Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane: evidence for a chondroitin sulfate proteoglycan.
The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.
DiMartino
S J
SJ
Department of Pathology, School of Medicine, State University of New York, Stony Brook 11794, USA.
Kew
R R
RR
D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D011508:Chondroitin Sulfate Proteoglycans / Q000097:blood* / Q000737:chemistry*; D003432:Cross-Linking Reagents; D003902:Detergents; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D012995:Solubility; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000737:chemistry*
D011508:Chondroitin Sulfate Proteoglycans; D003432:Cross-Linking Reagents; D003902:Detergents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D014809:Vitamin D-Binding Protein