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 tha t can modulate certain immune and inflammatory responses. The diverse cellu lar functions of DBP appear to require cell surface binding to mediate thes e processes. Numerous reports have detected DBP bound to the surface of sev eral cell types and would support the concept of a cell surface binding sit e for DBP, However, direct evidence for such a molecule has been lacking an d essentially nothing is known about its basic biochemical properties. In t he present study, radioiodinated DBP was used as a probe to characterize bi ochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitati on of DBP binding to either intact cells or purified plasma membranes showe d nonsaturable (linear) binding with positive cooperativity, possibly sugge sting DBP oligomer formation. Solubilization of cell bound I-125-DBP with v arious nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fractio n. 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 thes e 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 prot eases or chondroitinase ABC completely abrogated membrane binding of DBP, s uggesting that the protein binds to a chondroitin sulfate proteoglycan.