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

Abstract

The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.

Crystal structures of the vitamin D-binding protein and its complex with actin: Structural basis of the actin-scavenger system

“…In agreement with a study that used limited proteolysis to identify the sterol-binding site (35), Verboven et al localize this site to the N terminus of DBP (34). Such a location is distal to the actin-binding interface identified in this work, which provides a structural understanding of previous reports that the sterol-binding and actin-binding properties of DBP are independent (35)(36)(37)(38). It also seems reasonable to propose that the large structural differences between DBP and HSA are directed toward its actin-binding function, which involves specific interactions with all three domains of DBP and not toward its sterol-binding property, which is confined to the first four ␣-helices of DBP.…”

“…The structure of DBP complexed with a vitamin D metabolite has been reported very recently (34). In agreement with a study that used limited proteolysis to identify the sterol-binding site (35), Verboven et al localize this site to the N terminus of DBP (34). Such a location is distal to the actin-binding interface identified in this work, which provides a structural understanding of previous reports that the sterol-binding and actin-binding properties of DBP are independent (35)(36)(37)(38).…”

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

“…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 D 3 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 three DBP domains 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).…”

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

“…A 1.8-kb SalI-XhoI fragment containing the PGK-promoter/neomycin phosphotransferase (PGK-neo r ) cassette was blunt-end ligated into exon 5 of the mDBP fragment at the BamHI site to generate pDBPN5. To shorten the construct, pDBPN5 was digested with SalI and KpnI by removing a 2.3-kb fragment containing exons 2 and 3 generating pDBPN5 (4)(5)(6)(7)(8). The diphtheria toxin A chain gene (DTA) cassette (17) (generous gift from Barbara Knowles, The Jackson Laboratory, Bar Harbor, Maine, USA) on a 2.7-kb SalI-KpnI fragment was ligated to the KpnI-SalI pDBPN5(4-8) fragment, and circularized recombinants were selected.…”