Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

Trine Tamberg,Zebin Hong,Daphné De Schepper,Signe Skovbjerg,Daniel M Dupont,Lars Vitved,Christine R Schar,Karsten Skjoedt,Lotte K Vogel,Jan K Jensen

doi:10.1074/jbc.ra118.004126

Abstract

Matriptase is a member of the type-II transmembrane serine protease (TTSP) family and plays a crucial role in the development and maintenance of epithelial tissues. As all chymotrypsin-like serine proteases, matriptase is synthesized as a zymogen (proform), requiring a cleavage event for full activity. Recent studies suggest that the zymogen of matriptase possesses enough catalytic activity to not only facilitate autoactivation, but also carry out its in vivo functions, which include activating several proteolytic and signaling cascades. Inhibition of zymogen matriptase may therefore be a highly effective approach for limiting matriptase activity. To this end, here we sought to characterize the catalytic activity of human zymogen matriptase and to develop mAb inhibitors against this enzyme form. Using a mutated variant of matriptase in which the serine protease domain is locked in the zymogen conformation, we confirmed that the zymogen form of human matriptase has catalytic activity. Moreover, the crystal structure of the catalytic domain of zymogen matriptase was solved to 2.5 Å resolution to characterize specific antibody-based matriptase inhibitors and to further structure-based studies. Finally, we describe the first antibody-based competitive inhibitors that target both the zymogen and activated forms of matriptase. We propose that these antibodies provide a more efficient way to regulate matriptase activity by targeting the protease both before and after its activation and may be of value for both research and preclinical applications.

Highlights

A peptide inhibitor, biotin-RQRR-CMK [23], was included as a positive control. These results strongly suggest that antibodies aZ-mAb-6 and aZ-mAb-7 inhibit matriptase-catalyzed cleavage of a physiologically relevant protein substrate
We used a zymogen-locked form of matriptase produced by mutating arginine 614 of the activation motif in the activation loop to an alanine (R15A), termed zSPD
The zymogenicity factor was calculated to be 33 Ϯ 11, equivalent to the zymogen preparation having ϳ3% of the activity of an activated matriptase preparation. This result is consistent with the reported zymogenicity factor of 27 for rat matriptase [20]

Summary

Results

To characterize the catalytic activity of matriptase in its zymogen form, we prepared a zymogen variant of the serine protease domain (SPD) by recombinant expression in yeast (Pichia pastoris). The catalytic activity of zSPD or acSPD toward a peptide substrate was measured in the presence of each antibody (Fig. 2B). No inhibition was observed toward the mouse ortholog of matriptase (Fig. 5), whose catalytic domain shares 87% identity to the human protein Together, these observations strongly suggest that antibodies aZ-mAb-6 and aZ-mAb-7 have high specificity toward human matriptase. Both antibodies aZ-mAb-6 and aZ-mAb-7 but not the control antibody inhibited the proteolytic turnover of the broad-spectrum serine protease substrate; this activity was interpreted as derived from unopposed matriptase in either the active zymogen form or the activated form. When matriptase was co-expressed with HAI-2 C47F, we observed a clear proteolytic activity, which could be silenced by aZ-mAb-6 and aZ-mAb-7 but not by the control antibody (anti-uPA), strongly suggesting the presence of unopposed matriptase activity under these conditions. ␮M acSPD No antibody aZ-mAb-1 aZ-mAb-2 aZ-mAb-3 aZ-mAb-4 aZ-mAb-5 aZ-mAb-6 aZ-mAb-7 aZ-mAb-9 aZ-mAb-10

39 Ϯ 4 18 Ϯ 4 35 Ϯ 2 15 Ϯ 3 35 Ϯ 6 34 Ϯ 6 37 Ϯ 4 37 Ϯ 2 28 Ϯ 6 31 Ϯ 4

Discussion

Experimental procedures