCALVKAYAKLAGCYQALD 177
GIFRFRFbJRFGKWEVVIDDLLPTRDGKLLFARSI(TPNEFIJSALLEKAFAXLYGCYENLV 178
***:*.**:t
House Capn5
Rat Capn5
.
*:*:
:*:
:
:*:*
. :*: : . :*: :
***:
238
238
GTADALVDFTGVSEPIDLTEGDFMDETKRNQLFERHLKVHSRGGLISASIKAVTAA 237
GGHL5DALQDVZGVAETLH1ThXFLKDDPNDTELRLFNDLKTAFDKGALVVAAIAARTKE 238
:**: .
:** *.* * *
**: :*
:***:*.: :
:
GG'ITADALVDFTGGVSEPIDLTEGDLATDEAKRNQLFERVLKVHSRGGLISASIKkVTAA
GGTADALVDFTGGVSEPIDLTEGDLATDEAKRSQLFERVLKVHSRGGLISASIKAHTAA
.
House Capn5
Rat Capn5
Human Capn5
Tra-3
House Capns
Rat Capn5
Human Capns
Tra-3
*
* ***t*******:
Domain II]
House Capn5
Human Capn5
Tra-3
Human Capn5
Tra-3
:.:
.:
:*:
::***:*****:
Domain III
[
***:****
..**
:
348
348
347
358
*:::***
:*****
S IRKT1PEEARLHGA JTRID---PQQNR5GGC INHKDTFFQNPQYVFEV0PEDEVL 151 405
SIHKTbERLRWTRHED---PQQNRSGGCINHKDTFFQNPQYIFEVKKPEDEVLICI 405
S IHKTVEEARLHGAWTLD---PRQNRGGGC INHKDTFFQNPQYIFVKKPEDEVL IC I 404
418
SFSRSYDEQIVFSEWTTNGKKSGAFDDRAGGCIThIFKATFCNNPQYIFD IPSPNCSVHFAL
* ......
**
. .* *** * * ** .****.*.
*.
*.
.
House Capns
Rat Capn5
*
GPWSDT5EETJQKVSKSREKHGVTVQ----DDGEFIJMTFEDMCRYFTD I IKCRL ThITSYL
GPbJSDTSEEWQKVSKSEREKHGVWQ----ODGEFWMTYEDHCRYFTDIIKCRLIMTSYL
GPtJSDTSEEWQI<VZKSEREKHGVWQ----DDGEFbJHTTEDVCRYFTDIIKCRV]NTSHL
GAbT5DDSPEPQNV5A5QL5THGVQpAJq5D5DDGDFWHpilESFVHyFTDISLCQLFNTSVF
*
Rat Capns
292
292
291
298
DARLACGLVKfAYAVTDVRKVRLG----HGLLAFFKZEK--LDMIRLRNPtJGEREJT
D1TRLACGLVKG{AYAVTDVRRVRLG----HGLLAFFKSEK--LDHIRLRNPtJGERV1JT
DMARLACGLVKIAYAVTDVRKVRLG- ---HGLLAFFESEK--LDHIRLRNPiGERE1dN
EIEESLDCGLVKc{AYAVVCTIDVTNPNERSFTSFLHGSKR0JILIRLQNPb1GEKEbJN
.
.
QQRPKRSTRREGKGENLAIGFD IYKVEENRQYRNHSLQHKAASS IYINSRSVFLRTELF 464
QQRPKRSTRREGKGENLAIGFD IYKVEENRQYRNHSLQ1JKAASS IYINSPSVFLRTE-LP 464
QQRPKRSTRREGI<ENLAIGFD IYKVEENRQYRMHSLQHKAASS IYINSPSVFLRTD-QP 463
Ir-DP5EGLKREpyVTIGHHHJENJIRQyRvHTAHl IAISDYASGRSITYLHLQSLP 477
*
::**:.
:
*******
Domain III]
(
* * *
*
:
*
Domain T
House Capn5
Rat Capri5
EGRYVIIPTTFEPGHTGEFLLRVFTDVPSNCRELRLDEPPRTCWSSLCGYPQQVAQVHVL
Human CapnS
EGRYVI IPTTFEPGHTGEFLLRVFTDVPSNCRELRLDKPPHTCI5SLCGYPQLVTQVHVL 523
RGRYLLIPTTFAPKEQTLFHLRVYSDEHIHFSpLTKHAPKLGLLK--CKSAQSVTRLTIH 535
Tra-3
House CapoS
Rat Capn5
Human Cap n5
Tra-3
.***: :*****
Human Capn5
Tra-3
*
.
:
***: :*
*
:
*
::
.*
*
GAAGLKDSPTGANHYVI IRCEGEKSJRSAVQRGTSTPEYNVIGIFYRKKLAQP ITVQVtThIH 584
GAJiGLKDSSTGANSYVI IKCEGEFNRSAVQRGTSTPEYNVI'ZGIFYRKKLSQP ITVQVWNN 584
GAJLGL KD HP TGJNZ WI IKCEGDRVRS AVQKGTSTP E YNVKGIFYRKKL SOP ITVQVINH 583
GVDFNSASTGT1ThJVYAILKDSRKBFRTKThSGVpS IQWDEQFLFHKSKNRQQYKIEVWED 595
*
House Capn5
Rat Capn5
524
EGRVIIPTTFEPGHTGELLLRVFTDVPSNCRELRLDEPPRTCISSLCGYPQQVVHVL 524
*
*
*.*
*.
.......... *
*
*
R-VLKDEFLGQVfflKTAPDDLQDLHTLiuQDRS5RQPSDLPGIVAVPVLCSASLTAV 640
R-VLKDEFLGQVHLKTAPDDLQDLHSLHLQDRSGRQPSDLPGIVAVRVLCSASLTAV 640
R-VLKDEFLGQVKADPDNLQHTLthRDpSRQP5NLPGTVAVHILSSTSL!V 639
RKMARDHLLJLQSVI IAL IDNENRDTTLQLTDPRG-.---TVIGTVZVTVSAFDDPHYL 648
:*:**
.
:
* *:*
:
.
.
28
Figure 4. Calpain-5 alignment. The amino acid sequences of mouse, rat, human and
C. elegan were compared by Clustaiw Sequence Alignments at EMBL-EBI (European
Bioinformatics Institute; http ://www.ebi .ac.uklToolsf). All proteins contained a
conserved domain structure. Catalytic residues are indicated by underlining. The
predicted EF hand motif is double-underlined.
In this study, we expressed human calpain-5 in insect cells to yield active
protein. We cloned capn5 gene into a baculovirus expression vector with polyhistidine
and the cMyc at C-terminal as tags. The expression system utilized viral-mediated
protein expression in insect cells to ensure proper post-translational processing. The
protein then was collected and purified. We used various approaches to prepare calpain5 to study its biology. Calcium-sensitivity of the enzyme was assayed as well its
temperature sensitivity, p1, effects of inhibitors and physiological substrates. The work
revealed several interesting physical attributes of calpain-5 and provides hints of its
function in
vivo.
IiJ
3. MATERIALS AND METHODS
3.1
Calpain-5 expression vector
Calpain-5 cDNA, cloned into pSEC4O vector (Figure 5), was obtained from Dr.
H. Sorimachi, Tokyo, Japan. pSEC4O-capn5 (1000 ng4xl) then was digested with EcoR
land Dra I at 37 °C in a 50 tL reaction containing lx
One-phor-A11TM
buffer (enzymes
and buffers were from Pharmacia, Piscataway, NJ). This digestion yielded a 1995 bp
fragment with protein initiation codon, human calpain-5 coding region (1935 bp), c-
Myc tag, and His tag at the 3'end and termination codon. The digested fragment was
then purified with ethanol purification and then inserted into a baculovirus expression
vector (pFastBac 1; Invitrogen, Carlsbad, CA).
3.2
Insect cell culture
Insect cell line, SF-9 was purchased from ATCC (Rockville, MD). Cells were
grown in IPL41 medium (GibcoBRL, Rockville, MD) with 10% bovine serum and 50
pg/m1 of gentamycin (GibcoBRL, Rockvill, MD) at 27 °C. Cells were maintained in
adherent culture and passaged at confluency. For large scale culture, cells were cultured
in a shaking flask with addition of 0.1% Pluronic F-68 (GibcoBRL, Rockvili, MD) at a
density of 0.7 to 1.0
x106
cells/mi. Suspension cultures were passaged before they
reached a density of 2.0 to 2.5 x106 cells/ml.
31
F;1iI
Xba I
I!
:
"Wi'
SR
rornoter
EcoR I
Xba I
TCTA&A
GCATC bAATl.
tCCCCM
EiI
Human CapnS cDNA
Initiation codon
Termination cadon TGA is changed to TCT (Ser.
Figure 5. Map and cloning site of pSEC4O and human calpain 5.
3.3
32
Baculovirus expression system
The Bac-to-Bac Baculovirus Expression System was purchased from Invitrogen
(Carlsbad, CA). Figure 6 depicts the generation of recombinant baculovirus and
expression of the gene of interest (ex, capn5) using this system.
Baculovirus expression vector pFastBacl was digested with EcoR
I and
Stu I
(within MCS; multiple clone site) and purified under the same conditions described
above. The capn5 fragment (30 ng) and digested pFastBacl(25 ng) were then ligated
(1mM ATP, lx One-phor-A11TM buffer, room temperature) with T4 ligase (Phamacia)
for 4 hours. The ligation reaction then was used for competent XL- 1 Gold E.
coli
(Stratagene, La Jolla, California) transformation using the heat shock method.
For transformation, competent XL-1 Gold E.
coli (100
pL) and 8 tL ligation
mixture were incubated together at 4 °C, 30 sec. The mixture was heated at 42 °C for 30
sec and put back on ice for 15 mm. The mixture then was incubated at 37 °C for 1 hour
and applied to an LB/ampicillin plate for 1 day or until colonies formed. E.
coli
containing cloned vector was checked by mini-preparation (kit from Qiagen, Valencia,
CA) and then restriction digestion. The Capn5 fragment was double-confirmed by
sequencing. This expression vector then was purified and transformed into competent
DH 1 OBacTM E. coli
cells for bacmid preparation.
DH1OBacTM E.coli competent cells were purchased from Invitrogen (Carlsbad,
CA). The pFastBacl-hCapns construct was incubated with DH1OBacTM E.coli
competent cells on ice for 30 mm. The cells were heat-shocked for 45 sec at 42 °C
33
oonm penmid
ot
T 78
Pfooneeen,
)-
omtntn
Competent OM1OC
Donor PlSm'd
E COICI4O
E, 00y
(LecZi
Ctitarntn Recomtimrg Bacnid
MnpropofHi9h
Detetflua Wt 1tor
Recomboant
by Plequn
ranofeoueof
000 0000000
/
cecto'Reooero
Renombkant
Baom DNA
Recombinant Gene Cimpresslon
or Wat M1peation
Figure 6. Diagram of the Bac-to-Bac Baculovirus Expression System. Figure is
modified from menu of Bac-to-Bac Baculovirus Expression System (Invitrogen).
34
without shaking and then immediately transferred back on ice for 2 mm. The cells
were then plated on a LB agar plate containing 100 tg!ml Bluo-gal, and 40 j.ig/ml IPTG
(tetracycline and IPTG were from Sigma, St. Louis, Mo; kanamycin, gentamycin, and
Bluo-gal were from Gibco-BRL). The plate was incubated for 24-48 hours at 37 °C.
White colonies were selected for PCR analysis and results were assayed by 2% agarose
gel electrophoresis.
PCR-confirmed capn5-expressing DH1O E.coli single colonies were picked and
inoculated in 2 mL LB medium containing 100 tg/m1 Bluo-gal, and 40 pjg/mI IPTG.
The cultured E. coli were then incubated at 37 °C in a shaking incubator until cells
reach stationary phase. The cells were pelleted by centrifugation at 14,000 x g for 1 mm
and then resuspended gently with 0.3 mL of Solution 1(15 mM Tris-Cl, pH 8.0, 10 mIVI
EDTA, 100 tg!mL RNase A). Solution 11(0.3 mL: 0.2N NaOH, 0.1 % SDS) was added
and gently mixed. The solution was incubated at room temperature for 5 mm. After this,
0.3 mL of 3M potassium acetate, pH 5.5, were added and then the mixture was
incubated on ice for 5 mm. DNA then was collected by adding isopropanol, 4 °C, 30
mm and by centrifuging at 14,000 x g forl5 mm. DNA was then recovered in TE
buffer, pH 8.0.
3.4
Expression of Calpain-5
We initiated calpain-5 expression by transfecting recombinant capn5 bacmid
into insect SF-9 cells as described in Bac-to Bac baculovirus expression system
(Invitrogen). SF-9 cells were cultured in a 6-well plate. When cell density reached
lx 106 cells/mi, the cells then were incubated with lipofectin (0.6%) and bacmid (1
35
i.tg) at 27 °C with imi fresh IPL41 medium for 5 hours. The cells were then replaced
with fresh IPL41 medium with 10% serum and 50 jig/mi of gentamycin and incubated
for 7 days to amplify viral particles. Virus was collected by centrifugation of the cell
medium at 2500 x g for 5 mm. The supematant containing virus particles was collected
and stored at 4 °C.
When transfecting the virus into SF-9 cells, 100 ml of cells were collected at a
density of about 1.66 xlO6 /ml. The cells were centrifuged at 1000 rpm for 5 mm. Cells
were then re-suspended in 4 ml of medium with 10% serum, 50 jig/mi of gentamicin,
0.1% F-68 (as shock absorbent) and 1 ml of virus solution. The cells were incubated at
room temperature for 1 hour and mixed gently every 15 mm. Then the cells were
transferred to a culture flask with 100 ml IPL-41 medium and incubated at 27 °C for 41-
43 hours for protein expression.
3.5
Cell lysis
Cells with expressed calpain-5 protein were collected by centrifugation at 2,500
x g for 5 mm. The cells then were resuspended in TED buffer (2Omn Tris-Cl pH7.5,
5mM EDTA, 1mM DTT), 0.5mM PMSF, 5OjiM leupeptin and 50 jiM chymostatin
(PMSF, leupeptin and chymostatin were from Sigma, St. Louis, MO). For large-scale
preparation, the cells were homogenized on ice for 5 mm using Brinkman PT 10/3 5
Polytron SP (Westbury, NY). For small-scale preparation, cells were freeze/thawed
three times in liquid nitrogen. The cell lysate was then incubated on ice for 15 mm and
36
centrifuged at 11,000 x g for 1 hour. The supernatant was then filtered (0.8 pm) into a
clear tube for further analysis.
3.6
Chromatography column test
Before applying the SF-9 cell lysate sample onto a large column for large-scale
preparation, a small amount of the sample was applied into a set of syringe columns to
decide which ion exchange ligand was best. Both ion exchanger and hydrophobic
interaction chromatography columns were tested. Ion exchange columns included
HiTrap SP Sepharose Fast Flow, HiTrap Q Sepharose Fast Flow, HiTrap DEAE
Sepharose Fast Flow, and HiTrap CM Sepharose Fast Flow (HiTrap IEX Selection kit,
Amersham Biosciences, Piscataway, NJ). Hydrophobic interaction columns included
Phenyl Sepharose Fast Flow, Butyl Sepharose Fast Flow, and Octyl Sepharose Fast
Flow (HiTrap HIC Selection kit, Amersham Biosciences). The start buffer for Q and
DEAE Sepharose columns was 40 mM Tris (pH 8.5), 5mM EDTA with 1mM DTT.
The start buffer for CM and SP Sepharose columnswas 40 mM Tris (pH 6.5), 5mM
EDTA with 1mM DTT. The start buffer for hydrophobic columns is 40 mM Tris (pH
7.5), 5mM EDTA, 1mM DTT with 0.5M NaC1.
Purification procedures are described in the instructions to selected kits. Briefly,
for ion exchange columns, after setup of the columns, the columns were washed and
equilibrated with start buffer after which sample was applied at 1 mL/min. The column
was then washed with 5 mL of start buffer or until no material appeared in the eluent
and finally eluted with 5-10 mL of elution buffer (start buffer with 0.4 M NaC1). For
37
hydrophobic columns, columns were pre-washed with start buffer and then samples
were applied. Columns then were washed with 10 ml of start buffer then eluted with
TED (pH7.5) containing 50% ethylene glycol.
3.7
Chromatography
About 400 ml of SF-9 cell culture pellet were resuspended in 50 ml TED buffer
with inhibitors (500 iM PMSF, 50 tM chymostatin, and 50 M leupeptin). After
filtration (0.8 pm), sample was chromatographed on a large DEAE-Sepharose (Sigma)
column (2 x 30 cm). The column was first equilibrated with TED buffer and eluted with
a 400 mL linear gradient buffer of NaCI (0 mM to 1M in TED) and collected in 5 ml
fractions. The column eluent was monitored for protein concentration and analyzed by
Western blot against c-Myc antibody. Highly inimunoreactive column eluents were
pooled and dialysed overnight and then filtered for Q-Sepharose (Sigma)
chromatography (1.5 x 67 cm). As above, the column was equilibrated and eluted with
400 mL TED/NaCl gradient buffer from 0 mM to 1M. The column eluent was
monitored for protein concentration and analyzed by Western blot. A pool of highly
immunoreactive column eluents were pooled and dialysed, then concentrated (by
Centriprep- 10 concentrator, Millipore, Bedford, MA) into 1 ml and filtered for
Superdex-200 (Pharmercia Biotech, Piscataway, NJ) chromatography (1.5 x 28 cm).
The Superdex column was eluted with TED buffer with no salt. Again, a pooi of highly
immunoreactive eluents were pooled and dialysed and then filtered for Source-Q
(Pharmacia) chromatography (1.5 x 20 cm). The Source -Q column was equilibrated
and eluted with a TED/NaCl gradient buffer from 0 mM to 1M at flow rate 0.1
mi/mm. The column eluant was monitored for protein concentration and analyzed by
Western blot. Only the fractions containing highly immunoreactive product were used
for Capn5 activity assays.
3.8
SDS-PAGE and Western blot
Protein was subjected to 10% SDS-PAGE according to Laemmli (1970). The
gel was run at 120V in SDS buffer (15.6 mM Tris, 120 mM glycine, 10% SDS, pH 8.3)
until the bromophenol blue dye front reached the bottom of the gel. After
electrophoresis was completed, the gel was either washed with water and then stained
with Biosafe
Coomassie stain (Biorad, Hercule, CA) or transferred by electroblotter
apparatus. The protein was transfered (25mM Tris, 192mM glycine, 20% methanol, 4
°C) onto a PVDF (Biorad) membrane overnight at 30 V. The membrane was then
blocked with 5% skim milk in TTBS (20mM Tris-Cl pH7.6, 137mM NaCl, 0.1%VIV
Tween-20) for 30 mm. After four washes with TTBS, the membrane then was incubated
with primary antibody [anti-c-Myc antibody (1:5000), anti-calpain-1 (1:1000) and anticalpain-2 (1:500) (Biomol, Plymouth meeting, PA; in 5% milk/TTBS)] for at least 1
hour. The membrane was then washed with TTBS four times and incubated with antimouse antibody conjugated with HRP (1:10000, Biorad) for another hour. After four
TTBS washes, the signal was detected using a chemiluminesecence method by Immu-
Star HRP substrate (Biorad). Signal was detected by exposure to Biomax film
(Eastman Kodak, Rochester, NY).
39
3.9
2D electrophoresis
An original protein sample (about 3-5 mg /mL) was diluted by rehydration
buffer (8M urea, 2% CHAPS, 10mM DTT, 0.2% Bio-lyte 3-10 ampholyte) to 0.3-0.5
mglmL. For first dimension isoelectric focusing electrophoresis, an 11 cm ReadyStrip
IPG strip @H 3-10; Bio-rad) was wetted in 200 pL solution containing sample and
buffer then overlapped with mineral oil. The strip was allowed to rehydrate for about 12
hours. The strip was then run in a Bio-rad PROTEAN IEF Cell (Bio-rad) at 6,000 V for
a total of 40,000 volt-hours. For the second SDS-PAGE dimension, the strip was first
equilibrated in 5 mL equilibrating buffer (50mM Tris, pH 8.8, 6M urea, 2% SDS, 30 %
glycerol, and 1% DTT). Then the strip was re-equilibrated in the same buffer containing
1.5% iodoacetamide. The strip was transferred onto a Criterion pre-cast gel (12.5%
Tris) overlaid with 0.5 % agarose then run for 2 hours at 100V. The gel then was then
either stained with SYPRO Ruby (Bio-rad) for 3 to 12 hours or transferred to a PVDF
membrane for Western blot. p1 of Calpain-5 was analyzed by PDQuest software
(BioRad).
3.10
His-tag protein purification
With His-tag at the C-terminal, the expressed Capn5 can be purified by TalonTM
Cell Thru resin (BD Biosciences, Palo Alto, CA). Briefly, cells in 25 ml of SF-9 culture
were lysed in Talon wash buffer (50mM sodium phosphate, 300mM NaCl, pH 7.0) by
the freeze/thaw method. Samples were incubated on ice for 15 mm and then
centrifuged at 11,000 x g, 4 °C for 1 hour. Supernatant was then incubated with preequilibrated Talon resin for at least 4 hours at 4 °C with frequent gentle shaking. The
samples were centrifuged at 700 xg for 5 mm to spin down the resin and collect the
supernatant. The resin then was washed with wash buffer (see above) 3 times. The resin
containing Capn5 was transferred onto the colunm and eluted with Talon elution buffer
(50mM sodium acetate, 300mM NaC1, pH 5.0). The eluent was collected in 500 L
fractions. The eluents were neutralized immediately with Tris-Ci pH 9 to a final pH of 7
and EDTA to a final concentration of 5 mM. The eluent was also sampled for
Coomassie blue staining and Western blotting (as described earlier).
3.11 c-Myc tagged protein purification
Anti-c-Myc agarose conjugate was purchased from Sigma. The procedure for
immunoprecipitation of c-Myc tagged fusion proteins basically followed Sigma product
information and was performed in a 1.5 microcentrifuge tube. Briefly, 100 tL of the 1:1
suspension of the anti-c-myc agarose conjugate was pre-washed with PBS 5 times.
Sample (either SF-9 cell lysate or Talon fraction) was added the agarose and incubated
at 4 °C overnight with frequent shaking. The resin was washed 4 times with PBS and
then the tagged protein was eluted in low pH buffer (TED buffer with 150mM NaC1,
lmg!mL glycine, pH 4). The eluent then was neutralized immediately to pH 7 with Tris
(3M, pH 8.8).
41
3.12 Calpain-5 activity assays
Calpain-5 samples were incubated with calcium (0 to 50 mM) or inhibitors at 37
°C for desired time (in buffer containing 100 mM Tris and 25 mM 2-ME). For casein
hydrolysis, 50 tg of casein was co-incubated with calpain-5 and various concentrations
of calcium at 37 °C for 8 hours (buffer containing 100 mM Tris and 25 mM 2-ME). For
spectrin hydrolysis, 2 tg of casein was co-incubated with calpain-5 and various
concentrations of calcium at 37 °C for 8 hours (buffer containing 100 mM Tris and 25
mM 2-ME). Reactions were stopped by adding SDS-loading buffer (8 X, 0.05M Tris,
pH 6.8, 50% glycerol, 2% SDS, 0.01 % BPB). Results of calpain-5 breakdown were
then analyzed by Western blot.
42
4. RESULTS
Calpain-5 was first reported in 1997. In that early study, Mugita and colleagues
found that over-expressed human calpain-5 (from E. coli) exhibited no proteolytic
activity in a caseinolytic assay which was applied as one of the standard calpain activity
assays (Mugita et al., 1997). This result indicated that bacterially-expressed calpain-5
might be folded incorrectly or may lack important eukaryotic post-translational
modifications needed for expression of activity. Therefore, we used a baculovirusmediated expression system to express calpain-5 in an insect cell line. In addition to
improved post-translational processing, advantages for insect-expressed protein include
large-scale expression and proper cellular compartmentalization. Consequently the
over-expressed protein has greater potential to exhibit proper function and biological
activity.
We obtained the calpain 5 cDNA cloned into pSEC4O vector (Figure 5) from Dr.
H. Sorimachi, Tokyo, Japan. The cDNA contained a protein initiation codon, the entire
calpain-5 coding region, replacement of the protein termination codon with a Ser, a c-
Myc tag, and a His tag at the 3'end and a termination codon. Total length of the cDNA
was 1995 bp. The fully-translated modified protein was 75.8 kDa with apT of 7.8 (result
was calculated by ExPASy, Peptide Mass).
We cloned the cDNA into baculovirus expression vector pFastBacl (4.7kb)
between the EcoR
I site
and the Stu I site. This insertion was first checked by PCR and
restriction digestion to ensure the correct insertion site and size (Figure 7). Then the
vectors with insertions were sequenced to verify the protein reading frame and to
ensure mutations were not present (sequence results are not shown). This ensured that
the translated protein could retain its function and possibly, its enzyme activity. The
final vector (pFastBacl-Capn5) contained the correct protein translation region.
Following, the pFastBacl-Capn5 was transformed into bacmid-producing cells. The
bacmid with calpain-5 cDNA was then collected and expressed in insect SF-9 cells.
Calpain-5 protein expression was checked by Western blot using anti-cMyc antibody
(Figure 8). From the Western blot, calpain-5 was expressed in transfected SF-9 cells at
the correct size (75.8 kDa).
To assay calpain-5 biological activity, the development of techniques and
methods for protein purification was an essential prerequisite. We designed the calpain5 purification strategies based on predicted protein sequence information and
experience from working with calpains. The approaches we used included both
traditional chromatography and affinity purification. Both the purified samples were
tested for calcium sensitivity, digestion patterns, inhibitor interactions and substrates to
gain insight in to calpain-5 ' s function.
4.1
Calpain-5 purification by chromatography
To search for appropriate chromatographic conditions, we first screened four
different ion exchange ligands and three hydrophobic interaction ligands for their ability
to retain calpain-5. For this, we used a HiTrap JEX Selection kit which included QSepharose (strong anion), DEAE-Sepharose (weak anion), SP-Sepharose (strong cation)
and CM-Sepharose (weak cation), and a HiTrap HIC Selection kit which included
Phenyl-Sepharose (strong hydrophobic interaction), Octyl-Sepharose (medium
hydrophobic interaction) and Butyl-Sepharose (weak hydrophobic interaction). The
buffer and elution conditions were modified according to the expressed protein's p1
(calpain-5 with His and cMyc tag, p1 = 7.8). The results showed that calpain-5 binds
both anion and cation exchange media (Figure 9, top). Calpain-5 was retained on all ion
exchange columns. This indicates that both anion and cation exchange medium can be
used in calpain-5 purification. The results also showed that calpain-5 did not bind to any
of the hydrophobic exchange columns (Figure 9, bottom), because its hydrophobicity is
low. This indicated that calpain-5 may not be a membrane-associated protein.
To prepare large scale quantities of calpain-5, SF-9 sample was applied onto a
DEAE-Sepharose column. The cytosol was prepared containing EDTA to prevent
calpain activity and autolytic degradation. Calpain-5 has a predicted p1= 7.8. However,
we decided to utilize a pH 7.5 buffer (physiological pH) because we had no information
for calpain-5 activity and pH sensitivity. Calpain-5 was then eluted in a NaC1 gradient
buffer. Analysis of the DEAE-purified fractions was completed by Coomassie Brilliant
Blue (CBB) staining and by Western blot analysis using a anti-cMyc antibody (Figure
10). The calpain-5 protein was eluted at a NaC1 concentration of 0.2 to 0.5 M. However,
this purification did not yield calpain-5 as major protein in the mix. We combined
DEAE fractions with peak calpain-5 signal for three more purification columns (Qsepharose, Superdex 200, and Source Q). These resins are either ion exchanger (Qsepharose and Source-Q) or gel filtration (Superdex-200). In each step, protein
concentration, Western blot and CBB (Figure 11) were completed. Only the fractions
50
with strong calpain-5 signal were utilized in subsequent chromatography. The
purification procedure was finally conducted as purification fold (method described in
Kinbara etal., 1998). The results of the purification procedure are presented by
comparing the ratio of densitometric quantification of imniunoblots (Figure 12) divided
by sample protein concentration and are summarized in Table 2.
These sequences of four columns did not completely yield calpain-5 as pure
protein. The three chromatographic steps following DEAE-Sepharose yielded a 13.7fold purification. We estimated the first DEAE column caused a 10-fold purification,
the total purification was near 100-fold. We believe this purification was sufficient for
biological assay because a similar approach in purifying calpain-3 yielded a 12.2-fold
purification (Kinbara et
al.,
1998). We used the final Source-Q fraction for the
following calpain-5 activity assay.
Calpain-5 preparations were first tested for their ability to "self-digest". Calpain5 samples from the Source-Q fraction were incubated with/out various concentration of
calcium at 37 °C for 8 hours. Results of the calcium sensitivity assay were analyzed by
immunoblot. The sample solution from the Source-Q fraction contained 5 mM of
EDTA to prevent auto-digestion during purification. When calcium concentration was
greater than 5mM, calpain-5 degradation was initiated in a calcium-dependent manner
(Figure 13). At 50 mM calcium, full-length calpain-5 could not be detected.
Approximately 40 mM calcium was required for clearance of calpain-5 in the 8 hour
52
Table 2. Purification of recombinant human calpain-5 from SF-9 cells. The protein
concentration was measured by BCA protein assay kit (Pierce, Rockford, IL). The
quantity of calpain-5 protein was estimated by densitometric quantification of the
Western blot shown in Figure 12.
Purification
step
Total protein
DEAEsepharose
Q-sepharose
Ratio (B/A)
1287.33
Total
desitometric
reading (B)
2421.85
530
2063.58
3.89
2.07
Superdex-200
174
544.68
3.88
2.02
Source-Q
44
1039.21
25.89
13.76
(!iWml; A)
Purification
1.88
Note: Fold purification of the DEAE column could not be calculated because we did not
have information on Western blot desitometry for initial sample. Usual analysis of CBB
stained gels indicates an approximate 10-fold purification at this step. Hence,
purification via four columns is about 100-fold.
54
assay. The degradation was from C-terminal because the immune epitope of c-Myc
was in C-terminal of calpain-5.
To verify that the calcium-dependent degradation of calpain-5 was not mediated
by other calcium-dependent proteases, we completed Western blot analysis for other
calcium-dependent proteases (calpains-1 and -2). Calpain-1 was detected in the DEAE-
Sepharose fraction but not in the following chromatographic steps (Figure 14). Calpain2 was not detected in the Source-Q fraction but could be found in Superdex fraction
(data not shown). These data indicate that calpain-5 degradation is calcium-dependent
and is not caused by conventional calpains.
We tested the time-dependence of calpain-5 degradation. We incubated calpain5 from the Source-Q fraction with 50 mM calcium at 37 °C for various times. The
results are shown in Figure 15. Calpain-5 degradation began quickly (within 1 minute)
and was almost completed following 30 minutes of incubation.
The next question we addressed was whether the degradation could be prevented
by calpain-specific inhibitors. We incubated the calpain-5 with various calpain specific
inhibitors and with 50 mM calcium at 37 °C. Western blot (Figure 16) showed that only
PD 150606 fully prevented the degradation. Because PD150606 inhibits calpain activity
by blocking calcium binding, this indicates that calpain-5 degradation is truly calcium-
dependent. Calpain's endogenous inhibitor, calpastatin, however, showed no protection.
4.2
Calpain-5 2D Electrophoresis
Calpain-5 has a predicted p1 of 8.3. With His and cMyc tags, the calculated p1
was 7.8. We assayed calpain-5 p1 to determine if there any post-translational
modification which could alter the p1. We ran two 2D gels with the same sample and
molecular weight/pI standards: one gel for SYPRO Ruby staining and the other one for
immunoblotting with anti-cMyc antibody. The two images were completed in parallel to
estimate the p1 of calpain-5 (Figure 17; PDQuest-7.1O; Biorad). The experimental p1
was calculated as 8.3. This confirmed that calpain-5 is a basic protease, which is
different from most of acidic calpains. Comparison of calpains' p1 are listed in Table 3.
4.3
Calpain-5 purification by polyhistidine affinity column
As presented previously, we purified recombinant calpain-5 from SF-9 cell
lysate using traditional chromatography (DBAE to Source-Q columns). Calpain-5
exhibited calcium-dependent protease activity. However, this preparation did not yield
pure calpain-5 protein or yield calpain-5 as major protein. It is possible that a protein in
the mixture might interfere with calpain-5 activity. Because the recombinant calpain-5
was contained a polyhistidine-tag, we used BD TalonTM metal affinity resins to purify
calpain-5.
SF-9 cells were homogenized in pH 7 buffer without EDTA. After
precipitation, the crude cell lysate was added to a polyhistidine affinity Talon colunm
and His-tagged protein was eluted in low pH (pH=5) buffer. To avoid denaturing
Table 3. Isoelectric points of calpains. pls of calpains were calculated with the ExPASy
p11MW tool.
calapin
Predicted p1 from cDNA/(experimental pI)(ref)
Calpain-1
5.44/5.3 (Yoshimura et al., 1983)
Calpain-2
4.88/4.6 (Yoshimura et al., 1983)
Calpain-3
5.81 (Sorimachi etal., 1989)
Calpain-5
7.8 /8.3 (with c-terminal tag)
Calpain-6
6.62 (Dear etal., 1997)
Calpain-9
5.37 (Lee et al., 1998)
Calpain-lO
6-8.7 (8 variants; Rasmussen etal., 2002)
61
calpain-5, we re-natured protein in the elution buffer immediately to pH 7. All eluted
fractions, as well as flow through and colunm wash buffer, were collected for CBB
staining and immunob lotting. Results are showed in Figure 18. Calpain-5 was eluted in
the pH 5 buffer at its correct size. However, the eluent also contained a mixture of
proteins.
4.3.1 Ca/pain -5 activity assay
To determine whether the calpain-5 Talon eluent retained protease activity, we
assayed calpain-5 calcium-dependent auto-degradation (Figure 19 and 20), time
dependence (Figure 21), and calpain specific inhibitor inhibition (Figure 22). Most of
calpain-5 was degraded in the presence of 20 mM of calcium (Figure 19). When
calcium concentration reached 50 mM, no detectable calpain-5 remained. The relative
immunoblot densities of each treatment were plotted in Figure 20. The calcium
concentration required for half-maximal activity at 37 °C was calculated to be 9.6 mM.
When the incubation temperature decreased into 30 °C, calpain-5 autolysis activity also
decreased. The calcium requirement for half-maximal activity at 30 °C is 26.5 mM.
These calcium requirements are much higher than these established for the conventional
calpains (approximately 0.4 mM for calpain-2; Masumato
et al.,
1998). Calpain-5 also
differs from conventional calpains in temperature sensitivity, because calpains- 1 and 2
were reported to exhibit their highest activities at lower temperature (activity is best in
25 °C compared to 30°C and 37°C; Goll et
al.,
2003). When testing calpain-5
degradation over time (Figure 21), calpain-5 degraded quickly when calcium was
67
present. Half of calpain-5 was degraded following 30 minutes of incubation and the
signal was undetectable after 2 hours. The calculated time for calpain-5 to degrade to
half was 1.4 hours. When testing calpain specific inhibitors (Figure 22), only calcium
binding inhibitor, PD 150606, prevented calpain-5 degradation. Again, calciumdependent activity was detected in the absence of maj or calpains (calpain- 1 and calpain-
2). As shown in Figure 23, neither calpain-1 nor calpain-2 was detectable in Talon
eluent. These results are similar to those with calpain-5 obtained from Source-Q
chromatography. These results confirmed that calpain-5 degradation is calcium
dependent.
4.3.2 Caseinolysis
In our next experiment, we tested the ability of calpain-5 to degrade casein.
Casein is one of the best-known calpain substrates and has been applied as a standard
index of calpain activity in vitvo. We applied results from previous tests which showed
calpain-5 maximal activity at 37 °C. Under this condition, calpain-5 calcium-dependent
caseinolytic activity was examined. Results are shown in Figure 24. Calpain-5 was
incubated with casein for 8 hours. Calpain-5 caseinolysis was also calcium-dependent.
The calcium concentration required for half-maximal activity was calculated to be close
to 10 mM. This concentration is very close to result from calpain-5 autolysis (Figure
20).
70
43.3 Spectrin degradation
Spectrin is a major component of cytoskeletal proteins. It is a high molecular
weight heterodimer, composed of two subunits (molecular weights of approximately
230 kD and 250 kD; Bennett and Baines, 2001). Spectrin is also a well known
calpain substrate (Goll et al., 2003). Therefore, we tested whether spectrin is also a
calpain-5 substrate. Results shown in Figure 25. Spectrin was degraded when coincubated with calpain-5 when calcium was present. Calpain-2 also degraded spectrin.
Degradation was almost complete when calcium concentration reached 20 mM. The
result showed that spectrin maybe a calpain-5 substrate. This result indicated that
calpain-5, like other calpains, might have a role in cell remodeling.
4.4
Calpain-5 purification and activity by double affinity
columns
To further purify calpain-5, we applied the Talon colunm eluent onto a second
affinity resin: cMyc-agarose. This resin recognized the recombinant calpain-5 Cterminal cMyc-tag and, with low pH buffer (1 mg!mL glycine, 0.15 M NaCl / TED, pH
4), calpain-5 can be eluted from the resin. The eluent was renatured to pH
7
immediately to prevent protein denaturation. The results are shown in Figure 26. One of
the two visible bands in the silver-stained gel corresponded to the signals in the
immunoblot. However, the protein yield following the two affinity columns was
extremely low (too low for functional assays). In an attempt to identify the calpain-5
72
degradation fragment, we used the double affinity colunm eluent for a calcium
activity assay and stained the SDS/PAGE gel with silver staining (Figure 27). Again,
degradation of calpain-5 was calcium-dependent. However, degradation products could
not be identified.
75
5. DISCUSSION
Proteolysis is an important function in all living cells. It is responsible for
normal protein turnover, modifying proteins and also for eliminating proteins whose
functions are no longer required during cell events such as cell cycle regulation and cell
death. There is growing evidence that calpain-related proteolysis is essential in
controlling gene expression, cell cycle, cell death and cell remodeling during
development or in response to environmental changes. Calpains cleave polypeptides at a
limited number of sites (Barnes and Gomes, 1995; Stabach et aL, 1997) and leave large
degraded fragments for "digestive enzymes" such as lysosomal proteases and the
proteasome (Creighton, 1993; Kisselev
et
al., 1998). This suggests that calpains have
regulatory functions or signaling functions in cells. Calpains' selective activities are
found universally in eukaryotes (Sorimachi et al., 1997; Mykles, 1998). In humans,
malfunction of calpains always relates to developmental problems or to degenerative
pathologies (Huang and Wang, 2001). Therefore calpain offers high potential for
therapeutic interventions.
Calpain-5 is a newly-described calpain in the calpain superfamily (Dear et al.,
1997; Mugita et al., 1997). It was cataloged as an atypical calpain which lacks a
calcium-binding domain (replaced with Domain T) and thus has no interaction with the
calpain small subunit (like typical calpains; calpains- 1 and 2). Calpain-5 contains a
protease domain (Domain 11) sequence which is similar to most calpains. Based on this,
it is predicted that calpain-5 retains protease function. mRNAs encoding mammalian
calpain-5 has been detected in many tissues (Dear
et
al., 1997) with highest levels found
76
in testis. Although the C. elegans calpain-5 homolog is involved in sex
determination, it is unclear whether calpain-5 plays any role in mammalian sex
determination, because calpain-5 mRNAs are also expressed in brain, colon, kidney,
liver, and trachea. So far, no calpain-5 protein has been reported in any tissue. Calpain-5
protease properties and physiological functions remain unknown.
It has been reported that calpain-5's C. elegans counterpart, TRA-3, is a
calcium-dependent protease (Sokol and Kuwabara, et al., 2000). TRA-3 was first
identified as a suppressor in the C. elegans sex deternimation cascade (Hodgkin, 1985)
and later was found to play a role in neurodegeneration in C. elegans (Syntichaki et al.,
2002). It is now clear that TRA-3 promotes female development in XX hermaphrodites
by proteolysis of its substrate, TRA-2A (Sokol and Kuwabara, et al., 2000). Like most
of calpains, TRA-3 is capable of autolysis when calcium is present. A unique aspect of
TRA-3 autolysis is that the cleavage site occurs in Domain III instead of at the Nterminal as in most of calpains.
With this background information, we hypothesized that calpain-5 is a
functional protease like its C. elegans homolog TRA-3. We developed a strategy to
investigate calpain-5 proteolysis to obtain information on how this protein exerts its
function. In this study, we purified and characterized recombinant human calpain-5
from baculovirus-infected insect cells. Two purification methods were used in purifying
calpain-5 and both methods yielded proteolytically active calpain-5. To examine
calpain-5 proteolytic activity, we measured activity in a time-dependent, calciumdependent maimer and with various inhibitors. The results are important because our
studies provide the first evidence that calpain-5 is an active protease. Like most
77
calpains, calpain-5 is a calcium-sensitive enzyme and which possesses an autoproteolysis (autolysis) feature when calcium is present. These fundamental properties
raise the possibility that calpain-5 is involved in cell function regulation through
specific proteolysis like its homologs, ex, TRA-3. Our studies on calpain-5 proteolytic
assay also provide insights into these vertebrate homologs, and will aid in the
understating of other atypical calpains in this sub-catalog.
5.1
Calpain-5 proteolytic activity
5.1.1 Calcium-dependency of calpain-5 autolysis
In this studies, we found that calpain-5 possesses calcium-regulated autolytic
activity (Figures 13 and 19) as has been reported for other calpains (Tompa
et al.,
1996). Calpain-5 from two preparations both showed calcium requirement for autolysis
in a dose-dependent manner. This proteolysis is clearly calpain-5 activity because
calpain- 1 and calpain-2 were not detectable in fractions used to assay activity and
because calcium required for calpain- 1 or -2 activity are much lower than that detected
in this study. The observation that calpain-5 is regulated by calcium is supported by
studies on TRA-3, which also possessed calcium-dependent activity (Sokol and
Kuwabara, 2000). While it is clear that calpain-5 digestion is activated by calcium, it is
not clear how calpain-5 binds calcium. Calpain-5 does not contain a calmodulin-like
calcium-binding domain IV. Instead, it contains two putative calcium-binding sites in
Domain 1111 and one predicted EF-structure in Domain III (Mugita et
al.,
1997). Studies
78
on the calpain- 1 protease core (containing only Domain 1111) showed that there are
two non-EF-hand sites which mediate cooperative calcium binding: one in Domain I
and the other one in Domain II (Moldoveanu et al., 2002). Calcium ions bind to these
two non-EF-hand sites weakly (compared to wild-type calpain-1) but the binding is
necessary for the truncated protein to exert its proteolytic function. Evidence from a
calpain-2 study supported the finding that Domain II protease activity is calciumdependent even when the calcium-binding domain is absent (Hata et al., 2001).
Residues that coordinate calcium binding in the Domain 1111 are highly-conserved in the
calpain family, including calpain-5. Hence, it is possible that the calcium cooperative
binding sites are functional in calpain-5. Besides these non-EF-hand sites in Domain
Jill, calpain-5 has a predicted EF-hand structure in Domain III (Mugita et al., 1997).
Previous studies with Schistosoma calpain showed that the conserved EF-structure in
Domain III binds calcium ions (Andresen et al., 1991). This evidence indicates that
calcium may bind to the calpain-5 protease core as well as to Domain III. However,
more information on the calpain-5 crystal structure is needed to elucidate the exact
calcium-binding site of calpain-5.
Unlike typical calpains, calpain-5 requires higher concentrations of calcium for
autolysis (i.e., calpains- 1 and -2 require less than mM concentrations of calcium for in
vitro half-maximal activity, while calpain-5 requires up to 30 mM of calcium; Figure 13
and 19). The calculated calcium concentration requirements for half-maximal activity
are 26.5 mM at 30 °C and 9.6 mM at 37 °C. The higher calcium concentration
requirements are confirmed in calpain-5 caseinolysis (Figure 24). Considering the
chelator (5 mM of EDTA) present in the buffer system, these calcium requirements are
79
still much higher than those needed by most calpains (< 1 mM) and are much higher
than physiological calcium concentrations (< 1 pM; Guyton, 1991). Data from calpain- 1
and calpain-2 protease core constructs showed that the calcium requirement was higher
when Domain IV was not present. For example, Domain II of calpain-2 required 0.9
mM of calcium for half-maximal activity instead of 0.4 mM for the full-length protein
(Hata
et al.,
2001). It is suspected that interaction of protease core domains with other
domains reduces the calcium requirement. Typical calpains activities are regulated also
by calcium-binding conformation changes, autolysis, and cellular relocation (Goll et
2003; Jia
et al.,
al.,
2002). Studies have proven that interaction within domains, autolysis
after calcium binding and binding with phospholipids at the plasma membrane also
reduce the calcium requirement for calpain autolysis (Edmund et
2002; Saido
et al.,
al.,
1991; Jia et
al.,
1991). It is unknown whether calcium binding to calpain-5 causes
calpain-5 conformational changes or cellular relocation. How calpain-5 activity is
regulated in vivo still remains mystery. A hypothesis was given that some calpains use
an alternative activation mechanism, different from traditional calpains (Ma
et al.,
2001), for instance, by interaction with a distinct cofactor/activator. It is possible that
Domain T is responsible for the interaction (Dear
et al.,
1997).
The weak calcium-dependent proteolytic activity was also detected during
prolonged assays (Figures 15 and 20). Because 50 mM calcium was required for the
maximum calpain-5 activity, we employed this condition to test the time course for
calpain-5 activity. Calpain-5 auto-degradation was not completed within two hours of
incubation, while calpain-1 autolysis was almost completed within 30 mm of incubation
when excess calcium was present at 30 °C (Cong et
al.,
1989). It is unusual that calpain-
5 remains stable and active for such a long period because intact calpains- I and -2 are
inactivated rapidly when temperature is above 30 °C (Dayton et
al.,
1976). Our results
for calpain-5 are similar to those with calpains- 1 and 2 protease core constructs (the
calpain- 1 protease core construct required more than 20 hours to complete its
degradation; Moldoveanu et
al.,
2002). This suggests that interaction within calpain
domains is very important for calpain autolysis. Because the most difference between
calpain-5 and calpains is Domain T. This indicated that Domain T might have different
function.
5.1.2 Inhibitors test
To gain insight into the nature of calpain-5 'S proteolytic activity, we examined
ability of a variety of inhibitors to inhibit it. In this study, we found that only a calciumbinding inhibitor, PD 150606, inhibited calpain-5 activity (Figure 16 and 21). Calpain
endogenous inhibitor, calpastatin, and calpain active site inhibitor, E-64, did not inhibit
calpain-5 activity. These results may have been predicable. First, effective inhibition of
calpain by calpastatin requires that calpastatin binds to calpain at three sites when
calcium is present: two at calcium-binding domains in both large and small subunit and
one at protease Domain II (Takano and Maki, 1999). If protease Domain II is expressed
alone, calpastatin binds calpain poorly and does not inhibit protease activity
(Moldoveanu et
al.,
2002). Because calpain-5 lacks the calmodulin-like Domain N, it
should not interact with the small subunit. Hence calpain-5 carmot interact with
calpastatin to form an effective enzyme:inhibitor complex. As a result, it is not
F;"
surprising that calpastatin did not inhibit calpain-5 activity. These results confirm the
predicted mechanism by which calpastatin inhibits the house-keeping calpains.
B-64 is an active site-directed cysteine protease inhibitor (Barrett et at., 1982). It
does not specifically inhibit calpains but is used widely in calpain purification and
activity assays. It is surprising that E64 failed to prevent calpain-5 activity. A possible
explanation for the inability of E64 to inhibit calpain-5 was given by Thompson
et
at.
(1990). They proved that conformation of the cysteine protease active site, in the
absence of calcium (i.e. during purification), retarded the action of E64. It is possible
that this observation explains the inability of E64 to block calpain-5 activity.
The only calpain inhibitor which blocked calpain-5 activity was PD 150606.
PD 150606 inhibits calpain by binding to calpain's calcium-binding sites. The binding
disrupts the conformational change of calpain that is necessary to activate the protease
domain (Wang et at., 1996). Calpain-5 does not contain a calcium-binding Domain IV.
Hence, the inhibition by PD 150606 indicated that there is a calcium-binding site in
calpain-5, either the predicted EF-hand structure in Domain III or putative calciumbinding site in Domain Till. This result also confirmed that calpain-5 autolysis is
calcium-dependent.
5.1.3 Domain T and its suspected function
It is well accepted that autolysis has an important role in calpain function.
Calpains- 1, -2 and -4 undergo autolysis to "activate" the enzyme (McClefland et
al.,
1989). Calpain-3 was also documented to undergo autolysis but this was regarded as a
degradative step rather than a step required for its activation (Kimbara
et al.,
1998).
In our study, calpain-5 exhibited autolytic activity when calcium was present. The
degradation is from the C-terminal because the antibodies we used are directed against
the C-terminal of recombinant calpain-5 (Figure 15). No smaller fragments could be
detected in the Western blots. Therefore, the cleavage sites are unknown. The
degradation was confirmed in two sets of affinity purification samples (Figure 27)
where calpain-5 degradation was clearly shown in silver staining. These results agree
with results of Mugita et
al.,
1997, in which the C-terminal of calpain-5 was removed
and generated a 55kD product. In TRA-3, the predicated cleavage site for autolysis is
located in Domain III. In other words, autolysis may remove Domain T. However, it is
still not clear whether autolysis cause enzyme activation or for degradation.
Domain T is a very interesting domain. Domain T is the only domain which
shows no homology to other calpains. Domain T is rich in basic amino acids. In human
calpain-5, Domain T has a high p1(9.44) while pls for Domains I, II, and III are 6.49,
6.8, and 6.04 respectively (pls were calculated with the ExPASy p11MW tool). This is
the probable reason for calpain-5 '5 ability to interact with both anion and cation
exchangers (Figure 9). The high basic amino acid residue content of Domain T
underlies one of the major differences of calpain-5 from other acidic calpains. However,
the function of Domain T is still not clear. In TRA-3, Domain T is not critical for TRA3 function in sex determination (Barnes and Hodgkin, 1996). It was speculated that
Domain T interacts with other proteins in regulating its function. The putative
regulatory protein may not be calpain-4, the small subunit, because it lacks the calciumbinding domain for small subunit binding. In other words, atypical calpains, such as
calpain-5, are regulated through a different mechanism from conventional calpains.
An example maybe obtained from calpain- 10, another atypical calpain which possesses
a Domain T. When intracellular calcium levels increase, typical calpains, which are
cytoplamic proteins, relocate to membranes. Conversely, calpain-lO was found to
increase its expression in the nucleus. Calpain-5 will probably not bind to membranes
either, because it was shown to have very limited hydrophobic ability (Figure 9). This
evidence indicates that calpain-5 is regulated differently from classic calpains. Domain
T may account for this difference. As state previously, Domain T may not function in
accelerating proteolytic activity. Hence, it is highly possible that Domain T is involved
in functional regulation.
5.2
Calpain-5 substrates and physiological function
Assay of calpain proteolytic activity typically employs a protein substrate such
as casein. Wild type calpain-1 degrades casein rapidly in 30 minutes (Hitomi et
al.,
1998). However, in our study, calpain-5 did not digest casein even after 8 hours of
incubation at 37 °C (Figure 24). This result raises two possibilities. First, lack of
caseinolytic ability might be due to the lack of a calcium-binding domain. The low
specific activity of caseinolysis was also observed when Domain II of calpain-2 was
expressed alone (Hata et
al.,
2001). The authors concluded that other domains might
function in assisting proteolytic activity. Second, casein may be a poor calpain-5
substrate. Because substrates for calpain-5 have not been identified, it is not clear if
clapain-5 has preferred substrate following PEST rule.
Cleavage of cyto skeletal/membrane attachments is likely the best documented
function of the calpains. Calpains modulate the function of cytoskeletal proteins such as
spectrin, talin, integrin and so on (Schoenwaelder et al., 2000), in response to different
cellular events, for example, cell remodeling or apoptosis. The TRA-3 substrate, TRA2A, is also a membrane-associated protein (Sokol and Kuwabara, 2000). This leads to
the suspicion that calpain-5 degrades membrane-associated proteins. As a result, we
tested whether spectrin is calpain-5 substrate. As shown in Figure 25, calpain-5 digested
spectrin in a calcium-dependent manner in vitro. No spectrin degradation was observed
when calcium concentration was low (lower than 5 mM). This is consistent with
previous results that calpain-5 required higher concentrations of calcium for its
activation. When calcium concentration reached 20 mM, the degradation of spectrin
was almost complete. These results demonstrate that spectrin is a calpain-5 substrate (at
least in vitro). Hence, calpain-5 might have a role in cell remodeling.
The two lines of evidence of calpain-5 substrates presented above do not give
too much insight into calpain-S's physiological function. In search of calpain-5
physiological function, early studies have shown that calpain-5 mRNA was highly
expressed in human testis, and other tissues including brain, colon, and intestine
(Mugita et al., 1997). This information together with its homology with sexdetermination gene tra-3, calpain-5 was thought to participate in sex-related functions.
However, later studies considered a new thought. First, calpain-5 mRNA was found in
all the fetal tissues and was highly expressed in mouse embryonic brain, suggesting that
calpain-5 has a role in embryogenesis (Dear et al., 1997; Dear and Boehm, 1999).
Second, studies with TRA-3, the calpain-5 counterpart in C. elegans, showed that TRA-
3 is expressed in the C.
elegans
nervous system and participated in
neurodegeneration (Syntichaki et
al.,
2002). Our studies also show that calpain-5 has
general proteolytic ability and might also be involved in cell remodeling. These
observations indicate calpain-5 could be a multi-functional protease like calpains- 1 and
2. This needs to be elucidated when more substrate information becomes available.
5.3
Limitations
We would like to address the limitations of the project.
1) The vector was designed with both polyhistidine and cMyc tags in the C-terminal.
As a result, the protein was easy to isolate from cell lysates and was proved to
demonstrate proteolytic activity. However, this design did not allow us to identify
the cleavage site of autolysis.
2) We used a pH 7.5 buffer in chromatography. This is not the optimal condition for
calpain-5 and anion resin to bind. Calpain-5 has an p1 of 8.3. The calculated p1 for
calpain-5 with its C-terminal tag is 7.8. Theoretically, the best operating (buffer) pH
for anion exchangers is greater than the p1. In other words, the buffer pH should be
greater than 7.8 (Ausubel et
al.,
1987). However, we used pH 7.5 buffer to mimic
physiological conditions. The buffer conditions we used were not best for separating
calpain-5 and SF-9 proteins. Therefore, the yield was low.
3) We used 1.5 x 28 cm Superdex-200 as gel filtration resin in separating proteins by
size. This resin was designed to separate 1 x 1
to 6 x 1 0 Da proteins. After
completing Superdex chromatography, the fraction we collected still contained
mixture of many proteins (Figure 11). A longer filtration column might be needed
to obtain better separation.
4) The recombinant calpain-5 we used encoded a His-tag as well as a cMyc tag. The
first affinity purification we applied was a nickel-chelating resin and was washed
with imidazole. This did not yield detectable calpain-5 (data not shown) and
indicated calpain-5 is denatured in high concentrations of imidazole. Following this
we tried a Talon colunm which utilizes a cobalt-chelating resin and used a very mild
wash (pH 7.0 buffer) and elution (pH 5.0 buffer but immediately renatured to pH
7.0). This condition elutes a mixture of proteins and did not yield calpain-5 as a
major protein. We did not apply stringent wash (eg, washing in low pH buffer) to
avoid denaturation of the protein.
5) The cMyc and His tags could have altered calpain-5 calcium-sensitivity/kinetics.
5.4
Future work
With the work presented here, we have shown that calpain-5 is an active
protease which is regulated by calcium. Calpain-5 shows diverse proteolytic activity
from typical calpains in its calcium requirements, temperature sensitivity, digestion time
and selective inhibitors. Our work supports the hypothesis that atypical calpains,
including calpain-5, are regulated differently from house-keeping calpains. However,
many questions need to be answered. For example, what is calpain-5's physiological
function? Is there a co-factor which regulates or assists calpain-5 activity? And, what is
the relationship of calpain-5 to its "endogenous dominant negative", calpain-6?
To address these questions, first, it will be important to identify the in vivo
expression pattern of calpain-5 protein. Calpain-5 mRNA was found in all embryonic
cells line and was rich in mice embryonic brain (Dear et
al.,
1997; Dear and Boehm,
1999). However, the highest signal of calpain-5 mRNA in adult tissue was in testis.
Whether calpain-5 participates in the testis development at a critical time point or is
consistently expressed as a routine protease is unknown. Calpain-5 specific antibody is
now commercially available. Hence, it is now possible to examine calpain-5 expression
at different stages of development or different tissues. This information will facilitate
understanding of function. Identification of calpain-5 'S in vivo substrates will also assist
in elucidating its physiological function.
We hypothesize that Domain T is responsible for calpain-5 activity regulation.
Domain T is a unique domain. Domain T exists only in TRA-3, and in calpains-5 and 6. Highly homologous sequences are found in calpain-lO, and some nuclear proteins
like DNA-directed RNA polymerase subunit B, and steroid-related protein like steroid
1 1-beta-hydroxylase. Domain T's function is still unknown. To test if Domain T is
involved in regulating calpain-5 activity by interaction with its substrate, we propose a
scheme of express truncated calpain-5 (lack Domain T) to determine whether calpain-5
retains its protease activity and digest its substrate.
Another interesting question is the relationship of calpain-5 to calpain-6.
Calpain-5 and calpian-6 share sequence homology but calpain-6 lacks an active
protease Cys residue (Dear et
al.,
1997). Based on sequence information, calpain-6
appears as an endogenous dominant negative of calpain-5. In other words, calpain-6
was suspected to participate in regulatory processes by competing substrates or
cofactors. Calpain-5 was found high in testis while calpain-6 was found only in
placenta (Dear et al., 1997). The different location of two proteins indicates that these
two proteins have related role in sex determinationldevelopment. To test the possible
inhibition of calpain-6 on calpain-5, we propose co-incubation the calpain-5 and 6 in
the proteolytic assay to answer this question.
In summary, this study shows that calpain-5 is an active calcium-dependent
protease. It is unique because it needs higher concentrations of calcium for activation
and presents less temperature sensitivity than housekeeping calpains. Calpain-5 is not
regulated by calpain-4 or calpastatin. It was also found to have a potential role in cell
remodeling. Comparison of calpain-5 and house-keeping calpains resulting from this
and other studies is listed in Table 4. More information about calpain-5 substrates, in
vivo expression pattern, and on binding proteins/cofactors is needed to understand its
physiological function.
Table 4. Differences between house-keeping calpains and calpain-5.
House-keeping calpains
Calpain-5
Domain
Calcium-binding domain
Domain T
PT
4.5 5.5
8.3
MW
8OKD
75 KD
Activity
Fast
Slow
Calcium requirements
Less than 1 mM
10-30 mM
Autolysis
N-terminal
C-terminal
Preferred temperature
25°C
37°C
Inhibition by calpastatin
Yes
No
Binding to calpain-4
Yes
No
Hydrophobicity
Yes
No
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