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

Arch Biochem Biophys. 1999 Mar 15;363(2):356-60. doi: 10.1006/abbi.1998.1090.

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.

MeSH terms

  • Amino Acids / analysis
  • Animals
  • CHO Cells
  • Carbohydrate Metabolism
  • Chromatography, Ion Exchange
  • Cricetinae
  • Cysteine / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Gas Chromatography-Mass Spectrometry
  • Growth Substances / chemistry*
  • Growth Substances / isolation & purification
  • Hepatocyte Growth Factor*
  • Humans
  • Kallikreins / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Precursors / metabolism
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins*
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / isolation & purification

Substances

  • Amino Acids
  • Growth Substances
  • Protein Precursors
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • macrophage stimulating protein
  • Hepatocyte Growth Factor
  • Kallikreins
  • Cysteine