Limited proteolysis of human alpha2-HS glycoprotein/fetuin. Evidence that a chymotryptic activity can release the connecting peptide.
alpha2-HS glycoprotein is a major protein of human plasma whose function is still obscure. A proteolytically processed form of alpha2-HS glycoprotein lacking a segment of 40 amino acid residues bridging its heavy and light chain portions ("connecting peptide") has been described suggesting that this peptide is released by post-translational processing to fulfill biological role(s) of alpha2-HS glycoprotein. To test this hypothesis we investigated how the connecting peptide is released from the parental molecule by limited proteolysis. We developed monoclonal antibodies to various portions of the connecting peptide and its NH2-terminal flanking region which cross-react with the native alpha2-HS glycoprotein. Purified alpha2-HS glycoprotein from human plasma was subjected to limited proteolysis by proteinases including trypsin, chymotrypsin, elastase plasmin, kallikrein, thrombin, and renin. Immunoprint analysis of the proteolytic digests indicated that alpha2-HS glycoprotein is readily cleaved in its connecting peptide region. NH2-terminal amino sequence analysis of the generated fragments demonstrated that a single proteinase, chymotrypsin, cleaves the critical Leu-Leu bond flanking the NH2-terminal portion of the connecting peptide 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 region is cleaved under pathological conditions. Our results indicate that the connecting peptide and/or fragments thereof are readily releasable from alpha2-HS glycoprotein in vitro and in vivo.
Nawratil
P
P
Department for Clinical Chemistry and Clinical Biochemistry, The University of Munich, Nussbaumstrasse 20, 80336 Munich, Germany. Willi.Jahnen@UNI-MAINZ.DE
Lenzen
S
S
Kellermann
J
J
Haupt
H
H
Schinke
T
T
Müller-Esterl
W
W
Jahnen-Dechent
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D060749:alpha-2-HS-Glycoprotein; D000509:alpha-Fetoproteins / Q000378:metabolism*
C556354:AHSG protein, human; C556357:Ahsg protein, mouse; D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D060749:alpha-2-HS-Glycoprotein; D000509:alpha-Fetoproteins; D002918:Chymotrypsin