Affinity Purification of Human Plasma Vitamin-D-Binding Protein

Swamy, Narasimha; Roy, Arnab; Chang, Roger L.; Brisson, Marni; Ray, Rahul

doi:10.1006/prep.1995.1023

Cited by 13 publications

(8 citation statements)

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“…Receptor-associated protein (RAP) was produced in Escherichia coli (9); 3 H-25(OH)D 3 was from Amersham Pharmacia, and 25(OH)D 3 was from Dr. A.-M. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). Biotin-25(OH)D 3 was synthesized by coupling 25(OH)D 3 -3-(3Ј-aminopropyl)ether (10) with aminocaproic acid-biotin-4-nitrophenyl ester (Pierce) (11). Sterol-DBP complexes were prepared by incubating DBP with 10 to 100-fold excess labeled or unlabeled 25(OH)D 3 (2).…”

Section: Methodsmentioning

confidence: 99%

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

Nykjaer

Fyfe²,

Kozyraki

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

298

248

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Steroids destined for intracellular metabolic conversion or binding to nuclear receptors are believed to cross cell membranes by passive diffusion. According to this free hormone hypothesis, steroids bound to plasma carrier proteins are inactive because they cannot reach their intracellular targets (1). However, recent data show that carrier proteins may greatly facilitate steroid uptake by endocytosis of steroid-carrier complexes followed by intracellular release of the steroid (2, 3). Megalin, a member of the low density lipoprotein receptor family abundant in kidney proximal tubules, mediates endocytic uptake of complexes between the steroid 25 ( Megalin binds a large number of structurally unrelated ligands, and coreceptors may confer ligand specificity by sequestering and presenting their cargo to megalin (4). For example, intrinsic factorvitamin B 12 complex ) is taken up in the intestine by a tandem receptor-mediated mechanism; the complex is first bound to a receptor, cubilin, anchored to the outer leaflet of the plasma membrane possibly by an amphipathic helix (5), followed by endocytosis of cubilin and its cargo mediated by megalin (6, 7). The pivotal role of intestinal cubilin is underscored by the vitamin B 12 deficiency observed in patients with Imerslund-Gräsbeck disease characterized by defective cubilin incapable of binding IF-B 12 (8). These patients have low molecular weight proteinuria in addition to megaloblastic anemia, indicating dysfunction of cubilin coexpressed with megalin in kidney proximal tubules. However, whereas the role of cubilin in the intestine is well characterized, the physiological role in the kidney remains elusive.Here, we identify cubilin as an important coreceptor in the endocytic pathway for retrieval of 25(OH)D 3 -DBP complexes by megalin-mediated endocytosis in the kidney. We show that absence of cubilin or inhibition of its function markedly reduces cellular uptake of the steroid-carrier complex, and animals or patients lacking functional cubilin are characterized by abnormal vitamin D metabolism. This study identifies patients with mutations in an endocytic pathway that regulates steroid hormone metabolism. Materials and MethodsLigands, Receptors, and Antibodies. DBP was purified from human serum (2). Receptor-associated protein (RAP) was produced in Escherichia coli (9); 3 H-25(OH)D 3 was from Amersham Pharmacia, and 25(OH)D 3 was from Dr. A.-M. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). Biotin-25(OH)D 3 was synthesized by coupling 25(OH)D 3 -3-(3Ј-aminopropyl)ether (10) with aminocaproic acid-biotin-4-nitrophenyl ester (Pierce) (11). Sterol-DBP complexes were prepared by incubating DBP with 10 to 100-fold excess labeled or unlabeled 25(OH)D 3 (2). Uncomplexed steroid was removed by gel filtration or dialysis. Human retinolbinding protein (RBP) was from Dr. G. Alexander (University of Oslo, Norway). Rabbit megalin and cubilin were purified as reported (5).The primary antibodies used were rabbit anti-human DBP and anti-human RBP (Dako), goat anti-h...

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Section: Methodsmentioning

confidence: 99%

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

Nykjaer

Fyfe²,

Kozyraki

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

298

248

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“…Purified human DBP was obtained from commercially available pooled human serum (American Red Cross, Dedham, MA) by a ligand affinity chromatographic method developed in our laboratory [21]. Defatted bovine serum ALB (BSA) and all chemicals were purchased from Sigma-Aldrich, Milwaukee, WI, except 1-14 C-palmitic acid (specific activity 56 mCi/ mmol) which was a product of NEN-DuPont, Boston, MA.…”

Section: Methodsmentioning

confidence: 99%

Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

Swamy

Ray

2008

Bioorganic Chemistry

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Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14 Cpalmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14 C-palmitic acid specifically labeled DBP; but p-nitrophenyl-and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14 C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. KeywordsFatty acid-binding by vitamin D binding protein (DBP) and albumin (ALB); serum transport of fatty acids; affinity labeling analogs of palmitic acid; affinity labeling of fatty acid binding sites of DBP and ALB

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“…Purified human DBP was obtained from commercially available pooled human serum (American Red Cross, Dedham, MA) by a ligand affinity chromatographic method developed in our laboratory [29]. The recombinant C-terminal domain of hDBP (domain III, hDBP 277-458) was expressed in bacteria by our published procedure [25,30].…”

Section: Methodsmentioning

confidence: 99%

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

Swamy

Ray

2008

Biochemical and Biophysical Research Communications

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Serum vitamin D-binding protein (DBP) is structurally very similar to serum albumin (ALB); both have three distinct structural domains and high cysteine-content. Yet, functionally they are very different. DBP possesses high affinity for vitamin D metabolites and G-actin, but ALB does not. It has been suggested that there may be cross-talk among the domains so that binding of one ligand may influence the binding of others. In this study we have employed 2-p-toluidinyl-6-sulfonate (TNS), a reporter molecule that fluoresces upon binding to hydrophobic pockets of DBP. We observed that recombinant domain III possesses strong binding for TNS, which is not influenced by 25-hydroxyvitamin D(3) (25-OH-D(3)), yet TNS fluorescence of the whole protein is quenched by 25-OH-D(3). These results provide a direct evidence of cross-talk among the structural domains of DBP.

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Affinity Purification of Human Plasma Vitamin-D-Binding Protein

Cited by 13 publications

References 0 publications

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

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Affinity Purification of Human Plasma Vitamin-D-Binding Protein

Cited by 13 publications

References 0 publications

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D 3

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D 3

Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

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Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃