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*

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 abun-dance 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 2 Y 112 , thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr 112 . The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp 119 and at Asn 130 ) and, next, to the three inactive ectodomain forms (N terminus at Thr 222 , at Gly 284

Recent evidence indicates that membrane type-1 matrix metalloproteinase (MT1-MMP 1 or MMP-14) is a key enzyme in tumor cell migration and invasion (1)(2)(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)(14)(15)(16) discuss the existence of the alternative activation pathways, the cleavage of the 108 RRKR2Y 112 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 Tyr 112 (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 selfconvertase capable of cleaving the prodomain at the 108 RRKR2Y 112 site and generating the mature enzyme commencing from the N-terminal Tyr 112 through self-proteolysis, rather than via the cleavage by furin.

MATERIALS AND METHODS
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). ␣ 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-⌬TM⁄CT-The cDNA fragment coding for peptide Ala 21 -Ser 538 of the full-length MT1-MMP (the GenBank TM accession number U41078) was merged with the His 6 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.
The purified MT1-MMP-⌬TM/CT proenzyme samples were relatively unstable. When incubated at 45-50 g/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 M) (Fig. 2A). Serine proteinase inhibitors failed to affect MT1-MMP-⌬TM/CT (data not shown).
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 Tyr 112 . It is likely that this form was further processed to the intermediate with the N terminus at Trp 119 . The next autolytic cleavage  produced the form with the N terminus at Asn 130 . MT1-MMP with the N terminus at Trp 119 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 Thr 222 , Gly 284 , and Thr 299 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 M) 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 furindeficient 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 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 QQLY2GG 284 rather than at QQLYG2G 284 as earlier reported by Fridman and co-workers (22). Fridman and co-workers (22) have also directly suggested that the cleavage at the SDPSA2I 256 site requires the attachment of MT1-MMP to the plasma membrane. In agreement, the cleavage at the SDPSA2I 256 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 RRKR2Y 112 and generating the mature enzyme with N terminus at Tyr 112 . 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 Tyr 112 . 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 P 4 position or characteristic Pro at the P 3 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 au-tocatalytic cleavage at the R 4 RKR2Y 112 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.