MAGP2 is a small extracellular protein with both tumor angiogenesis and cell signaling activity. MAGP2 was originally isolated biochemically from microfibril-rich connective tissue. The localization of MAGP2 to microfibrils has been confirmed by both immunohistochemistry and immunogold electron microscopy. Whether MAGP2 binding to microfibrils is regulated post-translationally is still unclear, however, and a better understanding of this process would be instructive to understanding the angiogenesis and signaling functions ascribed to MAGP2. Here we show via immunofluorescence studies that the T3 cell line, derived from ovarian mouse tumor cells, produces abundant fibrillin-2 microfibrils to which MAGP2 can bind. Co-localization of MAGP2 and fibrillin-2 can be detected either when MAGP2 is overexpressed in, or exogenously introduced to, the cells. As expected, matrix association of MAGP2 required its conserved Matrix Binding Domain. Matrix association was positively regulated by proprotein convertase (PC) cleavage of MAGP2; mutation of the MAGP2 PC consensus site reduced the amount of matrix-associated MAGP2. Deletion analysis of the C-terminal 20-amino acid domain that is defined by the PC cleavage site suggests that this domain also positively modulates matrix localization of MAGP2, in a manner that requires the amino-terminal half of the protein. Together, our data indicate that matrix localization of MAGP2 by its Matrix Binding Domain is promoted by PC cleavage and the presence of its C-terminal 20 amino acids.
Microfibrils are extracellular matrix fibers whose major components are the fibrillin proteins (Fbn‐1, ‐2, ‐3), which are important for tissue mechanical stability and elasticity. MAGP2 is a secreted protein found on specific microfibrils. Following MAGP2 secretion, PC cleavage occurs at the RLRR consensus site near the MAGP2 carboxyl‐terminus (CT). Since PC cleavage of Fbn‐1 is required for microfibril incorporation, we investigated the role PC cleavage plays in the localization of MAGP2 on microfibrils using immunocytochemistry. Transient expression of wild‐type MAGP2 in mouse ovarian epithelial cells that produce abundant Fbn‐2 microfibrils resulted in weak, but detectable extracellular MAGP2 fibers. Abundant MAGP2 fibers were generated when only the CT half of MAGP2 was expressed, confirming that the matrix‐binding domain in the CT half of MAGP2 is responsible for fiber localization. Mutation of the CT half of MAGP2 to prevent PC cleavage showed a small, but significant, decrease in extracellular MAGP2 fibers, indicating that PC cleavage promotes MAGP2‐microfibril binding. Preliminary data suggest that MAGP2 may bind preferentially to Fbn‐2 fibers, and that soluble MAGP2 can bind to microfibrils. Understanding the normal maturation and function of MAGP2 may be relevant to both angiogenesis and ovarian carcinoma progression, since previous studies implicate MAGP2 in both processes.
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