Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G.
Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.
Maison
C M
CM
Department de Biologie, Moleculaire et Structural/Laboratoire d'Immunochimie, Unité INSERM 238, Grenoble, France.
Villiers
C L
CL
Colomb
M G
MG
D000595:Amino Acid Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000302:isolation & purification*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003176:Complement C3 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000302:isolation & purification; D012697:Serine Endopeptidases
D003176:Complement C3; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; D010196:Pancreatic Elastase