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Papers by Dale Cumming
[17] Chemical synthesis of fructose 2,6-bisphosphate
Methods in Enzymology, 1982
Publisher Summary This chapter describes the chemical synthesis of fructose 2,6-bisphosphate. Fru... more Publisher Summary This chapter describes the chemical synthesis of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase. The method of chemical synthesis of fructose 2,6-bisphosphate is based on the synthesis of fructose 2-phosphate. The treatment of fructose 1-phosphate with dicyclohexylcarbodiimide (DCC) in aqueous pyridine leads to the formation of fructose cyclic 1,2-phosphate. Alkaline hydrolysis of that compound yields fructose 2-phosphate as one product and fructose 1-phosphate as the other. Base-catalyzed ring opening of fructose 1,2-cyclic 6-bisphosphate (II) yields both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate. Fructose 1,6-bisphosphate is destroyed by heating in alkali, whereas the fructose 2,6-bisphosphate is stable and is purified by anion-exchange chromatography. 6-Phosphofructo-1- kinase activity and the activation of 6-phosphofructo-1-kinase by fructose 2,6- bisphosphate are determined with the aldolase-coupled assay. Purified rat liver 6-phosphofructo-1-kinase is used in the activation assay. The overall yield of this method is limited primarily by the base-catalyzed ring opening reaction, but the key to purification is heating in alkali.
Biochemistry, 1988
Human @-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal Nacetylhexosa... more Human @-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal Nacetylhexosamines from G M 2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure a(@,@b), and hexosaminidase B, 2(@,@b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the @b chain, and one non concanavalin A binding glycopeptide, localized to the @, chain, were found associated with the @-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the a subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the a and one of the @b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the @b chain was composed of a mixture of Mans-,-GlcNAc2 glycans. The unique glycopeptide associated with the @, chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the a and @ subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the a chain we found only one possible site for in vivo phosphorylation. In the @ it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an a1,2-linked mannose residue. Only the single Man6-, (of the ManS-,-GlcNAc2 glycans) containing site on the @b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase. fi-Hexosaminidase (EC 3.2.1.52) is a lysosomal hydrolase. There are two major isozymes in normal human tissues, hexosaminidase A and B. Hexosaminidase A is composed of two subunits, an a subunit, consisting of a single polypeptide chain, and a p subunit containing two peptides, pa and fib, derived by cleavage of a common pro-@ precursor. Its structure is ff(pa&). Hexosaminidase B, on the other hand, contains
Core 2 β1,6‐N‐acetylglucosaminyltransferases and α1,3‐fucosyltransferases regulate the synthesis of O‐glycans on selectin ligands on oral cavity carcinoma cells
APMIS, 2008
Selectin‐dependent cell binding has importance in the extravasation of blood‐circulating tumor ce... more Selectin‐dependent cell binding has importance in the extravasation of blood‐circulating tumor cells and in the generation of metastases. Cell surface glycoproteins decorated with sialylated, fucosylated epitopes, such as sialyl Lewisx (sLex), are ligands for selectins. Not only terminal sLex moieties but also proximal core structures contribute to the formation of binding epitopes for selectins. Core 2 β1,6‐N‐acetylglucosaminyltransferases (C2GnT) and α1,3‐fucosyltransferases (α1,3‐FucT) have been suggested to be the rate‐limiting enzymes in the synthesis of selectin ligands. We analyzed oral cavity epithelial carcinoma cell lines and showed their expression of RNA transcripts for C2GnT and α1,3‐FucT, identified α1,3‐FucT enzyme activities, and analyzed the cell surface sLex expression levels. Neither the pattern of expressed enzymes nor the α1,3‐FucT activity directly predicted the binding capacity of E‐selectin. However, only the sLex‐expressing cell lines were capable of binding...
Oligosaccharide-Protein Interactions: A Three-Dimensional View
Novartis Foundation Symposia, 2007
For carbohydrates to serve as recognition elements in cellular function, there must be 'recep... more For carbohydrates to serve as recognition elements in cellular function, there must be 'receptors' which are capable of distinguishing between the multitude of oligosaccharide structures generated by a cell. Generally these receptors are assumed to be proteins, and the plant lectins have been used as model systems to examine the molecular basis for specificity in such interactions. Three aspects of the specificity of oligosaccharide-protein interactions will be discussed: (1) the conformational flexibility of oligosaccharides will be demonstrated through a quantitative analysis of nuclear magnetic resonance measurements; (2) a comparison of the measured and calculated values for the entropy barrier to oligosaccharide binding will be used to argue that the barrier arises from a loss of this conformational flexibility upon binding to the lectin (this conclusion is also supported by X-ray crystallographic studies); and (3) the thermodynamic model can be extended to the binding of glycoproteins to receptors and the high affinity of these interactions explained by either multivalency or fixation of the oligosaccharide in the 'correct' three-dimensional structure through interaction with the protein moiety.
Oligosaccharide-protein intercations : a three-dimensional view
CIBA Foundation Symposium, Nov 15, 1988
Physiological relevance of protein glycosylation
Developments in biological standardization, 1992
The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It... more The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It is also a deterministic pathway dependent upon protein sequence, cellular phenotype, and the physiological environment. Two principal physiological roles have emerged within the past decade for protein-linked glycans: as recognition determinants and as modulators of various protein attributes such as bioactivity, pharmacokinetics, folding, and immunogenicity. All these attributes are crucial to development and application of protein-based pharmaceuticals. However, protein glycosylation represents a difficult structure/function problem since most glycoproteins exhibit microheterogeneity and oligosaccharides frequently contribute to this heterogeneity. Nevertheless, recent data suggest that different members of the heterogeneous ensemble exhibit distinguishable intrinsic properties, suggesting that the microheterogeneity of protein glycosylation represents a sophisticated mechanism of bio...
Journal of Immunology, Jul 1, 1996
Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathwa... more Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells.
Proteines ligands de p-selectine
Nouvelle glycoproteine ligand de P-selectine, qui comporte la sequence d'acides amines presen... more Nouvelle glycoproteine ligand de P-selectine, qui comporte la sequence d'acides amines presentee dans SEQ ID NO:2 ou par la sequence d'acides amines presentee dans SEQ ID NO:4. Des sequences d'ADN codant la proteine ligand de P-selectine sont egalement decrites, ainsi que des vecteurs, des cellules hotes et des procedes permettant de fabriquer ladite proteine ligand de P-selectine. Des compositions pharmaceutiques contenant cette proteine et des procedes de traitement d'etats inflammatoires caracterises par des adherences intercellulaires induites par la P-selectine et la E-selectine sont egalement decrites.
Method of inhibiting P-selectin ligand activity
Nucleic acids encoding P-selectin ligand fusion proteins
P-selectin ligand protein
A human colon carcinoma cell line exhibits adhesive interactions with P-selectin under fluid flow via a PSGL-1-independent mechanism
The American journal of pathology, 1996
It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment... more It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment play a role in metastasis. Using an in vitro flow model, we studied the adhesion of the human colon carcinoma cell line KM12-L4 to P-selectin, an inducible endothelial-expressed adhesion molecule involved in leukocyte recruitment. Recombinant forms of P-selectin and Chinese hamster ovary cells stably expressing P-selectin supported attachment and rolling of KM12-L4 cells at 1 to 2 dynes/cm2. The adhesive interactions to P-selectin were abolished by pretreatment of the KM12-L4 cells with neuraminidase but were unaltered by pretreatment of the KM12-L4 cells with O-sialoglycoprotein endopeptidase, an enzyme that cleaves mucin type glycoproteins such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is the only counter-receptor for P-selectin known to mediate myeloid cell adhesion to P-selectin under flow. Flow cytometric and Northern blot analyses revealed that KM12-L4 cells did not exp...
Canadian Journal of Chemistry, 1990
We describe a simple and efficient method for the preparation of the trisaccharide GlcNAc(β1-4)-[... more We describe a simple and efficient method for the preparation of the trisaccharide GlcNAc(β1-4)-[Fuc(α 1-6)-]GlcNAc(β 1-) (1) and of the protected form of GlcNAc(β 1-4)-[Fuc(α 1-6)-]GlcNAc(β1-Asn) (2). The key intermediate is benzyl 4,6-benzylidene chitobioside 5 giving the desired trisaccharide by insitu anomerization–glycosylation reaction with 2,3,4-tribenzylfucosyl bromide. The benzyl glycoside in the trisaccharide 6 has been replaced by acetate and then bromine; this glycosylating agent was used to prepare methyl and 8-methoxycarbonyloctyl glycosides as well as isothiocyanate 12, in a series of reactions. The latter compound gave, on reaction with 1-benzyl N-benzyloxycarbonyl-L-asparate, compound 13 (a protected derivative of 2), which should serve as a synthon for syntheses of glycopeptides. Keywords: glycopeptide, synthesis; oligosaccharide, synthesis; chitobiosides; fucosylated chitobiosides; N-linked oligosaccharides.
Mutants de la 2 b-1,6-n-acetylglycosaminyltransferase de nucleocapside
L'invention concerne un nouveau mutant d'acides nucleiques 2 GlcNAcT de nucleocapside, de... more L'invention concerne un nouveau mutant d'acides nucleiques 2 GlcNAcT de nucleocapside, des polypeptides codees par les acides nucleiques et leurs utilisations.
Mutants of Core 2 B-1,6-N-ACETYLGLYCOSAMINYLTRANSFERASE
Mutants of core 2 B-1, 6-N-acetylglycosaminyltransferase
Biochemistry
A major periodate-Schiff-positive component from milk-fat-globule membrane of human breast milk h... more A major periodate-Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2,17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.
C2 Domain of Cytosolic Phospholipase A2
Journal of Biological Chemistry, 1981
Chromatography on DEAE-cellulose of chloroform : methanolcwater (10 : 10 : 3) extracts obtained f... more Chromatography on DEAE-cellulose of chloroform : methanolcwater (10 : 10 : 3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[~-'HJmannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 m M HCl, 100 "C, 20 min), a low molecular weight watersoluble component (PO), which contained most of the "P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetarnido-2-deoxytetrose i s glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD, is able to incorporate deuterium into the tetrose. Since the pioneering work of Leloir and co-workers (1-3), considerable experimental work has been directed toward elucidating the role of polyprenol derivatives in glycoprotein biosynthesis (4-8). The discovery by Natowicz et al. ( ) of a phosphomannose moiety in certain N-linked glycoproteins (acid glycohydrolases) has prompted us to investigate the occurrence of lipid-linked oligosaccharides containing 6-phosphomannosyl residues. Although such compounds were not detected, our study led to the identification of a novel sugar, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Service Training Grant T32 GM-07319. The data reported in this paper are taken from a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Journal of Biological Chemistry, 1986
[17] Chemical synthesis of fructose 2,6-bisphosphate
Methods in Enzymology, 1982
Publisher Summary This chapter describes the chemical synthesis of fructose 2,6-bisphosphate. Fru... more Publisher Summary This chapter describes the chemical synthesis of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is an allosteric activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase. The method of chemical synthesis of fructose 2,6-bisphosphate is based on the synthesis of fructose 2-phosphate. The treatment of fructose 1-phosphate with dicyclohexylcarbodiimide (DCC) in aqueous pyridine leads to the formation of fructose cyclic 1,2-phosphate. Alkaline hydrolysis of that compound yields fructose 2-phosphate as one product and fructose 1-phosphate as the other. Base-catalyzed ring opening of fructose 1,2-cyclic 6-bisphosphate (II) yields both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate. Fructose 1,6-bisphosphate is destroyed by heating in alkali, whereas the fructose 2,6-bisphosphate is stable and is purified by anion-exchange chromatography. 6-Phosphofructo-1- kinase activity and the activation of 6-phosphofructo-1-kinase by fructose 2,6- bisphosphate are determined with the aldolase-coupled assay. Purified rat liver 6-phosphofructo-1-kinase is used in the activation assay. The overall yield of this method is limited primarily by the base-catalyzed ring opening reaction, but the key to purification is heating in alkali.
Biochemistry, 1988
Human @-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal Nacetylhexosa... more Human @-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal Nacetylhexosamines from G M 2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure a(@,@b), and hexosaminidase B, 2(@,@b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the @b chain, and one non concanavalin A binding glycopeptide, localized to the @, chain, were found associated with the @-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the a subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the a and one of the @b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the @b chain was composed of a mixture of Mans-,-GlcNAc2 glycans. The unique glycopeptide associated with the @, chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the a and @ subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the a chain we found only one possible site for in vivo phosphorylation. In the @ it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an a1,2-linked mannose residue. Only the single Man6-, (of the ManS-,-GlcNAc2 glycans) containing site on the @b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase. fi-Hexosaminidase (EC 3.2.1.52) is a lysosomal hydrolase. There are two major isozymes in normal human tissues, hexosaminidase A and B. Hexosaminidase A is composed of two subunits, an a subunit, consisting of a single polypeptide chain, and a p subunit containing two peptides, pa and fib, derived by cleavage of a common pro-@ precursor. Its structure is ff(pa&). Hexosaminidase B, on the other hand, contains
Core 2 β1,6‐N‐acetylglucosaminyltransferases and α1,3‐fucosyltransferases regulate the synthesis of O‐glycans on selectin ligands on oral cavity carcinoma cells
APMIS, 2008
Selectin‐dependent cell binding has importance in the extravasation of blood‐circulating tumor ce... more Selectin‐dependent cell binding has importance in the extravasation of blood‐circulating tumor cells and in the generation of metastases. Cell surface glycoproteins decorated with sialylated, fucosylated epitopes, such as sialyl Lewisx (sLex), are ligands for selectins. Not only terminal sLex moieties but also proximal core structures contribute to the formation of binding epitopes for selectins. Core 2 β1,6‐N‐acetylglucosaminyltransferases (C2GnT) and α1,3‐fucosyltransferases (α1,3‐FucT) have been suggested to be the rate‐limiting enzymes in the synthesis of selectin ligands. We analyzed oral cavity epithelial carcinoma cell lines and showed their expression of RNA transcripts for C2GnT and α1,3‐FucT, identified α1,3‐FucT enzyme activities, and analyzed the cell surface sLex expression levels. Neither the pattern of expressed enzymes nor the α1,3‐FucT activity directly predicted the binding capacity of E‐selectin. However, only the sLex‐expressing cell lines were capable of binding...
Oligosaccharide-Protein Interactions: A Three-Dimensional View
Novartis Foundation Symposia, 2007
For carbohydrates to serve as recognition elements in cellular function, there must be 'recep... more For carbohydrates to serve as recognition elements in cellular function, there must be 'receptors' which are capable of distinguishing between the multitude of oligosaccharide structures generated by a cell. Generally these receptors are assumed to be proteins, and the plant lectins have been used as model systems to examine the molecular basis for specificity in such interactions. Three aspects of the specificity of oligosaccharide-protein interactions will be discussed: (1) the conformational flexibility of oligosaccharides will be demonstrated through a quantitative analysis of nuclear magnetic resonance measurements; (2) a comparison of the measured and calculated values for the entropy barrier to oligosaccharide binding will be used to argue that the barrier arises from a loss of this conformational flexibility upon binding to the lectin (this conclusion is also supported by X-ray crystallographic studies); and (3) the thermodynamic model can be extended to the binding of glycoproteins to receptors and the high affinity of these interactions explained by either multivalency or fixation of the oligosaccharide in the 'correct' three-dimensional structure through interaction with the protein moiety.
Oligosaccharide-protein intercations : a three-dimensional view
CIBA Foundation Symposium, Nov 15, 1988
Physiological relevance of protein glycosylation
Developments in biological standardization, 1992
The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It... more The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It is also a deterministic pathway dependent upon protein sequence, cellular phenotype, and the physiological environment. Two principal physiological roles have emerged within the past decade for protein-linked glycans: as recognition determinants and as modulators of various protein attributes such as bioactivity, pharmacokinetics, folding, and immunogenicity. All these attributes are crucial to development and application of protein-based pharmaceuticals. However, protein glycosylation represents a difficult structure/function problem since most glycoproteins exhibit microheterogeneity and oligosaccharides frequently contribute to this heterogeneity. Nevertheless, recent data suggest that different members of the heterogeneous ensemble exhibit distinguishable intrinsic properties, suggesting that the microheterogeneity of protein glycosylation represents a sophisticated mechanism of bio...
Journal of Immunology, Jul 1, 1996
Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathwa... more Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells.
Proteines ligands de p-selectine
Nouvelle glycoproteine ligand de P-selectine, qui comporte la sequence d'acides amines presen... more Nouvelle glycoproteine ligand de P-selectine, qui comporte la sequence d'acides amines presentee dans SEQ ID NO:2 ou par la sequence d'acides amines presentee dans SEQ ID NO:4. Des sequences d'ADN codant la proteine ligand de P-selectine sont egalement decrites, ainsi que des vecteurs, des cellules hotes et des procedes permettant de fabriquer ladite proteine ligand de P-selectine. Des compositions pharmaceutiques contenant cette proteine et des procedes de traitement d'etats inflammatoires caracterises par des adherences intercellulaires induites par la P-selectine et la E-selectine sont egalement decrites.
Method of inhibiting P-selectin ligand activity
Nucleic acids encoding P-selectin ligand fusion proteins
P-selectin ligand protein
A human colon carcinoma cell line exhibits adhesive interactions with P-selectin under fluid flow via a PSGL-1-independent mechanism
The American journal of pathology, 1996
It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment... more It has been postulated that endothelial cell adhesion molecules involved in leukocyte recruitment play a role in metastasis. Using an in vitro flow model, we studied the adhesion of the human colon carcinoma cell line KM12-L4 to P-selectin, an inducible endothelial-expressed adhesion molecule involved in leukocyte recruitment. Recombinant forms of P-selectin and Chinese hamster ovary cells stably expressing P-selectin supported attachment and rolling of KM12-L4 cells at 1 to 2 dynes/cm2. The adhesive interactions to P-selectin were abolished by pretreatment of the KM12-L4 cells with neuraminidase but were unaltered by pretreatment of the KM12-L4 cells with O-sialoglycoprotein endopeptidase, an enzyme that cleaves mucin type glycoproteins such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is the only counter-receptor for P-selectin known to mediate myeloid cell adhesion to P-selectin under flow. Flow cytometric and Northern blot analyses revealed that KM12-L4 cells did not exp...
Canadian Journal of Chemistry, 1990
We describe a simple and efficient method for the preparation of the trisaccharide GlcNAc(β1-4)-[... more We describe a simple and efficient method for the preparation of the trisaccharide GlcNAc(β1-4)-[Fuc(α 1-6)-]GlcNAc(β 1-) (1) and of the protected form of GlcNAc(β 1-4)-[Fuc(α 1-6)-]GlcNAc(β1-Asn) (2). The key intermediate is benzyl 4,6-benzylidene chitobioside 5 giving the desired trisaccharide by insitu anomerization–glycosylation reaction with 2,3,4-tribenzylfucosyl bromide. The benzyl glycoside in the trisaccharide 6 has been replaced by acetate and then bromine; this glycosylating agent was used to prepare methyl and 8-methoxycarbonyloctyl glycosides as well as isothiocyanate 12, in a series of reactions. The latter compound gave, on reaction with 1-benzyl N-benzyloxycarbonyl-L-asparate, compound 13 (a protected derivative of 2), which should serve as a synthon for syntheses of glycopeptides. Keywords: glycopeptide, synthesis; oligosaccharide, synthesis; chitobiosides; fucosylated chitobiosides; N-linked oligosaccharides.
Mutants de la 2 b-1,6-n-acetylglycosaminyltransferase de nucleocapside
L'invention concerne un nouveau mutant d'acides nucleiques 2 GlcNAcT de nucleocapside, de... more L'invention concerne un nouveau mutant d'acides nucleiques 2 GlcNAcT de nucleocapside, des polypeptides codees par les acides nucleiques et leurs utilisations.
Mutants of Core 2 B-1,6-N-ACETYLGLYCOSAMINYLTRANSFERASE
Mutants of core 2 B-1, 6-N-acetylglycosaminyltransferase
Biochemistry
A major periodate-Schiff-positive component from milk-fat-globule membrane of human breast milk h... more A major periodate-Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2,17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.
C2 Domain of Cytosolic Phospholipase A2
Journal of Biological Chemistry, 1981
Chromatography on DEAE-cellulose of chloroform : methanolcwater (10 : 10 : 3) extracts obtained f... more Chromatography on DEAE-cellulose of chloroform : methanolcwater (10 : 10 : 3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[~-'HJmannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 m M HCl, 100 "C, 20 min), a low molecular weight watersoluble component (PO), which contained most of the "P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetarnido-2-deoxytetrose i s glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD, is able to incorporate deuterium into the tetrose. Since the pioneering work of Leloir and co-workers (1-3), considerable experimental work has been directed toward elucidating the role of polyprenol derivatives in glycoprotein biosynthesis (4-8). The discovery by Natowicz et al. ( ) of a phosphomannose moiety in certain N-linked glycoproteins (acid glycohydrolases) has prompted us to investigate the occurrence of lipid-linked oligosaccharides containing 6-phosphomannosyl residues. Although such compounds were not detected, our study led to the identification of a novel sugar, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Service Training Grant T32 GM-07319. The data reported in this paper are taken from a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Journal of Biological Chemistry, 1986