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Bellur Seetharam - One of the best experts on this subject based on the ideXlab platform.

  • Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane
    The Journal of Membrane Biology, 2003
    Co-Authors: Raghunatha R Yammani, Shakuntla Seetharam, Nancy M Dahms, Bellur Seetharam
    Abstract:

    Purified human Transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, Ka, of 66 nM and boundmax of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, Transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. 35[S]-TC II complexed with cobalamin (Cbl; Vitamin B12) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of 35[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immunecomplex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.

  • Function and stability of human Transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187
    American Journal of Physiology-Cell Physiology, 2003
    Co-Authors: Seema Kalra, David H. Alpers, Shakuntla Seetharam, Bellur Seetharam
    Abstract:

    The current studies have investigated the role of three disulfide bonds of human Transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When...

  • Transcobalamin II and its cell surface receptor.
    Vitamins & Hormones, 2000
    Co-Authors: Bellur Seetharam
    Abstract:

    Transcobalamin II (TC II), a nonglycoprotein secretory protein of molecular mass 43 kDa, and its plasma membrane receptor (TC II-R), a heavily glycosylated protein with a monomeric molecular mass of 62 kDa, are essential components of plasma cobalamin (Cbl; vitamin B12) transport to all cells. Evidence from studies over the past 10 years has provided some important information on their structure, regulation of expression, and function. Some of the specific findings include (a) identification of the structural relationship of the ligand TC II with other members of the Cbl-binding family of proteins, intrinsic factor (IF) and haptocorrin (HC), (b) regulation of TC II gene expression, (c) molecular basis for human TC II deficiency in patients with a lack of plasma TC II, (d) membrane expression, interactions, and dimerization of TC II-R, and (e) targeting and function of TC II-R in polarized epithelial cells. It is hoped that some of the recent findings presented in this review will provide new insights into the structure and function of these two fascinating proteins and stimulate future research in this area.

  • Purification, membrane expression, and interactions of Transcobalamin II receptor.
    Methods in Enzymology, 1997
    Co-Authors: Santanu Bose, Bellur Seetharam
    Abstract:

    Publisher Summary This chapter discusses purification, membrane expression, and interactions of Transcobalamin II (TCII) receptor. Transcobalamin II receptor activity has been detected in many human tissues. However, owing to unrestricted availability and relatively high levels of TCII-R activity, human placenta has been the primary source for its purification. The major step in the purification of TCII-R is ligand (TCII) affinity chromatography. The source of TCII is rabbit plasma/serum. Transcobalamin II receptor has an unusual property that enables it to remain a dimer even after boiling it with sodium dodecyl sulfate (SDS) buffer both in the presence and absence of a reducing agent. This property has been used to study the expression and distribution of TCII-R monomer and dimer in tissue membranes. Recognition of the unusual property of TCII-R dimers to remain dimers following treatment with SDS and the preparation of monospecific antiserum to TCII-R have enabled the authors to study the tissue expression of TCII-R and the mechanism of its dimerization. With the ease of preparing several hundred micrograms of pure placental TCII-R, combined with the availability of its mono-specific antiserum, further studies on the structure and regulation of expression of this important vitamin B 12 receptor are now possible.

  • In Vitro and in Vivo Inactivation of Transcobalamin II Receptor by Its Antiserum
    Journal of Biological Chemistry, 1996
    Co-Authors: Santanu Bose, Shakuntla Seetharam, Richard A. Komorowski, Brian M. Gilfix, David S. Rosenblatt, Bellur Seetharam
    Abstract:

    Rabbits injected with pure human placental Transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, Transcobalamin II, in their plasma. Exogenously added Transcobalamin II-[57Co]cyanocobalamin bound very poorly (2-5%) to the affected rabbit liver, kidney, and intestinal total or intestinal basolateral membrane extracts relative to the binding by membrane extracts from normal rabbit tissues. The activity was restored to normal values following a wash of affected rabbit tissue membranes with pH 3 buffer containing 200 mM potassium thiocyanate. Immunoblot analysis of normal and affected rabbit kidney and liver total membranes revealed similar amounts of 124-kDa TC II-R dimer protein. The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II-[57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl-dependent enzymes. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes. Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and thus cause severe Cbl deficiency and the noted failure to thrive.

Sheldon P. Rothenberg - One of the best experts on this subject based on the ideXlab platform.

  • congenital Transcobalamin II deficiency due to errors in rna editing
    Blood Cells Molecules and Diseases, 2002
    Co-Authors: Lian Qian, A Regec, Edward V. Quadros, Jacqueline Zittoun, Sheldon P. Rothenberg
    Abstract:

    Transcobalamin II (TCII) is a plasma protein essential for the transport and cellular uptake of vitamin B12 (B12; cobalamin, Cbl). Congenital deficiency of functional TCII is an autosomal recessive genetic disorder that results in clinical B12 deficiency usually within several months following birth. In this report, we describe the molecular basis for TCII deficiency in two patients who developed a megaloblastic anemia in early infancy. The serum of both patients contained immunoreactive TCII that did not bind [57Co]Cbl. The fibroblasts from each patient secreted a similarly nonfunctional TCII, yet full-length TCII transcripts were identified by Northern blot. Overlapping cDNA fragments were generated by reverse transcription–polymerase chain reaction and several mutations were identified in the coding region of the cDNA, one of which was common to both patients. However, amplification of the corresponding regions of the gene from genomic DNA failed to identify these mutations. These findings were confirmed by replicate analyses and support the proposal that a variance in RNA editing is the likely mechanism for the mutations that resulted in the expression of a nonfunctional TCII protein in these patients.

  • Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood
    American Journal of Physiology-Gastrointestinal and Liver Physiology, 1999
    Co-Authors: Edward V. Quadros, A Regec, E.v. Quadros, K. M. Faisal Khan, Sheldon P. Rothenberg
    Abstract:

    This study was designed to identify the cellular component of the intestinal villus where Transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intesti...

  • QUANTITATIVE METHODS FOR MEASUREMENT OF Transcobalamin II
    Methods in Enzymology, 1997
    Co-Authors: Sheldon P. Rothenberg, Edward V. Quadros
    Abstract:

    Publisher Summary This chapter describes analytical methods to quantify Transcobalamin II (TCII) in plasma and other biological fluids. The recombinant TCII has the functional and immunoreactive properties of native TCII. TCII is a plasma protein that binds and transports cobalamin (Cbl, vitamin B 12 ) to tissues where cellular uptake of the TCII-Cbl complex occurs by receptor-mediated endocytosis. There are two major plasma proteins that bind Cbl—TCII and Transcobalamin I (TCI). A third Cbl-binding protein, Transcobalamin III (TCIII), is a minor component of plasma that comprises a small fraction of protein-bound Cbl, because it is rapidly cleared from plasma by the hepatic asialoglycoprotein receptor pathway. Although TCIII and TCI cross-react with an antiserum to TCI, they differ in carbohydrate composition. Most of the unsaturated Cbl-binding protein in normal plasma is apo-TCII. To quantify apo-TCII in plasma, sufficient [ 57 Co]Cbl is added to an aliquot of plasma to saturate both TCII and TCI. The unbound [ 57 Co]Cbl is removed by adsorption to protein-coated charcoal. The bound [ 57 Co]Cbl remaining in the plasma is the total Cbl-binding capacity. The plasma is then treated with QUSO G-32, which adsorbs only the TCII-[57Co]Cbl. The remaining [57Co]Cbl is bound to TCI in the sample.

  • 4 Transcobalamin II and the membrane receptor for the Transcobalamin II-cobalamin complex
    Baillière's Clinical Haematology, 1995
    Co-Authors: Sheldon P. Rothenberg, Edward V. Quadros
    Abstract:

    Summary Transcobalamin II is a plasma protein that binds vitamin B 12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues. The circulating Transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis. A number of genetic abnormalities are characterized either by a failure to express Transcobalamin II or by synthesis of an abnormal protein. These disorders result in cellular cobalamin deficiency and megaloblastic anaemia. In this chapter we review the structural and functional properties of Transcobalamin II, the receptor for the Transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating Transcobalamin II. In addition, we provide emerging data about the molecular genetics of Transcobalamin II which has emanated from our own and other laboratories.

Edward V. Quadros - One of the best experts on this subject based on the ideXlab platform.

  • congenital Transcobalamin II deficiency due to errors in rna editing
    Blood Cells Molecules and Diseases, 2002
    Co-Authors: Lian Qian, A Regec, Edward V. Quadros, Jacqueline Zittoun, Sheldon P. Rothenberg
    Abstract:

    Transcobalamin II (TCII) is a plasma protein essential for the transport and cellular uptake of vitamin B12 (B12; cobalamin, Cbl). Congenital deficiency of functional TCII is an autosomal recessive genetic disorder that results in clinical B12 deficiency usually within several months following birth. In this report, we describe the molecular basis for TCII deficiency in two patients who developed a megaloblastic anemia in early infancy. The serum of both patients contained immunoreactive TCII that did not bind [57Co]Cbl. The fibroblasts from each patient secreted a similarly nonfunctional TCII, yet full-length TCII transcripts were identified by Northern blot. Overlapping cDNA fragments were generated by reverse transcription–polymerase chain reaction and several mutations were identified in the coding region of the cDNA, one of which was common to both patients. However, amplification of the corresponding regions of the gene from genomic DNA failed to identify these mutations. These findings were confirmed by replicate analyses and support the proposal that a variance in RNA editing is the likely mechanism for the mutations that resulted in the expression of a nonfunctional TCII protein in these patients.

  • Transcobalamin II synthesized in the intestinal villi facilitates transfer of cobalamin to the portal blood
    American Journal of Physiology-Gastrointestinal and Liver Physiology, 1999
    Co-Authors: Edward V. Quadros, A Regec, E.v. Quadros, K. M. Faisal Khan, Sheldon P. Rothenberg
    Abstract:

    This study was designed to identify the cellular component of the intestinal villus where Transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intesti...

  • QUANTITATIVE METHODS FOR MEASUREMENT OF Transcobalamin II
    Methods in Enzymology, 1997
    Co-Authors: Sheldon P. Rothenberg, Edward V. Quadros
    Abstract:

    Publisher Summary This chapter describes analytical methods to quantify Transcobalamin II (TCII) in plasma and other biological fluids. The recombinant TCII has the functional and immunoreactive properties of native TCII. TCII is a plasma protein that binds and transports cobalamin (Cbl, vitamin B 12 ) to tissues where cellular uptake of the TCII-Cbl complex occurs by receptor-mediated endocytosis. There are two major plasma proteins that bind Cbl—TCII and Transcobalamin I (TCI). A third Cbl-binding protein, Transcobalamin III (TCIII), is a minor component of plasma that comprises a small fraction of protein-bound Cbl, because it is rapidly cleared from plasma by the hepatic asialoglycoprotein receptor pathway. Although TCIII and TCI cross-react with an antiserum to TCI, they differ in carbohydrate composition. Most of the unsaturated Cbl-binding protein in normal plasma is apo-TCII. To quantify apo-TCII in plasma, sufficient [ 57 Co]Cbl is added to an aliquot of plasma to saturate both TCII and TCI. The unbound [ 57 Co]Cbl is removed by adsorption to protein-coated charcoal. The bound [ 57 Co]Cbl remaining in the plasma is the total Cbl-binding capacity. The plasma is then treated with QUSO G-32, which adsorbs only the TCII-[57Co]Cbl. The remaining [57Co]Cbl is bound to TCI in the sample.

  • 4 Transcobalamin II and the membrane receptor for the Transcobalamin II-cobalamin complex
    Baillière's Clinical Haematology, 1995
    Co-Authors: Sheldon P. Rothenberg, Edward V. Quadros
    Abstract:

    Summary Transcobalamin II is a plasma protein that binds vitamin B 12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues. The circulating Transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis. A number of genetic abnormalities are characterized either by a failure to express Transcobalamin II or by synthesis of an abnormal protein. These disorders result in cellular cobalamin deficiency and megaloblastic anaemia. In this chapter we review the structural and functional properties of Transcobalamin II, the receptor for the Transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating Transcobalamin II. In addition, we provide emerging data about the molecular genetics of Transcobalamin II which has emanated from our own and other laboratories.

L Kierat - One of the best experts on this subject based on the ideXlab platform.

  • indication against genetic localisation of the human Transcobalamin II gene tc2 on chromosome 16
    Clinical Genetics, 2008
    Co-Authors: Martin Gallmann, Marijke Fraterschroder, Wolfgang Scheffrahn, Jurg Ott, Balthasar Schmid, Esther Butler, Vreni Biedermann, L Kierat
    Abstract:

    The genetic locus of human Transcobalamin II (TC2) is not yet known. The mouse Transcobalamin II gene has been assigned to mouse chromosome 11, linked to hemoglobin A. This fact suggested a similar linkage of Transcobalamin II in man, assigning it thus to human chromosome 16. Our linkage investigation in a family material of more than 600 individuals demonstrated absence of linkage between Transcobalamin II and phosphoglycolate phosphatase, which is very closely linked to hemoglobin A on chromosome 16. Additionally we confirmed absence of linkage with the chromosome 16 gene marker system haptoglobin. These two gene marker systems are located far from each other, and the total length of chromosome 16 is estimated only about 100 cM. Together with recent results of investigations in somatic mouse-man cell hybrids, we conclude that TC2 is not located on chromosome 16. Additionally we found absence of linkage between Transcobalamin II and 6-phosphoglucona-te dehydrogenase, rhesus blood group (both on chromosome 1), GC (chromosome 4), Esterase D (chromosome 13) and AG; absence of close linkage with “debrisoquin polymorphism”.

Richard C. Chu - One of the best experts on this subject based on the ideXlab platform.

  • synthesis and secretion of Transcobalamin II by cultured astrocytes derived from human brain tissue
    Journal of the Neurological Sciences, 1994
    Co-Authors: James A Begley, Pamela D. Colligan, Richard C. Chu
    Abstract:

    Astrocytes derived from human brain tissue secreted a single cobalamin (vitamin B12, Cbl) binding protein over a 4 day period in culture. Cycloheximide reversibly inhibited the release, and the binding protein was identified as Transcobalamin II (TCII) based on molecular size, reaction with anti-human TCII antiserum, precipitation with 2.0 M ammonium sulfate and its ability to bind radioactive cyanocobalamin. It also enhanced the cellular incorporation of the vitamin. Our data show that cultured cells from human brain synthesize and secrete TCII and suggests that at least some of the TCII known to be present in cerebrospinal fluid may originate from within the central nervous system.

  • Transcobalamin II Mediated Delivery of Albumin-Bound Hydroxocobalamin to Human Liver Cells
    Experimental Biology and Medicine, 1993
    Co-Authors: James A Begley, Pamela D. Colligan, Richard C. Chu
    Abstract:

    AbstractWe show that hydroxocobalamin bound to human serum albumin can dissociate and bind to Transcobalamin II present in serum. Human liver cells in culture exposed to hydroxocobalamin bound to albumin incorporated less of the vitamin than when similar amounts of unbound hydroxocobalamin or cyanocobalamin were present. In the presence of Transcobalamin II, a 4.5-fold increase in cellular uptake occurred, but this amount was less than when hydroxocobalamin or cyanocobalamin were added to Transcobalamin II. These results indicate that albumin, by binding hydroxocobalamin, can alter the dynamics of binding to Transcobalamin II and the subsequent cellular incorporation of this form of the vitamin.