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Biomedical subjects

K Panneerselvam

Publications and source records attributed to K Panneerselvam.

At least 19 recordsLinked to original sources
PubMed2001

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Amino Acid Sequence↗
PubMed2001

A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses.

We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

Acute Disease↗
PubMed2000

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG).

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.

Carbohydrate Metabolism, Inborn Errors↗
PubMed2000

Structure determination of porcine haemoglobin.

To investigate a potential candidate material for making artificial red blood cells to supplement blood transfusion, the X-ray structure of porcine haemoglobin at 1.8 A resolution was determined as part of research towards synthesizing human blood. Porcine haemoglobin was crystallized by the vapor-diffusion method, producing crystals of dimensions 0.3-0.5 mm after successive seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.10, b = 72.27, c = 114.85 A. The initial phase was determined by the molecular-replacement method, using human oxyhaemoglobin as a model. The final R factor was 21.1% for 36 820 reflections after validation of 574 water molecules. The r.m.s. deviations of bond lengths, angles, torsion angles and improper angles from their ideal values are 0.017 A, 3.0, 20.6 and 1.8 degrees, respectively. The average B factor is 33.63 A(2) for the haemoglobin molecule and 50.53 A(2) for the water molecules. The structure could be superimposed on a 2.8 A resolution structure with an r.m.s. difference of 0.59 A in main-chain atomic positions and 1. 27 A in side-chain atomic positions. Porcine and human haemoglobins are compared. A tentative model for artificial blood is proposed based on the complementarity relationship of the surface charges between haemoglobin and the surrounding cell membrane.

Amino Acid Sequence↗
PubMed2000

Crystallization of agglutinin from the seeds of Abrus precatorius.

Agglutinin protein purified from the seeds of Abrus precatorius has a high antitumour activity and was crystallized at room temperature with polyethylene glycol 8000 as the precipitant. The agglutinin crystal diffracted to 3.45 A and belongs to one of two possible tetragonal space groups, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 141.91, c = 105.63 A. The asymmetric unit contains a heterotetrameric protein molecule of molecular weight 134 kDa and has a solvent content of approximately 38%.

Crystallization↗
PubMed1998

Direct utilization of mannose for mammalian glycoprotein biosynthesis.

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2-3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic-derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.

Administration, Oral↗
PubMed1997

Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose.

Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI). The discovery of mammalian mannose-specific transporters that function at physiological concentrations suggested that mannose might directly contribute to oligosaccharide synthesis. To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-3H]mannose or [1,5,6-3H]glucose at the same specific activity, and the N-linked chains were released by PNGase F digestion. Most of the trichloroacetic acid-precipitable [3H]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid-precipitable material was released from cells labeled with [1,5,6-3H]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-3H]mannose contributed 65-75% of the [3H]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose. Mannose consumption and [2-3H]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-3H]mannose is used for glycosylation, while the rest (86-93%) is catabolized to 3H2O via PMI. Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-3H]mannose into 3H2O. Long term labeling of cells with [2-3H]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-3H]mannose added to the medium. These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis. PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis.

Biological Transport↗
PubMed1997

Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis.

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

Alleles↗
PubMed1997

Consequences of disrupting the gene that encodes alpha-glucosidase II in the N-linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum.

We have identified and disrupted the gene coding for alpha-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two alpha 1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks alpha-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to alpha-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in alpha-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme.

Amino Acid Sequence↗
PubMed1997

Abnormal metabolism of mannose in families with carbohydrate-deficient glycoprotein syndrome type 1.

Patients with carbohydrate-deficient glycoprotein syndrome (CDGS) Type 1 underglycosylate many glycoproteins by failing to add entire N-linked carbohydrate chains to them. The primary defect in these patients has been reported as a > 90% deficiency in phosphomannomutase activity (PMM), the enzyme that converts mannose-6-phosphate to mannose-1-phosphate. This lesion reduces both the amount and the size of the lipid-linked oligosaccharide precursor. We have now analyzed the activity of PMM and the level of glycosylation in cultured fibroblasts as well as the level of blood mannose in seven CDGS Type 1 patients and their parents. All of these patients were approximately 95% deficient in PMM activity and their parents had an average of 51% of control PMM activity. Furthermore, parental fibroblasts showed reduced glycosylation and a higher proportion of truncated N-linked chains compared to those made by control fibroblasts. Addition of 0.25 mM mannose to the culture medium corrected both the underglycosylation and size of the oligosaccharide chains in CDGS Type 1 patients and their parents. Finally, serum from CDGS patients had considerably reduced mannose levels (5-40 microM) compared to normal controls (40-80 microM) and some parents were below normal (16-103 microM). These results suggest that the reduced blood mannose level is a consequence of the PMM deficiency. This is the first inherited disorder in human metabolism that shows a decrease in available mannose. Increasing blood mannose levels might correct some protein underglycosylation in these patients.

Carbohydrate Conformation↗
PubMed1996

Mannose enters mammalian cells using a specific transporter that is insensitive to glucose.

The concentration of D-mannose in serum is 20-50 micron, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K(uptake) of about 30-70 micron and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2-deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mM D-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis.

Animals↗
PubMed1996

Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts.

Type I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins. Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities. First, they incorporate 3-10-fold less [3H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls. Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [3H] mannose incorporation and the size of LLO. These corrections are not permanent, and the defects immediately reappear when mannose is removed. To explore further the basis of mannose correction, we analyzed the amount of 3H-labeled LLO intermediates. Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10-fold decrease in CDGS cells. Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [3H]mannose appeared to be metabolized normally. Initial velocities of [3H]mannose uptake were two- to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [3H]mannose incorporation in CDGS cells. Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways. Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets.

Cell Line↗
PubMed1995

Characterization of a novel epidermal-growth-factor-receptor-related 200-kDa tyrosine kinase in tumor cells.

We have detected a tyrosine-phosphorylated 200-kDa protein in two human tumor cell lines, A1235 glioma and A172 glioblastoma. The protein is an integral plasma membrane sialoglycoprotein with tyrosine kinase activity. The interesting characteristic of this protein (gp200) is that it is recognized by a number of monoclonal and polyclonal antibodies to the 170-kDa epidermal-growth-factor (EGF) receptor; however, it lacks detectable EGF-binding activity. gp200 differs from three other EGF-receptor-related proteins, erb-B-2, erb-B-3 and erb-B-4 gene products, and hence appears to be yet another member of the EGF-receptor family of proteins. This is further strengthened by the fact that both gp200 and the EGF receptor contain a common epitope which is recognized by an anti-peptide IgG to the beta-type platelet-derived-growth-factor (PDGF) receptor. Our previous studies [Bishayee, S., Majumdar, S., Scher, C.D. & Khan, S. (1988) Mol. Cell. Biol. 8, 3696-3702] have demonstrated that this epitope in the PDGF receptor is highly susceptible to the phosphorylation state of the receptor and that such a conformational change appears to be important in biological message transmission. The expression of gp200, which appears to have tyrosine kinase activity and is immunologically related to the EGF receptor in tumor cells, suggests its possible involvement in cell growth.

Amino Acid Sequence↗
PubMed1995

A conformation-specific anti-peptide antibody to the beta-type platelet-derived growth factor receptor also recognizes the activated epidermal growth factor receptor.

Earlier we reported the generation of a conformation-specific antibody (Ab P2) to the beta-type platelet-derived growth factor receptor (Bishayee, S., Majumdar, S., Scher, C. D., and Khan, S. (1988) Mol. Cell. Biol. 8, 3696-3702). Ab P2 is directed to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the receptor, and it recognizes the phosphorylated platelet-derived growth factor receptor but not the unphosphorylated receptor. We now report that Ab P2 also interacts with the epidermal growth factor receptor and that the recognition is specific for a conformation induced by phosphorylation of the receptor; however, Ab P2 is not directed to phosphotyrosine. Studies conducted with P2-derived peptides suggest that the conformation-specific antibody is directed to an acidic tripeptide, Asp-Glu-Glu, and this sequence is also present in the cytoplasmic domain of the epidermal growth factor receptor. With respect to the C terminus amino acid or ATP-binding site, Asp-Glu-Glu is located in different regions in these receptors; nevertheless, this tripeptide along with the surrounding amino acids is cryptic in the unphosphorylated receptor, and tyrosine phosphorylation uncovers this site. This suggests that the Asp-Glu-Glu sequence may be important in receptor functions.

Amino Acid Sequence↗