Identification of two distinct cell binding sequences in the vitamin D binding protein

Abstract

The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with β2-microglobulin or BSA. The cell-DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1–191) and III (379–458), but not domain II (192–378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150–172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379–402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.

Cofactor regulation of C5a chemotactic activity in physiological fluids. Requirement for the vitamin D binding protein, thrombospondin-1 and its receptors

“…4A, Control) but binding is completely abolished if cells are first pretreated with soluble DBP (Fig. 4A, DBP), confirming our previous report (Zhang et al, 2010). Treatment of cells with anti-CD44 reduces cell binding to DBP by about 50%, this is almost the exact percent inhibition of binding we reported previously using a different binding assay (McVoy and Kew, 2005).…”

“…Formation of a DBP binding site complex in leukocytes is a dynamic, multi-step and transient process requiring cell activation (DiMartino et al, 2007) and perhaps several distinct macromolecules. This complex also appears to function independent of C5a interacting with the C5a receptor (C5aR1/CD88) since DBP does not alter C5a receptor-ligand interactions (Perez, 1994), and DBP does not bind to C5a or the C5a receptor (DiMartino et al, 2001), (Zhang et al, 2010). Previously, we reported that C-activated serum has significantly greater leukocyte chemotactic activity than C-activated plasma; this difference was due to thrombospondin-1 (TSP-1) released into serum from activated platelets (Trujillo and Kew, 2004).…”

Behind the scenes of vitamin D binding protein: More than vitamin D binding

“…CD44 (a major cell surface CSPG on leukocytes) and the associated annexin A2 (a cell membrane Ca 2þ /phospholipid binding protein) are part of the putative cell surface DBP binding site complex and mediate the chemotactic cofactor effect [104]. The binding capacity of DBP is not influenced by the C5a receptor (C5aR1/CD88)-ligand interactions [103,105]. DBP does not alter the neutrophil C5a receptor number or the Kd for C5a [97,106].…”

Comparison of Two ELISA Methods and Mass Spectrometry for Measurement of Vitamin D-Binding Protein: Implications for the Assessment of Bioavailable Vitamin D Concentrations Across Genotypes

“…2–5 The vast majority of vitamin D metabolites circulate bound to DBP (85–90%) or albumin (10–15%), with <1% circulating in the free form. The binding affinity of DBP for vitamin D metabolites is more than 1000-fold stronger than that of albumin, and therefore, the albumin-bound and free fractions together are considered bioavailable.…”