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the domain. The residues that comprise the catalytic triad, His-656, Asp-711, and Ser-805, corresponding to His-57, Asp-102, and Ser-195 in chymotrypsin, are observed in MT-SP1 (for reviews, see refs. 39 and 40). The sequence Ser214Trp215Gly216 (Ser825Trp826Gly827), which is thought to interact with the side chains of the substrate for properly orienting the scissile bond is present. Gly-193 (Gly-803) and Gly-196 (Gly-805), which are thought to be necessary for proper orientation of Ser-195 (Ser-805), also are present. Based on homology to chymotrypsin, three disulfide bonds are predicted to form within the protease domain at Cys-44–Cys-58, Cys-168–Cys-182, and Cys-191–Cys-220 (Cys-643–Cys-657, Cys-776–Cys-790, and Cys-801–Cys-830), and a fourth disulfide bond should form between the catalytic and the pro-domain Cys-122–Cys-1 (Cys-731–Cys-604), as observed for chymotrypsin. This predicted disulfide with the pro domain suggests that the active catalytic domain should still be localized to the cell surface via a disulfide linkage. The presence of the catalytic machinery and other conserved structural components described above suggest that all features necessary for proteolytic activity are present in the encoded sequence.

Substrate Specificity of the MT-SP1 Protease Domain. The S1 site specificity (41) of a protease is largely determined by the amino acid residue at position 189. This position is occupied by an aspartate in MT-SP1, suggesting that the protease has specificity for Arg/Lys in the P1 position. In addition, the presence of a polar Gln-192 (Gln-803), as in trypsin, is consistent with basic specificity. Furthermore, the presence of Gly-216 (Gly-827) and Gly-226 (Gly-837) is consistent with the presence of a deep S1 pocket, unlike elastase, which has Val-216 and Thr-226 that block the pocket and thereby contribute to the P1 specificity for small hydrophobic side chains. The specificity at the other subsites is largely dependent on the nature of the seven loops A–E and loops 2 and 3 (Fig. 4). Loop C in enterokinase has a number of positively charged residues that are thought to interact with the negatively charged activation site in trypsinogen, Asp-Asp-Asp-Asp-Lys (26). One known substrate for MT-SP1 (as described below) is the activation site of MT-SP1, which is Arg-Gln-Ala-Arg (residues 611–614). Loop C contains two Asp residues that may participate in the recognition of the activation sequence.

One means of obtaining further data on substrate specificity is by characterization of the activity of the recombinant proteolytic domain. Enterokinase has been characterized from both recombinant (38, 42) and native (43, 44) sources. However, proteolytic activity for the other reported membrane-type serine proteases hepsin (25) and TMPRSS2 (32) are only predicted based on sequence homology. To produce active recombinant MT-SP1, a His-tagged fusion of the protease domain was cloned into an E.coli vector and expressed and purified to homogeneity. Fortuitously, the protease domain refolded and autoactivated after resuspension and purification from inclusion bodies. This activity, coupled with the lack of activity in the Ser195Ala (Ser805Ala) variant, demonstrates that the cDNA encodes a catalytically proficient protease. Autoactivation of the protease domain at the arginine-valine site (Arg614-Val615) shows that the protease has Arg/Lys specificity as predicted by the sequence homology to other proteases of basic specificity. Specificity and selectivity are confirmed by the lack of cleavage of AAPX-pNA substrates that do not have x=R, K. Further characterization with Spectrozyme tPA revealed an active enzyme with kcat=2.6×102 s–1. However, the His-tagged serine protease domain does not cleave H-Arg-pNA, showing that, unlike trypsin, there is a requirement for additional subsite occupation for catalytic activity. This suggests that the enzyme is involved in a regulatory role that requires selective processing of particular substrates rather than nonselective degradation.

MT-SP1 Function. In other studies, we have found that inhibition of serine protease activity by ecotin or ecotin M84R/M85R inhibits testosterone-induced branching ductal morphogenesis and enhances apoptosis in a rat ventral prostate model (F.Elfman, T.T., C.S.C., G.Cunha, and M.A.S., unpublished results). Moreover, the rat homolog of MT-SP1 is expressed in the normal rat ventral prostate (data not shown). Assays of the protease domain with ecotin and ecotin M84R/ M85R showed that the enzymatic activity is strongly inhibited (782±92 pM and 9.8±1.5 pM, respectively), suggesting that rat MT-SP1 is likely to be inhibited at the concentrations of these inhibitors used in our experiments. MT-SP1 inhibition may result in the observed inhibition of differentiation and/or increased apoptosis. Future studies are aimed at definitively resolving the role of MT-SP1 in prostate differentiation. The broad expression of MT-SP1 in epithelial tissues is consistent with the possibility that it is involved in cell maintenance or growth, perhaps by activating growth factors or by processing prohormones.

MT-SP1 may participate in a proteolytic cascade that results in cell growth and/or differentiation. Another structurally similar membrane-type serine protease, enteropeptidase (Fig. 3), is involved in a proteolytic cascade by which activation of trypsinogen leads to activation of downstream intestinal proteases (5). Enteropeptidase is expressed only in the enterocytes of the proximal small intestine, thus precisely restricting activation of trypsinogen. Thus, in contrast to secreted proteases that may diffuse throughout the organism, the membrane association of MT-SP1 should also allow the proteolytic activity to be precisely localized, which may be important for proper physiological function; improper localization of the enzyme, or levels of downstream substrates could lead to disease.

We have found subcutaneous coinjection of PC-3 cells with wild-type ecotin or ecotin M84R/M85R led to a decrease in the primary tumor size compared with animals in whom PC-3 cells and saline were injected (O.Melnyk, T.T., C.S.C. and, M.A.S., unpublished results). Because wild-type ecotin is a poor, micromolar inhibitor of uPA, serine proteases other than uPA likely are involved in this primary tumor proliferation. Both wild-type ecotin and ecotin M84R/M85R are potent, subnanomolar inhibitors of MT-SP1, raising the possibility that MT-SP1 plays an important role in progression of epithelial cancers expressing this protease.

Direct biochemical isolation of the substrates may be possible if MT-SP1 adhesive domains such as the CUB domains or LDLR repeats interact with the substrates. In addition, likely substrates may be predicted and tested for by using knowledge of extended enzyme specificity. For example, the characterization of the substrate specificity of granzyme B allowed the prediction and confirmation of substrates for this serine protease (45). Thus, these complimentary studies should further shed light on the physiological function of this enzyme.

We thank Marion Conn, Robert Maeda, Todd Pray, Ibrahim Adiguzel, and Ralph Reid for technical assistance and helpful discussions. T.T. was supported by a National Institutes of Health postdoctoral fellowship CA71097, and this work was supported by National Institutes of Health Grant CA72006.

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