Processing of syndecan-2 by matrix metalloproteinase-14 and effect of its cleavage on VEGF-induced tube formation of HUVECs

Abstract

Syndecans (SDCs) are transmembrane proteoglycans that are involved in cell adhesion and cell communication. Specifically, SDC2 plays a key role in tumorigenesis, metastasis, and angiogenesis. Previously, we found that rat SDC2 is shed by matrix metalloproteinase-7 (MMP-7) in colon cancer cells. Here, we analyzed the susceptibility of rat SDC2 to various MMPs. We found that the rat SDC2 ectodomain (ECD) fused to the C-terminal Fc region, which was expressed in mammalian cells, was cleaved more efficiently by MMP-14 than MMP-7. Likewise, when anchored on the surface of HeLa cells, rat SDC2 was cleaved more efficiently by the treatment of MMP-14 than MMP-7 and was shed more readily by membrane-anchored MMP-14 than soluble MMP-14. Furthermore, MMP-14 cleaved recombinant SDC2-ECD expressed in Escherichia coli into multiple fragments. Using N-terminal amino acid sequencing and the top-down proteomics approach, we determined that the major cleavage sites were S⁸⁸↓L⁸⁹, T⁹⁸↓M⁹⁹, T¹⁰⁰↓L¹⁰¹, D¹³²↓P¹³³, and N¹⁴⁸↓L¹⁴⁹ for rat SDC2-ECD and S⁵⁵↓G⁵⁶, S⁶⁵↓P⁶⁶, P⁷⁵↓K⁷⁶, N⁹²↓I⁹³ D¹²²↓P¹²³, and S¹³⁸↓L¹³⁹ for human SDC2-ECD. Finally, the rat and human SDC2-ECD lost the ability to suppress vascular endothelial growth factor-induced formation of capillary-like tubes by human umbilical vein endothelial cells following cleavage by MMP-14, but its major cleavage-site mutant of rat SDC2-ECD did not. These results suggest that MMP-14 is a novel enzyme responsible for degrading SDC2 and impairing its physiological roles including angiogenesis.

Keywords: angiogenesis; mass spectrometry; metalloproteases; protein degradation; syndecans.