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J. Biol. Chem., Vol. 279, Issue 51, 53282-53287, December 17, 2004

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Identification of a Region in the Vitamin D-binding Protein that Mediates Its C5a Chemotactic Cofactor Function^*

Jianhua Zhang and Richard R. Kew

From the Department of Pathology, Stony Brook University School of Medicine, Stony Brook, New York 11794-8691

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 [³H]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 Ca²⁺ 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 Ca²⁺ 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.

Received for publication, October 7, 2004

^* This work was supported by National Institutes of Health Grant GM 63769 (to R. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 631-444-3941; Fax: 631-444-3424; E-mail: rkew{at}notes.cc.sunysb.edu.

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