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 beta(2)-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.

Figure 1

Schematic representation of the DBP domain structure. The 458 amino acid sequence of human DBP with the three structural domains and known functional regions indicated. Domain I: amino acids 1–191; domain II: 192–378; domain III: 379–458; vitamin D binding: 35–49; C5a chemotactic cofactor: 130–149; G-actin binding: 373–403. The domain and functional regions are drawn approximately to scale.

Figure 2

Initial characterization of DBP-cell binding by SPR. Purified human DBP was covalently coupled to a CM5 sensor chip. Panel A: detection of immobilized DBP with soluble anti-DBP. Panel B: overlay plot of sensorgrams when different U937 cell concentrations were injected into the flow cell.

Figure 3

Effect of soluble DBP or DBP domains on cell binding to immobilized DBP. U937 cells (10⁶/ml) in HBSS were pretreated with either buffer (control) or 0.2 µM of native DBP or the indicated DBP domain for 15 min at 22°C. Data is presented as an overlay plot of response curves obtained from the original sensorgrams.

Figure 4

Cell binding to immobilized DBP domains. Purified recombinant DBP domains expressed in E. coli were individually coupled to separate CM5 sensor chips as described in Experimental Procedures. Panel A: detection of immobilized DBP domains with soluble anti-DBP. Panel B: overlay plot of response curves obtained from the original sensorgrams examining U937 cell binding to the indicated DBP domain.

Figure 5

Cell binding capacity of domain I DBP peptides. Panel A: U937 cells (10⁶/ml) in HBSS were pretreated with either buffer (control), 0.2 µM of native DBP, 0.2 µM DIp1 (amino acids 130–152) or 0.2 µM DIp2 (amino acids 150–172) for 15 min at 22°C. Cells were then injected into the flow cell to interact with immobilized DBP. Data is presented as an overlay plot of response curves obtained from the original sensorgrams. Panel B: Purified domain I peptides (DIp1 and DIp2) were individually coupled to separate CM5 sensor chips as described in Experimental Procedures. Data is shown as an overlay plot of sensorgrams examining U937 cell binding to the indicated domain I DBP peptides.

Figure 6

Cell binding capacity of DBP domain III sequences. Panel A: overlay plot of response curves obtained from the original sensorgrams examining U937 cell binding to the indicated DBP domain. Panel B: U937 cells (10⁶/ml) in HBSS were pretreated with either buffer (control), 0.2 µM DIIIp1 (amino acids 378–390), 0.2 µM DIIIp2 (amino acids 392–412) or 0.2 µM of both peptides (DIIIp1+2) for 15 min at 22°C. Cells were then injected into the flow cell to interact with immobilized DII + ΔDIII (192–402). Data is presented as an overlay plot of response curves obtained from the original sensorgrams.

Figure 7

Schematic representation of the DBP domain structure with proposed cell binding regions. The 458 amino acid sequence of human DBP with the three structural domains and known functional regions indicated. Domain I: amino acids 1–191; domain II: 192–378; domain III: 379–458; vitamin D binding: 35–49; C5a chemotactic cofactor: 130–149; cell binding #1 (cb1): 150–172; G-actin binding: 373–403; cell binding #2 (cb2): 379–402. The domain and functional regions are drawn approximately to scale.