Biomedical subjects

B Seetharam

Publications and source records attributed to B Seetharam.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vitro and in vivo inactivation of transcobalamin II receptor by its antiserum.

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.

Animals↗

PubMed1996

Expression of insulin-like growth factor (IGF)-I receptors, IGF-II/cation-independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells.

We have analyzed the surface distribution and functional expression of the insulin-like growth factor I (IGF-I) receptor and the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CI-MPR) in the polarized human colon adenocarcinoma cell line, Caco 2. Domain-selective biotinylation of the apical and basolateral surfaces of Caco-2 cells grown on filter supports revealed a 3-4-fold enrichment of these receptors on basolateral membranes. In addition, the biotinylation studies revealed the presence of the cation-dependent MPR on both membrane surfaces, with a 3.4-fold enrichment on basolateral membranes. Binding of 125I-IGF-I at 4 degrees C confirmed similar higher levels of expression of the IGF-I receptor at the basolateral surface than at the apical surface. Cell surface-specific binding of the iodinated lysosomal enzyme beta-glucuronidase was detected at 4 degrees C on both plasma membrane domains. However, significant uptake of beta-glucuronidase at 37 degrees C was observed only from the basolateral surface. These results indicate that the MPRs and the IGF-I receptor are expressed in a polarized fashion in Caco-2 cells and that the IGF-II/CI-MPR present on apical membranes, unlike the IGF-II/CI-MPR expressed on the basolateral surface, is not functional in endocytosing lysosomal enzymes.

Caco-2 Cells↗

PubMed1995

Regulation of expression of transcobalamin II receptor in the rat.

Surface and intracellular membrane distribution and hormonal regulation of transcobalamin II receptor (TC II-R) activity and protein levels have been studied in an effort to understand its regulation of expression in the rat. TC II-R activity and the levels of the 62 kDa monomeric and 124 kDa dimeric forms of TC II-R were highest in the rat kidney and intestine, and in these tissues the receptor expression was not dependent upon the postnatal development of the rat. TC II-R expression was uniform in the various regions of the gut. Surface membrane distribution of TC II-R in the kidney revealed the expression of the 124 kDa dimer form of TC II-R in the apical and basolateral membranes in the ratio of 1:10. Further subcellular distribution of TC II-R in the kidney revealed the expression of the 124 kDa dimer in the intermicrovillar clefts and clathrin-coated vesicles and the 62 kDa monomer in the microsomes. Neither the monomer nor the dimer could be detected in the early endosomes or lysosomes. Membrane TC II-R activity and TC II-R protein levels and cobalamin (Cbl; vitamin B12) transport in vivo were inhibited by about 90% in adrenalectomized rats and all three returned to normal levels by oral treatment of these animals with cortisone acetate. In contrast, thyroidectomy or experimentally induced diabetes had no effect on TC II-R activity or Cbl transport. Based on these observations, we suggest that TC II-R expression is not developmentally or regionally regulated in rat renal and intestinal membranes and its expression in the kidney is asymmetrically distributed between the apical (10%) and basolateral (90%) membranes. In addition, our results also show that the dimerization of TC II-R is a post-microsomal event and that the expression of TC II-R and plasma Cbl transport is regulated by cortisone.

Adrenalectomy↗

PubMed1995

Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.

Base Sequence↗

PubMed1995

Membrane expression and interactions of human transcobalamin II receptor.

Antiserum raised to purified 62-kDa human placental transcobalamin II receptor (TC II-R) has been used to study its synthesis and membrane expression. The antiserum immunoprecipitated a 45-kDa protein from the cell-free translation using human kidney mRNA and recognized a single 124-kDa band on immunoblotting of placental and other human tissue membranes, and quantitation of the blots revealed high levels of TC II-R expression in the human kidney followed by placenta, intestine, and liver. Triton X-100 extraction of placental membranes resulted in the complete (100%) solubilization of the receptor, and immunoblotting of the Triton X-100-soluble fraction revealed a single band of 62 kDa. Lipid extraction of placental membranes with a mixture of chloroformmethanol (2:1) followed by immunoblotting revealed a single band of molecular mass 62 kDa. The molecular mass of the pure Triton X-100-bound receptor increased on SDS-polyacrylamide gel electrophoresis from 62 to 124 kDa upon its insertion in liposomes prepared using egg phosphatidylcholine and cholesterol. Chemical cross-linking of native membrane-or lipid vesicle-bound TC II-R or detergent-soluble extracts of the membrane with 125I-TC II-cobalamin revealed that both the 124- and 62-kDa forms of the receptor were active in ligand binding. Based on these results we suggest that TC II-R is synthesized as a single polypeptide of 45 kDa, and following its maturation (involving N- and O-glycosylation) the 62-kDa mature receptor is expressed in plasma membranes as a noncovalent dimer of 124 kDa. The dimerization of TC II-R in the plasma membranes is due to its interactions with annular lipids.

Biopolymers↗

PubMed1995

Genomic structure of human transcobalamin II: comparison to human intrinsic factor and transcobalamin I.

Human transcobalamin II (TC II) gene was isolated and partially sequenced. The gene is composed of nine exons and eight introns spanning approximately 20 kb. Multiple potential transcription start sites were revealed by primer extension analysis. The 5'-flanking region of the gene contained no TATA-like motif, but a binding motif for HIP1, which is suggested to be important in the transcription of TATA-less housekeeping genes, was identified in a region very close to the initiator methionine codon. In addition, potential binding sites for a variety of transcription factors such as SP1, AP2, CF1, NF-IL6, Ets-1, Myb and E2A were also observed. Comparison of the genomic structure of TC II to other Cbl-binding proteins, human gastric intrinsic factor (IF) and transcobalamin I (TC I) revealed similar intron-exon organizations with respect to the number, position and size of exons. These results suggest that TC II, TC I and IF genes have originated by gene duplications of an ancestral gene and TC II, unlike the other two Cbl-binding proteins, is the product of a "housekeeping" gene.

Amino Acid Sequence↗

PubMed1995

Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells.

The mitogenic and metabolic effects of insulin-like growth factor-II (IGF-II) can be modulated by six distinct IGF binding proteins (IGFBPs). As a first step toward understanding the role of IGFs and their binding proteins in intestinal epithelial cell differentiation, the expression of IGF-II and IGFBPs was characterized in the human colon adenocarcinoma Caco-2 cell line. Northern blot analysis revealed two IGF-II transcripts of 5.4 and 4.5 kb, and ribonuclease protection assays indicated that IGF-II mRNA levels are regulated during Caco-2 differentiation. A specific radioimmunoassay detected IGF-II in serum-free conditioned medium, the level of which was three- to fivefold higher in proliferating cells than in differentiated cells. Immunoprecipitation and ligand blot analyses of conditioned medium demonstrated that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 are synthesized by Caco-2 cells, with IGFBP-2 and IGFBP-4 being the major IGFBPs secreted, and that the levels of IGFBP-2 and IGFBP-6 decreased as differentiation proceeded. These results indicate that the expression of IGF-II, IGFBP-2, and IGFBP-6 is regulated in a differentiation-dependent manner in Caco-2 cells.

Caco-2 Cells↗

PubMed1994

Effect of processing inhibitors on cobalamin (vitamin B12) transcytosis in polarized opossum kidney cells.

The main objectives of the current study were to investigate the effect of tunicamycin and other posttranslational processing inhibitors on the apical brush border expression of intrinsic factor-cobalamin receptor (IFCR) and the apical to basolateral transcytosis of cobalamin (Cbl). Because of the high and selective expression of IFCR in the apical brush border membrane of opossum kidney (OK) cells (K. S. Ramanujam, S. Seetharam, N. Dahms, and B. Seetharam, (1991) J. Biol. Chem. 266, 13135-13140), we have used cultured OK cells to address these issues. When polarized OK cells grown on culture inserts were incubated with tunicamycin, deoxynojirimycin, swainsonine, or cerulenin, the surface binding of the ligand, intrinsic factor-[56Co]Cbl was inhibited by tunicamycin but not by the other inhibitors. However, Cbl transcytosis was inhibited by both tunicamycin and cerulenin but not with deoxynojirimycin or swainsonine. Incubation of cells with tunicamycin decreased the half-life of IFCR from 48 to 24 h, thus causing faster degradation and depletion of the surface receptor. Incubation of cells with cerulenin resulted in the intralysosomal retention of internalized Cbl. Mature receptor labeled with either [35S]methionine or [3H]mannose was sensitive to digestion with both endoglycosidase H and peptide N-glycosidase F and revealed the presence of two or three N-linked oligosaccharides of the high mannose or hybrid type. Metabolic labeling of OK cells with [3H]palmitic acid revealed that IFCR was palmitoylated and the label was sensitive to treatment with hydroxylamine. Based on these results we suggest that IFCR expression in the apical membrane and Cbl transcytosis in polarized OK cells are regulated by core N-glycosylation but not by further processing of the terminal sugars. In addition, we also suggest that the inhibition of Cbl transcytosis by cerulenin is due to inhibition of postinternalization events.

1-Deoxynojirimycin↗

PubMed1994

Nonsense mutations in human transcobalamin II deficiency.

Reverse transcription-polymerase chain reaction has been used to amplify, clone and sequence transcobalamin II (TC II) cDNA from fibroblasts of three unrelated TC II deficient patients who had undetectable TC II protein and mRNA in their fibroblasts (Li et al., Biochem. J, 301, 585-590, 1994). One child of a consanguineous marriage contained a single nucleotide deletion at position 258 in both alleles, while the child from unrelated parents revealed a nonsense mutation at position 1206 in one allele and a single nucleotide deletion at position 483 in the other allele. Both the single nucleotide deletion mutations caused a frameshift and introduced a premature termination codon (indirect nonsense mutations). No mutation was detected in TC II cDNA from the third patient. Based on these results we suggest that TC II deficiency due to lack of TC II protein/mRNA in these patients is due to heterogeneous types of nonsense mutations.

Alleles↗

PubMed1994

Polymorphism of human transcobalamin II: substitution of proline and/or glutamine residues by arginine.

The sequence of transcobalamin II (TC II) cDNA amplified from human fibroblast and colon adenocarcinoma (Caco-2) and the electrophoretic mobility of TC II secreted by these cell lines were analyzed to get some insights into the structural basis for the expression of various polymorphic forms of human TC II. Based on relative anodic mobilities of TC II phenotypes expressed in human serum, TC II expressed in the fibroblast cell line studied and Caco-2 cells were assigned as the MX (medium/extremely slow) and S (slow) types, respectively. Nucleotide sequence analysis of TC II cDNA amplified from these cells revealed that residues Arg and Arg, Gln and Arg, and Gln and Pro were present at positions 234 and 259, respectively, in TC II alleles encoding the X, S and M types. Based on these results, we suggest that differences in the anodic mobilities of the various polymorphic forms of TC II such as the X, S and M types are due to charge difference on the protein caused by the replacement of uncharged residues by arginine at positions 234 and/or 259.

Arginine↗

PubMed1994

Expression of transcobalamin II mRNA in human tissues and cultured fibroblasts from normal and transcobalamin II-deficient patients.

Transcobalamin II (TCII) is an important plasma transporter of cobalamin (Cbl; vitamin B12). In the present study, TCII gene expression in human and rat tissues and in the fibroblasts of patients with TCII deficiency was investigated. Northern-blot analyses revealed expression of TCII mRNA in many human and rat tissues. In humans, this was 14-fold higher in the kidney than in liver, whereas in the rat the levels of expression were similar in the kidney and liver. Southern-blot analysis of genomic DNA from several species revealed sequence similarity in TCII across species. Metabolic labelling and ribonuclease protection assay revealed a 43 kDa TCII protein and a fully protected TCII mRNA band in normal fibroblasts but not in fibroblasts from three TCII-deficient patients. Southern-blot analysis of genomic DNA from all these fibroblasts revealed identical restriction patterns on BamHI, HindIII, KpnI, MspI and EcoRI digestion. On the basis of these results, we suggest that TCII is expressed in multiple tissues, and its level of expression in tissues varies within the same and across species. Furthermore, the TCII deficiency characterized in this study is due to the absence of TCII protein which in turn is due to the absence or extremely low levels of its mRNA and not to detectable gross alterations in the gene structure.

Animals↗

PubMed1994

Identification of two mutant alleles of transcobalamin II in an affected family.

Transcobalamin II (TC II) deficiency is a rare autosomal recessive disease leading to cobalamin (Cbl; Vitamin B12) deficiency characterized by failure to thrive, megaloblastic anemia, impaired immunodefence and neurological manifestations. By means of Southern blotting and sequence analysis of TC II cDNA amplified from fibroblasts of an affected child and his parents, we have identified two mutant TC II alleles, one with a gross deletion and the other with a 4 nucleotide deletion. Both the mutations caused TC II mRNA and protein deficiency and hence defective plasma transport of Cbl and the development of Cbl deficiency in the affected child. The present study has identified molecular defects that cause TC II deficiency and lead to intracellular Cbl deficiency in humans.

Alleles↗

PubMed1993

Regulated expression of intrinsic factor-cobalamin receptor by rat visceral yolk sac and placental membranes.

Intrinsic factor-cobalamin receptor (IFCR) activity in visceral yolk sac and placental membranes is regulated during pregnancy in rats. While the IFCR activity declined in the visceral yolk sac membranes by 15-fold, it rose nearly 20-fold in the placental membranes from fourteen to nineteen days of gestation. The visceral yolk sac membranes revealed a 230 kDa protein that co-migrated with pure rat renal IFCR. This 230 kDa band was also identified as IFCR in both the membranes by immunoblotting with anti-serum to rat renal IFCR. Immunoprecipitation of 35S labeled proteins obtained from in vitro translation using visceral yolk sac mRNA from 14-day pregnant rats, yielded on SDS-PAGE a single band of 220 kDa, while those obtained from 19-day pregnant rats did not. The binding of intrinsic factor-cyano[57Co]cobalamin complex to the visceral yolk sac membranes was inhibited by preincubation of these membranes with anti-serum to rat IFCR but not with anti-serum to rat asialoglycoprotein receptor or mannose or mannan or N-acetylglucosamine. Based on these results, we suggest that the IFCR activity, protein expression and mRNA levels in fetal membranes are regulated during pregnancy and may play an important role in the maternal-fetal transfer of cobalamin.

Animals↗

PubMed1993

Isolation and sequence analysis of variant forms of human transcobalamin II.

Two cDNA clones (1.9 kb and 1.5 kb, respectively) encoding full length human TC II have been isolated from a human endothelial cell cDNA library and sequenced. The differences between the two clones are the length of the 5' end and the 3' end non-coding regions and the codon at position 198 and 219. Both the clones differ from the recently isolated (human endothelial cell) cDNA for TC II (Platica, O., Janecko, R., Quadros, E.V., Regee, A., Romain, R. and Rothenberg, S.P. (1991) J. Biol. Chem. 266, 7860-7863) in codon 259 and 376 and in their calculated pI values. In vitro transcription followed by translation in a reticulocyte lysate system and SDS-PAGE revealed that the isolated cDNA clones encode a protein of 43 kDa. Upon treatment with canine pancreatic microsomes, the molecular mass of the in vitro translated product was reduced to 41.5 kDa, indicating the presence of an approximately 1.5 kDa signal peptide. This translation product was immunoprecipitated with rabbit anti-serum to human TC II and was able to bind to Cbl-Sepharose beads. The amino acid sequence alignment of TC II with that of other Cbl binding proteins (rat intrinsic factor, human transcobalamin I and porcine haptocorrin) revealed only 33% overall homology. However, there were four regions of greater than 80% homology and two regions of about 60% homology. These regions encompass the majority of the hydrophobic areas of the Cbl-binders. Based on these studies, we suggest that structural basis for the expression of different polymorphic forms of TC II may be due to single point mutations and that TC II, like other mammalian Cbl-binders, have evolved from a common ancestral gene. Furthermore, the Cbl-binding functional domain most probably resides in a hydrophobic pocket which is formed by all or some of the six regions of high homology.

Amino Acid Sequence↗

PubMed1993

Intrinsic factor-cobalamin receptor activity in a marsupial, the American opossum (Didelphis virginiana).

1. Significant and specific binding of intrinsic factor-cobalamin occurred in proximal but not in the distal half of the intestine in an adult marsupial, the American opossum. 2. The purified opossum kidney receptor, like rat and canine kidney receptors, revealed a single band of M(r) approximately 230 on SDS-PAGE. However, unlike the rat and canine receptors, the opossum receptor was sensitive to both Endoglycosidase H and peptide-N-glycosidase F. 3. The opossum intrinsic factor-cobalamin receptor demonstrated a ten-fold higher affinity for intrinsic factor-cobalamin complex when the source of IF was from the opossum pancreas, rather than rat stomach. 4. The opossum kidney receptor had low immune-crossreactivity with anti-serum raised to rat and canine kidney receptor. 5. These studies suggest that intrinsic factor-cobalamin receptor expressed in the American opossum, though conserved, appears to be structurally different from the rat and canine receptors.

Animals↗

PubMed1993

Characterization of mannose 6-phosphate receptors (MPRs) from opossum liver: opossum cation-independent MPR binds insulin-like growth factor-II.

Bovine, human, and rat cation-independent mannose 6-phosphate receptors (CI-MPRs) are capable of binding both mannose 6-phosphate and insulin-like growth factor-II (IGF-II). However, the receptor isolated from either chicken or frog lacks the high affinity IGF-II-binding site. To determine whether CI-MPRs isolated from a species that is closely related to placental mammals can bind IGF-II, the MPRs were purified from a marsupial, the American opossum (Didelphis virginiana), by phosphomannan-Sepharose affinity chromatography and then tested for their ability to bind IGF-II. Opossum liver expressed both the CI-MPR and the cation-dependent MPR (CD-MPR). Both receptors contained Asn-linked oligosaccharides. In contrast to CD-MPRs isolated from other species, the opossum CD-MPR displayed heterogeneity with respect to the number of Asn-linked oligosaccharide chains it contains. The CI-MPR isolated from opossum liver, like the CI-MPR from bovine liver, bound iodinated human recombinant IGF-II. However, Scatchard analysis revealed that the opossum CI-MPR bound IGF-II with a lower affinity (Kd = 14.5 nM) than the bovine receptor (Kd = 0.2 nM). The addition of excess IGF-II, but not IGF-I or insulin, inhibited binding to [125I]IGF-II, indicating that the opossum CI-MPR exhibits specificity for IGF-II. These results suggest that the emergence of a high affinity IGF-II-binding site in the CI-MPR occurred in evolution before the divergence of marsupials and placental mammals from their last common ancestor.

Animals↗

PubMed1992

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.

Animals↗

PubMed1992

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.

Ammonium Chloride↗