Proteolytic cleavage of Trop2 at Arg87 is mediated by matriptase and regulated by Val194

Abstract

Proteolytic processing is an important post-translational modification affecting protein activity and stability. In the current study, we investigate the N-terminal cleavage of Trop2, a protein which is overexpressed in many cancers. We demonstrate that Trop2 is cleaved at Arg87 by a transmembrane serine protease, matriptase. Homology modeling and site-directed mutagenesis of amino acids in close proximity to the matriptase cleavage site reveal the importance of Val194 in regulating Trop2 cleavage. Co-immunoprecipitation studies confirm that amino acid substitutions at Arg87, Thr88, Lys189, Val194, and His195 do not affect Trop2 dimerization. However, cleavage of wild-type Trop2 by matriptase is inhibited when it is allowed to dimerize with a V 194 A mutant monomer, further confirming the role of Val194 in matriptase-mediated N-terminal cleavage.

Structural insights into the cis and trans assembly of human trophoblast cell surface antigen 2

“…Tumor-specific proteolytic cleavage of TROP-2 occurs at the conserved cleavage site R87-T88 of the TY loop, which is similar to that of EpCAM at the typical dibasic site R80-R81 ( Kamble et al., 2020 ; Pavšič et al., 2014 ; Trerotola et al., 2021 ; Wu et al., 2017 ). Proteolytic cleavage activates the cell-growth stimulatory properties of TROP-2 ( Trerotola et al., 2021 ).…”

Trop2 Forms a Stable Dimer with Significant Structural Differences within the Membrane-Distal Region as Compared to EpCAM

“…Mapping of proteolytic cleavage sites reported in the literature reveals that all are at least partially accessible in Trop2 dimer ( Figure 4 c,d). First, the matriptase cleavage site at surface-exposed R87 [ 20 , 44 ] is located within the TY-loop, similarly as the equivalent matriptase cleavage site in EpCAM (R80–R81; Figure 1 ) [ 6 , 45 ]. Although the TY-loop is part of the dimer interface, the structural plasticity described above may (via temporal TY-loop disengagement from juxtaposed βCD) enable efficient cleavage by enhancing the accessibility of the cleavage site.…”

“…Although the TY-loop is part of the dimer interface, the structural plasticity described above may (via temporal TY-loop disengagement from juxtaposed βCD) enable efficient cleavage by enhancing the accessibility of the cleavage site. Interestingly, it was shown that mutation V294A (part of the βCD, shown in orange in Figure 4 c) prevents matriptase cleavage at R87 without affecting the Trop2 dimerization, and the observed effect was attributed to impaired binding of matriptase to the Trop2 as the substrate [ 44 ]. Since in our crystal structure side chains of V194 and the nearby V191 are both located on the opposite side of βCD than the juxtaposed TY-loop and are therefore not exposed, it is likely that side chain of V194 is not directly involved in matriptase interaction.…”

“…In EpCAM the interaction is mediated by 16 salt bridges compared to nine salt bridges in Trop2, and a triple TY-loop mutant of EpCAM ectodomain (K83D, P84D, L88D) was found to be constitutively monomeric [ 14 ]. No comparable mutations were yet reported for Trop2 dimer destabilization, however single alanine mutations (R87A within TY-loop, and K189A and H195A within βCD) did not affect alter Trop2 oligomeric state [ 44 ]. Still, since the latter mutations were introduced into the full-length protein direct comparison with isolated ectodomains is probably not possible due to different molecular context (additional dimer stabilization via TM dimerization as described above).…”

TROP2 represents a negative prognostic factor in colorectal adenocarcinoma and its expression is associated with features of epithelial-mesenchymal transition and invasiveness

“…Human specimen collection methods were performed in accordance with relevant national and EU directives and regulations. The study was approved by the Ethics Committee of the Third Faculty of Medicine of Charles University in Prague (ref. 26/2014 and 01/2020).…”