From the Cancer Research Center, The Burnham Institute, La Jolla, California 92037
Received for publication, December 30, 2002
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ABSTRACT |
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An understanding of the regulatory mechanisms
that control the activity of membrane type-1 matrix metalloproteinase
(MT1-MMP), a key proteinase in tumor cell invasion, is essential for
the design of potent and safe anti-cancer therapies. A unique
proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The
abundance of proteolytic enzymes in cancer cells makes it difficult to
identify the autocatalytic events in this pathway. To identify these
events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris.
Following secretion, the latent zymogen and active enzyme were each
purified from media by fast protein liquid chromatography. Trace
amounts of active MT1-MMP induced activation of the zymogen and its
self-proteolysis. This autocatalytic processing generated six main
forms of MT1-MMP, each of which was subjected to the N-terminal
microsequencing to identify the cleavage sites. Our data indicate that
MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR Recent evidence indicates that membrane type-1 matrix
metalloproteinase (MT1-MMP1
or MMP-14) is a key enzyme in tumor cell migration and invasion (1-3).
The expression of MT1-MMP was documented in many tumor cell types and
strongly implicated in malignant progression (4-6). Membrane-tethered
MT1-MMP is distinguished from soluble MMPs by a relatively short
transmembrane domain and a cytoplasmic tail, which associate the
protease with discrete regions of the plasma membrane and the
intracellular compartment, respectively (7). This protease functions in
cancer cells as the main mediator of proteolytic events on the cell
surface, including initiation of pro-MMP-2 and pro-MMP-13 activation
cascade (8), cleavage of cell surface receptors (9-11), and focused
pericellular proteolysis of extracellular matrix components (12).
Although several publications (13-16) discuss the existence of the
alternative activation pathways, the cleavage of the
108RRKR To elucidate the mechanisms that control the activity and structure of
distinct species of MT1-MMP at the cell surface, we expressed the
soluble, C-terminally truncated pro-MT1-MMP in Pichia pastoris yeasts, isolated the properly folded, secreted zymogen, stimulated its activation and autoproteolysis, and identified the
peptide sequence of the autolytic cleavage fragments. By using this
knowledge we reconstructed the multistep pathway by which multiple
molecular forms of MT1-MMP are likely to be generated in tumor cells.
The findings presented in this report support and extend the
observations by other groups (15, 16, 19, 20, 22). Furthermore, our
data provide evidence that MT1-MMP may function as a proprotein
self-convertase capable of cleaving the prodomain at the
108RRKR Reagents--
All reagents were from Sigma unless otherwise
indicated. A hydroxamate inhibitor GM6001 and rabbit polyclonal
antibodies AB815 against the hinge region of MT1-MMP were from Chemicon
(Temecula, CA). Expression of MT1-MMP- Purification of MT1-MMP- To isolate sufficient quantities of MT1-MMP, we synthesized in
methylotrophic yeast P. pastoris the recombinant soluble
MT1-MMP (MT1-MMP-Y112, thus
autocatalytically generating the mature MT1-MMP enzyme with an N
terminus starting at Tyr112. The mature enzyme undergoes
further autocatalysis to the two distinct intermediates (N terminus at
Trp119 and at Asn130) and, next, to the three
inactive ectodomain forms (N terminus at Thr222, at
Gly284, and at Thr299). These findings provide,
for the first time, a structural basis for understanding the
unconventional mechanisms of MT1-MMP activation and regulation.
Finally, our data strongly imply that MT1-MMP is a likely substitute
for the general proprotein convertase activity of furin-like
proteinases, especially in furin-deficient cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Y112 prodomain sequence of MT1-MMP by
furin, a Golgi-associated subtilisin-like serine proteinase, is still
considered as a singular functionally relevant mechanism involved in
the activation of newly synthesized MT1-MMP during its section pathway
from the Golgi compartment to the cell surface (17). The furin cleavage
was thought to generate active MT1-MMP commencing from the
Tyr112 (18-21). The activity of MT1-MMP is controlled by a
unique regulatory cleavage pathway (20, 22, 23), inhibition of the
tissue inhibitor of matrix metalloproteinases (4), and the trafficking and internalization mechanisms governing the presentation of MT1-MMP at
cell surfaces (14, 24, 25). In the critically important yet
inadequately understood cleavage pathway, the active MT1-MMP enzyme
undergoes a series of proteolytic events that regulate the nature and
functional activity of the enzyme forms at the cell surface and the
pericellular space.
Y112 site and generating the
mature enzyme commencing from the N-terminal Tyr112 through
self-proteolysis, rather than via the cleavage by furin.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1-Antitrypsin was obtained from
Calbiochem. The recombinant version of the catalytic domain of MT1-MMP
(MT1-MMP-CAT) was expressed in Escherichia coli, purified
from inclusion bodies, and refolded as described previously (26).
TM/CT--
The cDNA fragment
coding for peptide Ala21-Ser538 of the
full-length MT1-MMP (the GenBankTM accession number U41078)
was merged with the His6 tag and placed in the pPIC9
plasmid (Invitrogen) under control of the alcohol oxidase promoter and
-mating factor pre-propeptide. The resulting construct encoding the
soluble, secretory MT1-MMP without both the transmembrane domain and
the cytoplasmic tail (MT1-MMP-
TM/CT) was used to transform P. pastoris GS115 spheroplasts using Pichia expression kit
(Invitrogen). The clones were grown and selected according to the
manufacturer's instructions (Invitrogen). The expression of MT1-MMP
was examined in conditioned media samples by SDS-PAGE and Western
blotting using AB815 antibody. The most efficient clone, which produced
about 5 mg of total MT1-MMP per 1 liter of conditioned medium, was used
for further analysis.
TM/CT--
To produce
MT1-MMP-
TM/CT, P. pastoris transformant cells were grown
in 1 liter of BMGY medium containing 1% glycerol (Invitrogen) for 2 days at 30 °C. Next, the cells were collected and resuspended in 200 ml of BMMY medium containing 0.5% methanol. After 24 h, the cells
were removed by centrifugation. The medium was used to purify
MT1-MMP-
TM/CT by ammonium sulfate precipitation (80% saturation)
followed by FPLC of the precipitated material aliquots on a MonoQ HR
5/5 column (Amersham Biosciences) equilibrated with 20 mM
Tris, pH 8.0. After extensive washing to remove the impurities, MT1-MMP-
TM/CT was eluted with a 0-0.5 M NaCl gradient.
The fractions were analyzed by SDS-PAGE for the presence of the
MT1-MMP-
TM/CT species. The fractions containing the proenzyme and
the enzyme were pooled and used for further analysis.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
TM/CT) with both the transmembrane and cytoplasmic
domains deleted (Fig. 1A). The
expression of the
-factor-His6-MT1-MMP construct in the
pPIC9 plasmid was controlled by the alcohol oxidase promoter. The
presence of the
-factor sequence stimulated secretion of
MT1-MMP-
TM/CT from cells into the extracellular medium. Following the induction of the alcohol oxidase promoter, MT1-MMP-
TM/CT was
efficiently expressed by yeast cells and secreted in medium. In the
first 24 h following the gene induction, MT1-MMP was predominantly represented by the proenzyme and mature enzyme forms (Fig.
1B, inset, left lane). Further
cultivation of cells negatively affected MT1-MMP and predominantly
yielded its degraded forms (data not shown). Consequently, FPLC on a
MonoQ column was employed to isolate the purified proenzyme and enzyme
forms of MT1-MMP-
TM/CT from a 1-day medium (Fig. 1B,
inset, the middle and right
lanes, respectively).
View larger version (37K):
[in a new window]
Fig. 1.
Expression and isolation of functionally
active MT1-MMP from P. pastoris. A, the
domain structure of MT1-MMP and the peptide sequence of the cleavage
sites. Scissile bonds in MT1-MMP- TM/CT construct expressed in
P. pastoris (upper panel) and the wild type
MT1-MMP (lower panel; adopted from Refs. 19, 20, and 22) are
shown by arrows. The N-terminal sequences of the domains and
the cleavage fragments of MT1-MMP are shown below and
above the panel, respectively. The relative position of the
HE240LGHALGLEH zinc-binding site is
shown within the catalytic domain. PEX, hemopexin domain;
TM, transmembrane domain; CT, cytoplasmic tail.
B, purification and separation of the proenzyme and the
enzyme of MT1-MMP-
TM/CT by FPLC. Ammonium sulfate (80% saturation)
precipitated fraction was dissolved and dialyzed against 20 mM Tris, pH 8.0. This fraction was loaded onto a MonoQ HR
5/5 column and eluted with a 0-0.5 M NaCl gradient.
Elution of proteins was monitored by absorbance measurement
(A280). The fractions containing the
MT1-MMP-
TM/CT proenzyme and the enzyme are shown by hatching.
Inset, 10% SDS-PAGE of the aliquots of the ammonium sulfate
fraction, the purified MT1-MMP-
TM/CT proenzyme, and the enzyme
(left, middle, and right
lanes, respectively). C, the proteolysis of
1-antitrypsin by MT1-MMP.
1-Antitrypsin
(0.5 µg) was incubated with or without GM6001 (1 µM)
for 3 h with MT1-MMP-CAT (15 ng), the proenzyme, and the enzyme of
MT1-MMP-
TM/CT (50 ng each) in 15 µl of 50 mM HEPES, pH
6.8, containing 10 mM CaCl2, 50 µM ZnCl2, and 0.005% Brij 35. The samples
were separated by 10% SDS-PAGE and stained with Coomassie.
The enzyme of MT1-MMP-TM/CT demonstrated high proteolytic activity
in the cleavage of the peptide fluorescence substrates, gelatin
zymography, and in initiating the activation of the MMP-2 zymogen.
These activities of MT1-MMP-
TM/CT were comparable with those of
MT1-MMP-CAT derived from E. coli. Thus, specific
activity against fluorescence peptide substrate
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 where Mca is
7-methoxycoumarin, and Dpa is 3-(2,4-dinitrophenyl)diaminopropionic acid (Bachem, Torrance, CA) of MT1-MMP-
TM/CT and MT1-MMP-CAT was 20 and 60 arbitrary units/nm, respectively. Similarly, the catalytic quantities of the MT1-MMP-
TM/CT enzyme were capable of
efficiently cleaving
1-antitrypsin, a protein
substrate susceptible to MMPs, including MMP-2 and MT1-MMP (11),
thereby confirming the proteolytic potency of the purified construct.
The purified proenzyme of MT1-MMP-
TM/CT was inert in cleaving
1-antitrypsin. A wide range hydroxamate inhibitor of MMP
activity, GM6001 (1 µM), fully inhibited the cleavage
reaction (Fig. 1C, right panel).
The purified MT1-MMP-TM/CT proenzyme samples were relatively
unstable. When incubated at 45-50 µg/ml for 1-4 h at 37 °C, pro-MT1-MMP-
TM/CT readily generated the several distinct molecular forms of the enzyme. The conversion of the proenzyme to the enzyme was
fully blocked by co-incubation of the samples with GM6001 (1 µM) (Fig. 2A).
Serine proteinase inhibitors failed to affect MT1-MMP-
TM/CT (data
not shown).
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To investigate further the autolytic pathway that generates multiple
molecular forms of MT1-MMP frequently observed in cancer cells, we
concentrated the purified sample of the individual MT1-MMP-TM/CT proenzyme and incubated the concentrated material at 0.45-0.5 mg/ml
for 1 h at 37 °C. Extensive self-proteolysis of
MT1-MMP-
TM/CT generated six prominent proteolytic fragments that
were separated by SDS-PAGE and subjected to N-terminal microsequencing
(Fig. 2B). The results are summarized in Fig. 1A.
Thus, microsequencing confirmed the expected N-terminal sequence of the
MT1-MMP-
TM/CT construct (RFPSI which represent the N-terminal
sequence of the yeast
-factor). Self-activation of MT1-MMP then
generated the mature enzyme commencing from Tyr112. It is
likely that this form was further processed to the intermediate with
the N terminus at Trp119. The next autolytic cleavage
produced the form with the N terminus at Asn130. MT1-MMP
with the N terminus at Trp119 also missed the C-terminal
portion of the molecule, thereby generating the species with the lower
than expected molecular weight. Further cleavages generate the
functionally inert forms of MT1-MMP lacking the catalytic domain. These
three forms commencing from Thr222, Gly284, and
Thr299 correspond to the inactive ectodomain
membrane-tethered forms of MT1-MMP frequently found in cancer cells.
Thus, the similar forms of MT1-MMP were observed in MCF7 breast
carcinoma cells transfected with the full-length MT1-MMP gene and,
therefore, overexpressing MT1-MMP at the cell surface. The co-incubation of carcinoma cells with GM6001 (50 µM) for
48 h blocked the self-proteolysis of MT1-MMP and promoted
accumulation of the proenzyme and the enzyme in the cells (Fig.
3). Our findings suggest that the
proenzyme of MT1-MMP-TM/CT is susceptible to autocatalytic
activation. Similarly, pulse-chase experiments in furin-deficient LoVo
colon carcinoma cells (27) overexpressing MT1-MMP clearly demonstrated
that MT1-MMP was activated in this cell type and confirmed the presence
of two molecular species of the MT1-MMP enzyme on cell
surfaces.2
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Our data confirm and extend the observations by several groups (15, 16,
19, 20, 22, 28) who examined proteolysis and shedding of MT1-MMP in
tumor cells (Fig. 1A). In these studies, insufficient
amounts of MT1-MMP in tumor cells greatly complicated an extensive and
unambiguous structural analysis. Our data, however, indicate that the
MT1-MMP in its autolytic pathway cleaves the catalytic domain at
QQLYGG284 rather than at QQLYG
G284
as earlier reported by Fridman and co-workers (22). Fridman and
co-workers (22) have also directly suggested that the cleavage at
the SDPSA
I256 site requires the attachment of MT1-MMP to
the plasma membrane. In agreement, the cleavage at the
SDPSA
I256 site was not identified in our samples of
soluble MT1-MMP.
The observations reported here suggest that MT1-MMP is a proprotein
self-convertase capable of autocatalytically cleaving its prodomain at
the furin cleavage site RRKRY112 and generating the
mature enzyme with N terminus at Tyr112. These data
reinforce our earlier observations (28, 29) that there are alternative
pathways of MT1-MMP activation and maturation, such as
furin-independent autocatalytic and furin-dependent
pathways in cancer cells. Furthermore, our findings are also consistent with the earlier data that demonstrated that the full-length and soluble MT1-MMP constructs expressed in either the baculovirus (30)
system or in P. pastoris (31) were found not to contain the
prodomain and were largely represented by the active enzyme commencing
from Tyr112. Furthermore, a unique substrate binding mode
identified in our earlier work (32) discriminates MT1-MMP from other
MMPs; the presence of either Arg at the P4 position or
characteristic Pro at the P3 position of the substrate is
essential for efficient hydrolysis and for selectivity for MT1-MMP.
This unconventional feature is important to MT1-MMP biology because it
explains the autocatalytic cleavage at the
R4RKR
Y112 cleavage site. It is tempting to
hypothesize that in many cancer cell types and especially in
furin-deficient cancer cells, such as LoVo colon carcinoma (27),
MT1-MMP is a likely substitute for the general proprotein convertase
activity of furin-like proteinases. Altogether, our findings provide a
structural basis for understanding the unconventional regulation of
MT1-MMP at cancer cell surfaces and stimulate highly focused
mutagenesis for further elucidation of the structure-function
relationship of MT1-MMP in normal and pathophysiological conditions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA83017 and CA77470, California Breast Cancer Research Program Grant 5JB0094, and Susan G. Komen Breast Cancer Foundation Grant 9849 (all to A. Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: The Burnham Institute,
10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-713-6271;
Fax: 858-646-3192; E-mail: strongin@burnham.org.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M213246200
2 E. I. Deryugina and A. Y. Strongin, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
MT1-MMP, membrane
type-1 matrix metalloproteinase;
MMP, matrix metalloproteinase;
MT1-MMP-TM/CT, membrane type-1 matrix metalloproteinase without both
the transmembrane domain and the cytoplasmic tail;
FPLC, fast protein
liquid chromatography.
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REFERENCES |
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---|
1. | Egeblad, M., and Werb, Z. (2002) Nat. Rev. Cancer 2, 161-174[Medline] [Order article via Infotrieve] |
2. |
Hotary, K.,
Allen, E.,
Punturieri, A.,
Yana, I.,
and Weiss, S. J.
(2000)
J. Cell Biol.
149,
1309-1323 |
3. |
Hotary, K. B.,
Yana, I.,
Sabeh, F.,
Li, X.-Y.,
Holmbeck, K.,
Birkedal-Hansen, H.,
Allen, E. D.,
Hiraoka, N.,
and Weiss, S. J.
(2002)
J. Exp. Med.
195,
295-308 |
4. |
Nagase, H.,
and Woessner, J. F., Jr.
(1999)
J. Biol. Chem.
274,
21491-21494 |
5. |
Nabeshima, K.,
Inoue, T.,
Shimao, Y.,
Okada, Y.,
Itoh, Y.,
Seiki, M.,
and Koono, M.
(2000)
Cancer Res.
60,
3364-3369 |
6. | Overall, C. M., and Lopez-Otin, C. (2002) Nat. Rev. Cancer 2, 657-672[CrossRef][Medline] [Order article via Infotrieve] |
7. | Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65[CrossRef][Medline] [Order article via Infotrieve] |
8. | Murphy, G., Stanton, H., Cowell, S., Butler, G., Knauper, V., Atkinson, S., and Gavrilovic, J. (1999) Acta Pathol. Microbiol. Immunol. Scand. 107, 38-44 |
9. |
Belkin, A. M.,
Akimov, S. S.,
Zaritskaya, L. S.,
Ratnikov, B. I.,
Deryugina, E. I.,
and Strongin, A. Y.
(2001)
J. Biol. Chem.
276,
18415-18422 |
10. |
Kajita, M.,
Itoh, Y.,
Chiba, T.,
Mori, H.,
Okada, A.,
Kinoh, H.,
and Seiki, M.
(2001)
J. Cell Biol.
153,
893-904 |
11. |
Rozanov, D. V.,
Ghebrehiwet, B.,
Postnova, T. I.,
Eichinger, A.,
Deryugina, E. I.,
and Strongin, A. Y.
(2002)
J. Biol. Chem.
277,
9318-9325 |
12. |
Mori, H.,
Tomari, T.,
Koshikawa, N.,
Kajita, M.,
Itoh, Y.,
Sato, H.,
Tojo, H.,
Yana, I.,
and Seiki, M.
(2002)
EMBO J.
21,
3949-3959 |
13. |
Cao, J.,
Rehemtulla, A.,
Bahou, W.,
and Zucker, S.
(1996)
J. Biol. Chem.
271,
30174-30180 |
14. | Rozanov, D. V., Ghebrehiwet, B., Ratnikov, B., Monosov, E. Z., Deryugina, E. I., and Strongin, A. Y. (2002) FEBS Lett. 527, 51-57[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Sato, T.,
Kondo, T.,
Fujisawa, T.,
Seiki, M.,
and Ito, A.
(1999)
J. Biol. Chem.
274,
37280-37284 |
16. |
Yana, I.,
and Weiss, S. J.
(2000)
Mol. Biol. Cell
11,
2387-2401 |
17. | Pei, D., and Weiss, S. J. (1995) Nature 375, 244-247[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Strongin, A. Y.,
Collier, I.,
Bannikov, G.,
Marmer, B. L.,
Grant, G. A.,
and Goldberg, G. I.
(1995)
J. Biol. Chem.
270,
5331-5338 |
19. |
Hernandez-Barrantes, S.,
Toth, M.,
Bernardo, M. M.,
Yurkova, M.,
Gervasi, D. C.,
Raz, Y.,
Sang, Q. A.,
and Fridman, R.
(2000)
J. Biol. Chem.
275,
12080-12089 |
20. | Lehti, K., Lohi, J., Valtanen, H., and Keski-Oja, J. (1998) Biochem. J. 334, 345-353[Medline] [Order article via Infotrieve] |
21. |
Pei, D.,
and Weiss, S. J.
(1996)
J. Biol. Chem.
271,
9135-9140 |
22. |
Toth, M.,
Hernandez-Barrantes, S.,
Osenkowski, P.,
Bernardo, M. M.,
Gervasi, D. C.,
Shimura, Y.,
Meroueh, O.,
Kotra, L. P.,
Galvez, B. G.,
Arroyo, A. G.,
Mobashery, S.,
and Fridman, R.
(2002)
J. Biol. Chem.
277,
26340-26350 |
23. |
Stanton, H.,
Gavrilovic, J.,
Atkinson, S. J.,
d'Ortho, M. P.,
Yamada, K. M.,
Zardi, L.,
and Murphy, G.
(1998)
J. Cell Sci.
111,
2789-2798 |
24. |
Jiang, A.,
Lehti, K.,
Wang, X.,
Weiss, S. J.,
Keski-Oja, J.,
and Pei, D.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13693-13698 |
25. |
Uekita, T.,
Itoh, Y.,
Yana, I.,
Ohno, H.,
and Seiki, M.
(2001)
J. Cell Biol.
155,
1345-1356 |
26. | Ratnikov, B., Deryugina, E., Leng, J., Marchenko, G., Dembrow, D., and Strongin, A. (2000) Anal. Biochem. 286, 149-155[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Takahashi, S.,
Nakagawa, T.,
Kasai, K.,
Banno, T.,
Duguay, S. J.,
Van de Ven, W. J.,
Murakami, K.,
and Nakayama, K.
(1995)
J. Biol. Chem.
270,
26565-26569 |
28. |
Rozanov, D. V.,
Deryugina, E. I.,
Ratnikov, B. I.,
Monosov, E. Z.,
Marchenko, G. N.,
Quigley, J. P.,
and Strongin, A. Y.
(2001)
J. Biol. Chem.
276,
25705-25714 |
29. |
Ratnikov, B. I.,
Rozanov, D. V.,
Postnova, T. I.,
Baciu, P. G.,
Zhang, H.,
DiScipio, R. G.,
Chestukhina, G. G.,
Smith, J. W.,
Deryugina, E. I.,
and Strongin, A. Y.
(2002)
J. Biol. Chem.
277,
7377-7385 |
30. | Jo, Y., Yeon, J., Kim, H. J., and Lee, S. T. (2000) Biochem. J. 345, 511-519[CrossRef][Medline] [Order article via Infotrieve] |
31. | Roderfeld, M., Buttner, F. H., Bartnik, E., and Tschesche, H. (2000) Protein Expression Purif. 19, 369-374[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Kridel, S. J.,
Sawai, H.,
Ratnikov, B. I.,
Chen, E. I.,
Li, W.,
Godzik, A.,
Strongin, A. Y.,
and Smith, J. W.
(2002)
J. Biol. Chem.
277,
23788-23793 |
33. |
Bordier, C.
(1981)
J. Biol. Chem.
256,
1604-1607 |