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Characterization of free alpha- and beta-chains of recombinant macrophage-stimulating protein
W Yoshikawa1, H Hara, T Takehara
1Toyobo Co., Ltd., 2-1-1 Katata, Ohtsu, 520-02, Japan.
Abstract:
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 alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. 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 Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484.
Insights
Human serum macrophage-stimulating protein (MSP) is a growth factor for epithelial cells. This study characterizes the structure of recombinant MSP, including its unique cysteine residues and glycosylation patterns, revealing insights into its biological activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Human serum macrophage-stimulating protein (MSP) is a known growth and motility factor for epithelial cells and macrophages.
- MSP is secreted as an inactive single-chain precursor (pro-MSP) that is proteolytically cleaved to an active alpha-beta heterodimer.
- Recombinant pro-MSP processing revealed heterogeneity, including free alpha and beta chains, suggesting some molecules lack the interchain disulfide bond.
Purpose of the Study:
- To characterize the free alpha and beta chains of recombinant MSP.
- To analyze the disulfide bond formation and glycosylation patterns of MSP.
- To identify cleavage sites in pro-MSP.
Main Methods:
- Purification of free alpha and beta chains from recombinant pro-MSP.
- Disulfide bond analysis using mass spectrometry.
- Oligosaccharide structure determination via mass spectrometry.
- Identification of signal peptide and cleavage sites using sequence analysis.
Main Results:
- The beta-chain of MSP contains three unique cysteines (Cys527, Cys562, Cys672) not present in plasminogen.
- Disulfide bonds were identified between Cys527-Cys562 and Cys588-Cys672, with the latter preventing the expected alpha-beta chain disulfide linkage.
- Heterogeneous, sialylated fucosyl biantennary oligosaccharides were found at all three Asn-linked glycosylation sites.
- The pro-MSP signal peptide cleavage site was located at Gly18-Gln19, and the mature MSP cleavage site at Arg483-Val484.
Conclusions:
- The unique cysteine residues and disulfide bonding patterns in MSP influence its processing and potentially its function.
- The complex and heterogeneous glycosylation of MSP may play a role in its biological activity and stability.
- Precise identification of cleavage sites provides a foundation for understanding MSP activation and function.