Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity.
The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37 degrees C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.
DiMartino
S J
SJ
Department of Pathology, School of Medicine, State University of New York, Stony Brook, NY 11794, USA.
Shah
A B
AB
Trujillo
G
G
Kew
R R
RR
D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D002630:Chemotactic Factors / Q000037:antagonists & inhibitors / Q000502:physiology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000276:immunology*; D015936:Complement C5a / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000502:physiology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014809:Vitamin D-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology
D002630:Chemotactic Factors; D010618:Phenanthrolines; D015842:Serine Proteinase Inhibitors; D014809:Vitamin D-Binding Protein; D015936:Complement C5a; D019272:Leukocyte Elastase; C025205:1,10-phenanthroline