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Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.

The multiple physiological properties of vitamin D-binding protein (DBP) include organ-specific transportation of vitamin D(3) and its metabolites, manifested by its ability to bind vitamin D sterols with high affinity. In the present investigation we probed the vitamin D sterol-binding pocket of human DBP with affinity labeling analogs of 25-hydroxyvitamin D(3) ¿25-OH-D(3) and 1, 25-dihydroxyvitamin D(3) ¿1,25(OH)(2)D(3) containing bromoacetate alkylating probe at C-3 (A-ring), C-6 (triene), C-11 (C-ring), and C-19 (exocyclic methylene) of the parent sterol. Competitive binding assays with DBP showed approximately 22-, 68-, and 2000-fold decrease in the binding of 1,25(OH)(2)-D(3)-11-BE, 25-OH-D(3)-3-BE, and 25-OH-D(3)-6-BE, respectively, compared to that seen with 25-OH-D(3), while there was no significant difference in the DBP-binding affinity of 25-OH-D(3)-19-BE and 25-OH-D(3). Surprisingly, ¿(14)C25-OH-D(3)-11-BE and ¿(14)C1, 25(OH)(2)-D(3)-19-BE failed to label DBP despite high-affinity DBP-binding, indicating the absence of any nucleophilic amino acid in the vicinity of their bromoacetate moiety to form a covalent bond, while these analogs are inside the binding pocket. In contrast, ¿(14)C25-OH-D(3)-6-BE and ¿(14)C25-OH-D(3)-3-BE specifically labeled DBP. BNPS-skatole digestion of DBP labeled with ¿(14)C25-OH-D(3)-3-BE or ¿(14)C25-OH-D(3)-6-BE produced two peptides (M(r) 17,400 and 33,840), with radioactivity associated with the N- and C-terminal peptides, respectively, raising the possibility that either different areas of the same vitamin D sterol-binding pocket, or different domains of DBP might be labeled by these analogs. Successful affinity labeling of recombinant domain I (1-203) of DBP with both reagents indicated that different areas of the same vitamin D-binding pocket (domain I) were labeled. These affinity analogs are potentially useful for "mapping" the vitamin D sterol-binding pocket and developing a functional model., (Copyright 2000 Academic Press.)

Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3 beta-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25-OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25-OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25-hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25-OH-D3, the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3, of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE covalently modified the 25-OH-D3-binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3-binding pocket in rDBP.

Biochemical analyses revealed that a thyroxine (T4)-binding protein (TBP) in the blood of some turtles exhibits a high degree of structural homology to mammalian vitamin D-binding protein (DBP), instead of mammalian T4-binding protein (TBG). Like DBP, TBP is rich in Cys (ca. 28/molecule). The 31 NH2-terminal amino acids of TBP show 68% identity to the corresponding region of mammalian (rat, mouse, and human) DBP and one peptide from Lys-C proteolysis shows similar homology to residues 35-49 of DBP (the predicted sterol binding domain). Another peptide shows a 75% identity to a sequence corresponding to residues 134-154 of DBP, but seven others showed no homology to any proteins in GenBank. Digestion with N-glycosidase F reduced the M(r) of TBP by only 4.5K compared with 14K for TBG; T4-binding activity of TBP was unaffected. TBP (and DBP) also behaves as a much smaller molecule (approximately 57 kDa) than TBG (> 70 kDa) by size-exclusion chromatography, despite similarities in their estimated sizes from sodium dodecyl sulfate electrophoresis (approximately 60 kDa). Binding studies confirm that the turtle T4-binding protein likely also represents the major blood DPB. Thus, in the turtle, a single binding protein resembling DBP performs two major transport functions which are normally served by proteins representing two different multigene families in mammals.