Cell Surface Vitamin D-Binding Protein (GC-Globulin) is Acquired from Plasma*

Abstract

Vitamin D-binding protein (DBP) is an abundant plasma protein. The observation of immunodetectable, cell-associated DBP on peripheral blood mononuclear cells and placental cytotrophoblasts had presented the question of the origin, function, and precise subcellular localization of cell-associated DBP. Using anti-human DBP F(ab')2 with fluorescence-activated cytometric analysis and immunogold electron microscopy, we detected DBP on the plasmalemma of U937 cells, a monoblastic, histiocytic cell line grown in media supplemented with fetal calf serum (FCS). DBP was then removed from FCS by actin affinity chromatography followed by anti-DBP immunoaffinity chromatography. U937 cells in this DBP-free medium exhibited nearly identical growth rates to cells grown in medium containing native FCS. However, in contrast to cells grown with native FCS, those grown for seven to eight generations with DBP-free FCS exhibited less cell-surface DBP as quantified by fluorescence-activated cytometric analysis (73% decrease) and immunoelectron microscopy (88% decrease). DBP mRNA could not be detected in U937 cells, placental tissues, freshly prepared resting and stimulated B and T lymphocytes, or lymphocyte-derived cell lines by Northern analysis. In addition, using the sensitive reverse transcriptase/polymerase chain reaction assay no DBP fragments were detectable in U937 cells. We conclude that U937 cell-associated DBP is exogenously derived from plasma and is located on the plasmalemma. Based upon this conclusion, we postulate that specific binding sites for DBP may exist on the plasma membranes of certain cell types.

Crystal structures of the vitamin D-binding protein and its complex with actin: Structural basis of the actin-scavenger system

“…In agreement with a study that used limited proteolysis to identify the sterol-binding site (35), Verboven et al localize this site to the N terminus of DBP (34). Such a location is distal to the actin-binding interface identified in this work, which provides a structural understanding of previous reports that the sterol-binding and actin-binding properties of DBP are independent (35)(36)(37)(38). It also seems reasonable to propose that the large structural differences between DBP and HSA are directed toward its actin-binding function, which involves specific interactions with all three domains of DBP and not toward its sterol-binding property, which is confined to the first four ␣-helices of DBP.…”

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

“…The same has been shown for albumin, a member of the DBP gene family that shares significant amino acid and secondary structural similarities with DBP but that is not a highaffinity steroid/sterol binding protein (4). A variety of cell types have been shown to have DBP on their surfaces through immunohistochemical and immunofluorescent analyses (51)(52)(53)(54)(55), suggesting that DBP might mediate entry of vitamin D sterols into the cell. However, in its role as a circulating reservoir for the vitamin D sterols, DBP might alternatively slow the entry of 1,25(OH) 2 D into its target cells by regulating the amount of free sterol available.…”

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

“…In addition to functioning as a co-chemotactic factor for C5-derived peptides, DBP functions to transport vitamin D sterols and acts as a scavenger protein to clear extracellular G-actin released from necrotic cells, and a deglycosylated form of DBP has been shown to be a potent macrophage and osteoclast-activating factor (22). Plasma-derived DBP also binds to the surface of many cell types including neutrophils (23)(24)(25). DBP bound to the plasma membrane of neutrophils appears to play an essential role in enhancing chemotaxis to C5-derived peptides (26).…”