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Mouse transcobalamin has features resembling both human transcobalamin and haptocorrin.

Hygum K, Lildballe DL, Greibe EH, Morkbak AL, Poulsen SS, Sorensen BS, Petersen TE, Nexo E - PLoS ONE (2011)

Bottom Line: Subsequent sequencing identified the protein as TC.Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney.Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

ABSTRACT
In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

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Protein purity and Western blot analyses.(A) 12% reduced SDS-PAGE showing purity of the purified Cbl-binding protein from mouse submaxillary glands. Lane 1) marker; Lane 2) purified mouse TC (see Materials and Methods for details). (B) Western blot employing anti-mouse antibodies on proteins separated by 10% native PAGE. Lane 1) approx. 60 fmol purified mouse TC (approx. 40kDa); Lane 2) extract of the Cbl-binder from mouse submaxillary glands corresponding to a total amount of Cbl+UB12BC of 64 fmol; Lane 3) mouse serum corresponding to a total amount of Cbl+UB12BC of 72 fmol.
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pone-0020638-g002: Protein purity and Western blot analyses.(A) 12% reduced SDS-PAGE showing purity of the purified Cbl-binding protein from mouse submaxillary glands. Lane 1) marker; Lane 2) purified mouse TC (see Materials and Methods for details). (B) Western blot employing anti-mouse antibodies on proteins separated by 10% native PAGE. Lane 1) approx. 60 fmol purified mouse TC (approx. 40kDa); Lane 2) extract of the Cbl-binder from mouse submaxillary glands corresponding to a total amount of Cbl+UB12BC of 64 fmol; Lane 3) mouse serum corresponding to a total amount of Cbl+UB12BC of 72 fmol.

Mentions: The Cbl-binding protein was purified to homogeneity from an extract of 300 glands in a one-step procedure by affinity chromatography employing Cbl coupled to sepharose. A total of approx. 0.3 mg protein was purified with a purification recovery of approx. 85%. As judged from reduced SDS-PAGE, the purified protein had an approx. 40 kDa molecular mass (Figure 2A), which is similar to the molecular mass of human TC.


Mouse transcobalamin has features resembling both human transcobalamin and haptocorrin.

Hygum K, Lildballe DL, Greibe EH, Morkbak AL, Poulsen SS, Sorensen BS, Petersen TE, Nexo E - PLoS ONE (2011)

Protein purity and Western blot analyses.(A) 12% reduced SDS-PAGE showing purity of the purified Cbl-binding protein from mouse submaxillary glands. Lane 1) marker; Lane 2) purified mouse TC (see Materials and Methods for details). (B) Western blot employing anti-mouse antibodies on proteins separated by 10% native PAGE. Lane 1) approx. 60 fmol purified mouse TC (approx. 40kDa); Lane 2) extract of the Cbl-binder from mouse submaxillary glands corresponding to a total amount of Cbl+UB12BC of 64 fmol; Lane 3) mouse serum corresponding to a total amount of Cbl+UB12BC of 72 fmol.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105116&req=5

pone-0020638-g002: Protein purity and Western blot analyses.(A) 12% reduced SDS-PAGE showing purity of the purified Cbl-binding protein from mouse submaxillary glands. Lane 1) marker; Lane 2) purified mouse TC (see Materials and Methods for details). (B) Western blot employing anti-mouse antibodies on proteins separated by 10% native PAGE. Lane 1) approx. 60 fmol purified mouse TC (approx. 40kDa); Lane 2) extract of the Cbl-binder from mouse submaxillary glands corresponding to a total amount of Cbl+UB12BC of 64 fmol; Lane 3) mouse serum corresponding to a total amount of Cbl+UB12BC of 72 fmol.
Mentions: The Cbl-binding protein was purified to homogeneity from an extract of 300 glands in a one-step procedure by affinity chromatography employing Cbl coupled to sepharose. A total of approx. 0.3 mg protein was purified with a purification recovery of approx. 85%. As judged from reduced SDS-PAGE, the purified protein had an approx. 40 kDa molecular mass (Figure 2A), which is similar to the molecular mass of human TC.

Bottom Line: Subsequent sequencing identified the protein as TC.Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney.Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.

ABSTRACT
In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species.

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