Characterization of free alpha- and beta-chains of recombinant macrophage-stimulating protein

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alpha beta-chain heterodimer, However cleavage of recombinant pro-RLSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alpha beta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys(527), Cys(562), and Cys(672), which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys(527)-Cys(562), but also a Cys(588)-Cys(672). Coopting Cys(588) by Cys(627) prevented the expected formation of a disulfide between alpha-chain Cys(468) and beta-chain Cys(588). Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP, The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly(18)-Gln(19) and the scissile bond for formation of mature MSP at Arg(483)-Val(484). (C) 1999 Academic Press. [References: 27]

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