Membrane Type-1 Matrix Metalloproteinase Functions as a Proprotein Self-convertase

EXPRESSION OF THE LATENT ZYMOGEN IN PICHIA PASTORIS, AUTOLYTIC ACTIVATION, AND THE PEPTIDE SEQUENCE OF THE CLEAVAGE FORMS*

Dmitri V. Rozanov and Alex Y. StronginDagger

From the Cancer Research Center, The Burnham Institute, La Jolla, California 92037

Received for publication, December 30, 2002

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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 RRKRdown-arrow 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.

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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 108RRKRdown-arrow 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.

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 108RRKRdown-arrow Y112 site and generating the mature enzyme commencing from the N-terminal Tyr112 through self-proteolysis, rather than via the cleavage by furin.

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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). alpha 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).

Expression of MT1-MMP-Delta 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 alpha -mating factor pre-propeptide. The resulting construct encoding the soluble, secretory MT1-MMP without both the transmembrane domain and the cytoplasmic tail (MT1-MMP-Delta 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.

Purification of MT1-MMP-Delta TM/CT-- To produce MT1-MMP-Delta 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-Delta 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-Delta 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-Delta TM/CT species. The fractions containing the proenzyme and the enzyme were pooled and used for further analysis.

    RESULTS AND DISCUSSION
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To isolate sufficient quantities of MT1-MMP, we synthesized in methylotrophic yeast P. pastoris the recombinant soluble MT1-MMP (MT1-MMP-Delta TM/CT) with both the transmembrane and cytoplasmic domains deleted (Fig. 1A). The expression of the alpha -factor-His6-MT1-MMP construct in the pPIC9 plasmid was controlled by the alcohol oxidase promoter. The presence of the alpha -factor sequence stimulated secretion of MT1-MMP-Delta TM/CT from cells into the extracellular medium. Following the induction of the alcohol oxidase promoter, MT1-MMP-Delta 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-Delta TM/CT from a 1-day medium (Fig. 1B, inset, the middle and right lanes, respectively).


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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-Delta 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-Delta 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-Delta 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-Delta TM/CT proenzyme, and the enzyme (left, middle, and right lanes, respectively). C, the proteolysis of alpha 1-antitrypsin by MT1-MMP. alpha 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-Delta 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-Delta 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-Delta 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-Delta TM/CT and MT1-MMP-CAT was 20 and 60 arbitrary units/nm, respectively. Similarly, the catalytic quantities of the MT1-MMP-Delta TM/CT enzyme were capable of efficiently cleaving alpha 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-Delta TM/CT was inert in cleaving alpha 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-Delta TM/CT proenzyme samples were relatively unstable. When incubated at 45-50 µg/ml for 1-4 h at 37 °C, pro-MT1-MMP-Delta 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-Delta TM/CT (data not shown).


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Fig. 2.   Self-proteolysis of the proenzyme of MT1-MMP-Delta TM/CT. A, autolytic conversions of the MT1-MMP-Delta TM/CT. The proenzyme (700 ng) was incubated with or without GM6001 (1 µM) for the time indicated 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 further separated by 10% SDS-PAGE and analyzed by Western blotting with AB815. Molecular mass markers are indicated on the left. B, self-proteolysis of MT1-MMP-Delta TM/CT and N-terminal sequencing of the cleavage fragments. The MT1-MMP-Delta TM/CT proenzyme (7 µg; right lane) was incubated for 1 h at 37 °C in 15 µl of 50 mM HEPES, pH 6.8, containing 10 mM CaCl2, 50 µM ZnCl2, and 0.005% Brij 35. The sample was separated by 4-12% gradient SDS-PAGE. Following SDS-PAGE, the protein bands were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) and stained with Coomassie. Following destaining, the main bands were subjected to N-terminal microsequencing. The N-terminal amino acid residue of the respective cleavage fragments is shown on the right. The proenzyme has the N-terminal sequence RFPSI of the alpha -factor. The proenzyme (left lane) and the enzyme of MT1-MMP-Delta TM/CT (middle lane) are shown for comparison. Molecular mass markers are indicated on the left.

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-Delta 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-Delta 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-Delta TM/CT construct (RFPSI which represent the N-terminal sequence of the yeast alpha -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-Delta 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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Fig. 3.   Tumor cells express multiple forms of the wild type MT1-MMP at the cell surface. MCF7 breast carcinoma cells overexpressing MT1-MMP (28) were incubated with or without GM6001 (50 µM) for 48 h. The integral membrane proteins including MT1-MMP were partitioned into the Triton X-114 detergent phase (33). The final detergent phase (2 µg of total protein) was analyzed by Western blotting with AB815. Molecular mass markers are indicated on the left.

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 QQLYdown-arrow GG284 rather than at QQLYGdown-arrow G284 as earlier reported by Fridman and co-workers (22). Fridman and co-workers (22) have also directly suggested that the cleavage at the SDPSAdown-arrow I256 site requires the attachment of MT1-MMP to the plasma membrane. In agreement, the cleavage at the SDPSAdown-arrow 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 RRKRdown-arrow Y112 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 R4RKRdown-arrow 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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: MT1-MMP, membrane type-1 matrix metalloproteinase; MMP, matrix metalloproteinase; MT1-MMP-Delta TM/CT, membrane type-1 matrix metalloproteinase without both the transmembrane domain and the cytoplasmic tail; FPLC, fast protein liquid chromatography.

    REFERENCES
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REFERENCES

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