Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

Maternal intake of dietary methyl-micronutrients (e.g. folate, choline, betaine and vitamin B-12) during pregnancy is essential for normal maternal and fetal methionine metabolism, and is critical for important metabolic processes including those involved in developmental programming. Maternal obesity and nutrient excess during pregnancy influence developmental programming potentially predisposing adult offspring to a variety of chronic health problems. In the present study, we hypothesized that maternal obesity would dysregulate the maternal and fetal methionine cycle. To test this hypothesis, we developed a nulliparous baboon obesity model fed a high fat, high energy diet (HF-HED) prior to and during gestation, and examined methionine cycle biomarkers (e.g., circulating concentrations of homocysteine, methionine, choline, betaine, key amino acids, folate, and vitamin B-12). Animals were group housed allowing full physical activity and social interaction. Maternal prepregnancy percent body fat was 5% in controls and 19% in HF-HED mothers, while fetal weight was 16% lower in offspring of HF-HED mothers at term. Maternal and fetal homocysteine were higher, while maternal and fetal vitamin B-12 and betaine were lower in the HF-HED group. Elevations in circulating maternal folate were evident in the HF-HED group indicating impaired folate metabolism (methyl-trap) as a consequence of maternal vitamin B-12 depletion. Finally, fetal methionine, glycine, serine, and taurine were lower in the HF-HED fetuses. These data show that maternal obesity disturbs the methionine cycle in primate pregnancy, providing a mechanism for the epigenetic changes observed among obese pregnant women and suggesting diagnostic and therapeutic opportunities in human pregnancies complicated by obesity.

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“…Holotranscobalamin: Plasma holotranscobalamin was measured by the monoclonal antibody capture assay (Ulleland et al 2002).…”

Section: Metabolite Measurementsmentioning

confidence: 99%

Maternal obesity disrupts the methionine cycle in baboon pregnancy

et al. 2015

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“…Treatment of recombinant human TC y with CNBr cleaved the protein after the 11 Met residues, and the peptides obtained were named after the corresponding cleavage sites (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). According to the nomenclature used, the elementary peptide 4 corresponded to the fragment between the fourth and the fifth Met residues [MflGQLAL…DTAAM(102-198)].…”

Section: Binding Of Antibodies To Cnbr Peptides On Western Blotmentioning

confidence: 99%

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Fedosov

Örning²,

Løvli³

et al. 2005

The FEBS Journal

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Vitamin B 12 (cobalamin, Cbl) is absorbed in the distal ileum with the help of a specific binding protein intrinsic factor (IF) and appears in the circulation bound to another carrier transcobalamin (TC) [1]. Tissue uptake of the TCAECbl complex (holo-TC) is mediated by specific receptors on the surface of the plasma membrane [2]. Holo-TC represents Cbl available for cellular uptake and a decrease in its level would indicate reduced absorption of the vitamin as well as systemic Cbl deficiency. Two new methods have recently been described for the measurement of holo-TC in plasma samples [3,4]. Both methods employ TC-specific antibodies to capture the protein from plasma but lack the specificity needed for direct measurement of holo-TC in serum. The antigenic determinants and the functional domains of TC have not been identified.Cloning [5][6][7] and recent expression of several kindred Cbl-binding proteins [8][9][10][11] Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA…TAAM or the C-terminal peptide LEPA…LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS…QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA…HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment , which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor. Co]cyano-Cbl; IF, intrinsic factor; RU, resonance unit; SPR, surface plasmon resonance; TC, transcobalamin; TC p , recombinant human transcobalamin produced in a plant system; TC p11 , TC y31 ,…, the proteolytic fragments of TC p and TC y with the indicated molecular mass; TC y , recombinant human transcobalamin produced in yeast.

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“…Total unsaturated B12 binding capacity (UB 12 BC) and that caused by unbound TC (apo-TC) in serum were measured as previously described (Jacob et al, 1977;Djalali et al, 1990;Benhayoun et al, 1993). The fraction of TC saturated with Cbl (holo-TC) was determined using the holo-TC assay kit (ICN diagnostics, Orsay, France) (Ulleland et al, 2002).…”

Section: Methodsmentioning

confidence: 99%

Transcobalamin deficiency due to activation of an intra exonic cryptic splice site

Namour¹,

Helfer²,

Quadros

et al. 2003

Br J Haematol

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Summary. Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

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Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

Cited by 106 publications

References 29 publications

Maternal obesity disrupts the methionine cycle in baboon pregnancy

Maternal obesity disrupts the methionine cycle in baboon pregnancy

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Transcobalamin deficiency due to activation of an intra exonic cryptic splice site

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