Identification of a region in the vitamin D-binding protein that mediates its C5a chemotactic cofactor function.
The vitamin D-binding protein (DBP), also known as group-specific component or Gc-globulin, is a multifunctional plasma protein that can significantly enhance the leukocyte chemotactic activity to C5a and C5a des-Arg. DBP is a member of the albumin gene family and has a triple domain modular structure with extensive disulfide bonding that is characteristic of this protein family. The goal of this study was to identify a region in DBP that mediates the chemotactic cofactor function for C5a. Full-length and truncated versions of DBP (Gc-2 allele) were expressed in Escherichia coli using a glutathione S-transferase fusion protein expression system. The structure of the expressed proteins was confirmed by SDS-PAGE and immunoblotting, whereas protein function was verified by quantitating the binding of [(3)H]vitamin D. Dibutyryl cAMP-differentiated HL-60 cells were utilized to test purified natural DBP and recombinant expressed DBP (reDBP) for their ability to enhance chemotaxis and intracellular Ca(2+) flux to C5a. Natural and full-length reDBP (458 amino acid residues) as well as truncated reDBPs that contained the N-terminal domain I (domains I and II, residues 1-378; domain I, residues 1-191) significantly enhanced both cell movement and intracellular Ca(2+) concentrations in response to C5a. Progressive truncation of DBP domain I localized the chemotactic enhancing region between residues 126-175. Overlapping peptides corresponding to this region were synthesized, and results indicate that a 20-amino-acid sequence (residues 130-149, 5'-EAFRKDPKEYANQFMWEYST-3') in domain I of DBP is essential for its C5a chemotactic cofactor function.
Zhang
Jianhua
J
Department of Pathology, Stony Brook University School of Medicine, Stony Brook, NY 11794-8691, USA.
Kew
Richard R
RR
D000483:Alleles; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry; D002454:Cell Differentiation; D002465:Cell Movement; D002633:Chemotaxis; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D014809:Vitamin D-Binding Protein / Q000378:metabolism / Q000502:physiology*
C488033:C5AR1 protein, human; D004220:Disulfides; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014809:Vitamin D-Binding Protein; D005982:Glutathione Transferase; D002118:Calcium