Proteolytic conversion of single chain precursor macrophage-stimulating protein to a biologically active heterodimer by contact enzymes of the coagulation cascade.
Human serum macrophage stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. It is a member of the family of kringle proteins, which typically exist in extracellular fluid as single chain precursors that are activated by proteolytic cleavage. In this work, we expressed [35S]cysteine-labeled recombinant pro-MSP in MSP cDNA-transfected Chinese hamster ovary cells and studied proteolytic processing of pro-MSP and the requirement of cleavage for biological activity. In media containing heat-inactivated fetal bovine serum, the protein was secreted as single chain pro-MSP, which was cleaved over a period of hours to the mature heterodimer. Cleavage was prevented by serine protease inhibitors such as leupeptin or aprotinin; it did not occur if cells were cultured in serum-free medium. Nanomolar concentrations of coagulation proteases kallikrein, factor XIIa or factor XIa cleaved pro-MSP to MSP within 30 min. Pro-MSP had no biological activity. After cleavage by kallikrein, biological activity was quantitatively comparable to that of natural MSP isolated from human plasma. These results support our hypothesis that MSP circulates as the biologically inactive precursor and can be activated by enzymes of the intrinsic coagulation cascade.
Wang
M H
MH
Immunopathology Section, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702.
Yoshimura
T
T
Skeel
A
A
Leonard
E J
EJ
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000378:metabolism*; D016466:CHO Cells; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015956:Factor XIIa / Q000378:metabolism; D015945:Factor XIa / Q000378:metabolism; D006133:Growth Substances / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D017228:Hepatocyte Growth Factor; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D046911:Macromolecular Substances; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology*; D051379:Mice; D008970:Molecular Weight; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014162:Transfection
D001779:Blood Coagulation Factors; D006133:Growth Substances; D007976:Leupeptins; D046911:Macromolecular Substances; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; D007611:Aprotinin; D007610:Kallikreins; D015945:Factor XIa; D015956:Factor XIIa; C032854:leupeptin; D003545:Cysteine