Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Abstract

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.

Class-3 Semaphorins and Their Receptors: Potent Multifunctional Modulators of Tumor Progression

“…However, there are exceptions as in the case of sema3E in which the cleaved product retains full activity [12,13,14]. In contrast, cleavage in the basic domain potentiated the activity of sema3A [10] and there are reports suggesting that cleavage at this site may be essential for the anti-angiogenic activities of sema3F and sema3C [15,16].…”

Semaphorin-3C Is Upregulated in Polycystic Kidney Epithelial Cells and Inhibits Angiogenesis of Glomerular Endothelial Cells

“…Our results demonstrated that SEMA3C reduced angiogenesis in HRGECs by inhibiting migration. The negative effect of SEMA3C depended on its C-terminal portion since the basic domain is involved in its binding to NRPs [12,[16][17][18].…”

“…It has been known that cleavage of SEMA3C at 549 RSRR 552 by FPPCs diminishes SEMA3C function [12,[16][17][18], and we generated SEMA3C mutants, FR and truncated (p65) forms. Generation and validation of these mutant constructs are detailed in online suppl.…”

Semaphorin 3C as a Therapeutic Target in Prostate and Other Cancers

“…This nuance indicates that under any given biological context not only are levels of SEMA3C relevant but so too are the levels and repertoire of enzymes that modify SEMA3C. To this end, we and others have developed cleavage-resistant forms of SEMA3C in an effort to compensate for SEMA3C truncations [16,39,139,140,141]; however, a systematic analysis correlating various forms of SEMA3C and their activities would be informative. In two recent studies, Mumblat et al [139] and Yang et al [140] showed that a truncated version of SEMA3C harboring a deletion of the c-terminal 13 amino acids or wild type cleavable SEMA3C, respectively, inhibited angiogenesis but did not affect cell growth.…”