Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3

doi:10.1016/j.molimm.2005.07.023

Molecular Immunology

Volume 43, Issue 8, March 2006, Pages 1109-1115

https://doi.org/10.1016/j.molimm.2005.07.023 Get rights and content

Abstract

The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D₃, and G-actin plus 25(OH)D₃ bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)₂D₃) completely eliminated the chemotactic cofactor function of DBP. Dose–response curves demonstrated that as little as 1 pM 1,25(OH)₂D₃ significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)₂D₃ needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B₄ was not altered by 1,25(OH)₂D₃, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)₂D₃. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)₂D₃.

Introduction

Leukocytes are recruited to sites of inflammation by numerous chemoattractants, but one of the most potent and early-acting chemotactic factors is complement activation peptide C5a (and its stable serum form C5a des Arg) (Guo and Ward, 2005). The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the Vitamin D binding protein (DBP), a plasma protein also known as Gc-globulin (Binder et al., 1999, Kew and Webster, 1988, Metcalf et al., 1991, Perez et al., 1988, Zwahlen and Roth, 1990). DBP is a multifunctional and highly polymorphic plasma protein synthesized primarily in the liver, and has a molecular mass of approximately 56 kDa and circulates in plasma at 6–7 μM (Gomme and Bertolini, 2004, White and Cooke, 2000). DBP is a member of the albumin (ALB), α-fetoprotein (AFP), and α-albumin/afamin (AFM) gene family and has the characteristic multiple disulfide-bonded, triple domain modular structure (White and Cooke, 2000). Besides functioning as a circulating Vitamin D transport protein, it has been demonstrated that plasma DBP effectively scavenges G-actin released at sites of necrotic cell death and prevents polymerization of actin in the circulation (Gomme and Bertolini, 2004, White and Cooke, 2000). Distinct binding regions within the 458 amino acid sequence of DBP have been identified. Analysis of the crystal structure of DBP (bound to either Vitamin D₃ or actin) has confirmed previous studies that the Vitamin D sterol binding segment resides in the N-terminal domain (amino acids 35–49) (Haddad et al., 1992, Swamy et al., 1997), and also revealed that actin interacts with distinct amino acid sequences in all the three DBP domains (Head et al., 2002, Otterbein et al., 2002, Swamy et al., 2002, Verboven et al., 2002). Recently, our lab has identified a C5a chemotactic cofactor region in the N-terminal domain (amino acids 130–149), a sequence distinct from either the Vitamin D or G-actin binding regions (Zhang and Kew, 2004).

The precise mechanisms by which DBP acts as a chemotactic cofactor for C5a (co-chemotactic activity) are not known. However, previous studies have shown that neutrophils transiently generate co-chemotactic activity for C5a/C5a des Arg on the cell surface within 15–20 min of DBP binding (Kew et al., 1995a). These cells also utilize membrane-bound elastase to shed the DBP-binding site into the extracellular milieu (DiMartino et al., 2001). Both plasma membrane binding and subsequent shedding of DBP are essential for the protein to function as a chemotactic cofactor for C5a. Recent studies from our lab also have shown that DBP requires platelet-derived thrombospondin-1 for maximal co-chemotactic activity (Trujillo and Kew, 2004), and that cell surface CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP (McVoy and Kew, 2005). Nevertheless, a fundamental question that remains is what effect does ligation of DBP with its primary physiological ligands, Vitamin D and G-actin, have on the C5a chemotactic cofactor activity. The results demonstrate that DBP bound to G-actin, 25(OH)D₃ or G-actin plus 25(OH)D₃ did not alter either its binding to neutrophils or its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)₂D₃) bound to DBP completely eliminated the C5a chemotactic cofactor function and this inhibition requires cell surface alkaline phosphatase (AP) activity.

Section snippets

Reagents

Purified human Vitamin D binding protein was purchased from Biodesign International (Kennebunkport, ME). Purified recombinant human C5a, formyl norleucyl-leucyl-phenylalanine (fNLP), leukotriene B₄ (LTB₄) and zymosan A (yeast cell walls from S. cerevisiae) were purchased from Sigma-Aldrich (St. Louis, MO). Complement-activated serum was generated by incubating 1 ml of human serum with 10 mg zymosan A for 1 h at 37 °C. Particulate matter was removed by centrifugation (15,000 × g) and samples were

Results

Previous work from our laboratory has demonstrated that the binding of DBP to neutrophils is essential for the chemotaxis enhancement of C5a (Kew et al., 1995a, Kew et al., 1995b). More recent studies have shown that the region of DBP that mediates co-chemotactic activity resides in its N-terminal domain, distinct from the Vitamin D sterol or G-actin binding regions (Zhang and Kew, 2004). Therefore, to determine if ligation of DBP with either Vitamin D and/or G-actin alters the ability to bind

Discussion

This paper reports three major findings: (1) cell surface binding and co-chemotactic activity of DBP are not affected when bound to G-actin, Vitamin D or both ligands; (2) the hormonally active form of Vitamin D (1,25(OH)₂D₃), when bound to DBP, completely inhibits co-chemotactic activity; (3) 1,25(OH)₂D₃ may utilize cell surface alkaline phosphatase activity to inhibit the chemotactic cofactor function. The first conclusion provides a functional correlate to our recent study that identified a

Acknowledgements

This investigation was supported in part by National Institutes of Health grant GM 63769 (to R.R.K.). A.B.S. was supported in part by the M.D. with Recognition in Research Program, Stony Brook University School of Medicine. S.J.D. was supported in part by a Medical Scientist Training Program (MSTP) grant from the National Institutes of Health. G.T. was supported by a W. Burghardt Turner Fellowship and the National Science Foundation-funded AGEP Program (both at Stony Brook University), and by a

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Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3

Abstract

Introduction

Section snippets

Reagents

Results

Discussion

Acknowledgements

Mol. Immunol.

Trends Biotechnol.

Curr. Biol.

J. Biol. Chem.

Arch. Biochem. Biophys.

Trends Endocrinol. Metab.

J. Biol. Chem.

Granules and secretory vesicles of the human neutrophil

Clin. Exp. Immunol.

Vitamin D

Am. J. Physiol.

Functions of lipid rafts in biological membranes

Annu. Rev. Cell Dev. Biol.

Vitamin D binding protein (Gc-globulin)

Endocr. Rev.

Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane. Evidence for a chondroitin sulfate proteoglycan

J. Immunol.

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

J. Immunol

Steroid receptor co-activator-1 mediates 1,25-dihydroxyvitamin D(3)-stimulated alkaline phosphatase in human osteosarcoma cells

Calcified Tissue Int.

The role of annexin 2 in osteoblastic mineralization

J. Cell Sci.

Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis

J. Cell Biol.

Role of C5a in inflammatory responses

Annu. Rev. Immunol.

Identification of the sterol- and actin-binding domains of plasma Vitamin D binding protein (Gc-globulin)

Biochemistry

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution

Biochemistry

Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)₂ Vitamin D₃