Characterization of C1 inhibitor binding to neutrophils.
In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH.
Chang
N S
NS
University of Massachusetts Medical Center, Department of Biochemistry, Worcester.
Boackle
R J
RJ
Leu
R W
RW
D001667:Binding, Competitive / Q000276:immunology; D002460:Cell Line; D003174:Complement C1 Inactivator Proteins / Q000378:metabolism*; D015941:Complement Hemolytic Activity Assay; D010450:Endopeptidases / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008863:Microspheres; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D012794:Sialic Acids / Q000276:immunology
D003174:Complement C1 Inactivator Proteins; D012794:Sialic Acids; D010450:Endopeptidases