Abstract
Purpose :
Thrombospondin-1 (TSP-1) is an extracellular matrix molecule that mediates many cell-cell and cell-matrix interactions. We recently identified a single gene variant, rs2228262 N700S, in TSP-1 to be significantly associated with glaucoma. Previously, N700S TSP-1 has been shown to be differentially cleaved by various proteases, potentially impacting matrix organization and signaling events critical to intraocular pressure (IOP) homeostasis by the trabecular meshwork. In this study, we tested specific matrix metalloproteinases (MMPs) known to be involved in IOP regulation and the pathogenesis of glaucoma, for their ability to cleave wild-type (WT) and N700S TSP-1.
Methods :
WT and N700S human TSP-1, each bearing a C-terminal histidine tag, were over-expressed in HEK-293 cells in the presence of protease inhibitors. WT and mutant proteins were purified using nickel affinity resin and then digested separately with a panel of MMPs comprising MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, and 13. Each digest was analyzed by SDS-PAGE, followed by Coomassie staining or Western immunoblotting using antibodies against specific domains of TSP-1. In subsequent studies, various C-terminal truncations of TSP-1 were recombinantly expressed, purified, and subjected to similar digests to ascertain the domains required for MMP binding/cleavage. The novel N-termini of cleaved fragments were determined by Edman sequencing.
Results :
Of the MMPs tested, MMPs -1, -7 and -10 were found to cleave TSP-1 and MMP-1 was most effective. Cleavage was not affected by the N700S mutation. The MMP-1 cleavage site was identified by Edman degradation and determined to be within the N-terminal heparin binding domain of TSP-1, prior to the oligomerization domain. MMP-1 cleavage liberates an approximately 22 kDa monomeric fragment, leaving the remainder of the trimericTSP-1 molecule intact.
Conclusions :
We have identified TSP-1 as a novel substrate for MMPs -1, -7 and -10. This finding is significant given that upregulation of MMP-1 reduces IOP and prostaglandin analogues, which lower IOP, increase mRNA expression of MMPs -1 and -10. This supports the hypothesis that MMP-mediated TSP-1 cleavage contributes to IOP regulation. The potential role of the liberated 22 kDa fragment in the biology of TSP-1 and the trabecular meshwork, particularly in the context of glaucoma, remains to be elucidated.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.