LIMITED PROTEOLYSIS OF HUMAN ALPHA(2)-HS GLYCOPROTEIN FETUIN - EVIDENCE THAT A CHYMOTRYPTIC ACTIVITY CAN RELEASE THE CONNECTING PEPTIDE

alpha(2)-HS glycoprotein is a major protein of human plasma whose func tion is still obscure, A proteolytically processed form of alpha(2)-HS glycoprotein lacking a segment of 40 amino acid residues bridging its heavy and light chain portions (''connecting peptide'') has been desc ribed suggesting that this peptide is released by posttranslational pr ocessing to fulfill biological role(s) of alpha(2)-HS glycoprotein. To test this hypothesis we investigated how the connecting peptide is re leased from the parental molecule by limited proteolysis. We developed monoclonal antibodies to various portions of the connecting peptide a nd its NH2-terminal flanking region which cross-react with the native alpha(2)-HS glycoprotein, Purified alpha(2)-HS glycoprotein from human plasma was subjected to limited proteolysis by proteinases including trypsin, chymotrypsin, elastase plasmin, kallikrein, thrombin, and ren in. Immunoprint analysis of the proteolytic digests indicated that alp ha(2)-HS glycoprotein is readily cleaved in its connecting peptide reg ion, NH2-terminal amino sequence analysis of the generated fragments d emonstrated that a single proteinase, chymotrypsin, cleaves the critic al Leu-Leu bond flanking the NH2-terminal portion of the connecting pe ptide region. Most but not all of the other proteinase cleavage sites map to a short stretch of 9 residues located in the center portion of the connecting peptide region, Immunoprint analysis of plasma samples from patients with sepsis demonstrate that the connecting peptide regi on is cleaved under pathological conditions, Our results indicate that the connecting peptide and/or fragments thereof are readily releasabl e from alpha(2)-HS glycoprotein in vitro and in vivo.