RESUMEN
On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.
Asunto(s)
Antígenos CD , Galactosiltransferasas/metabolismo , Glucolípidos/biosíntesis , Riñón/metabolismo , Lactosilceramidos , Microsomas/metabolismo , Sistema del Grupo Sanguíneo P/biosíntesis , Secuencia de Carbohidratos , Globósidos/metabolismo , Glicoesfingolípidos/metabolismo , Humanos , Isoenzimas , Isomerismo , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
In earlier studies, the minimum structure which inhibited the binding of anti-i to an i-active glycoprotein was the linear trisaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-D-Gal. There was an increasing hierarchy of inhibitory activities in the linear tetrasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D -GlcNAc , its methyl beta-glycoside, and in the methyl beta-glycoside of the hexasaccharide. The linear octasaccharide methyl beta-glycoside in this series is approximately only half as active as the hexasaccharide methyl beta-glycoside. Analyses by high resolution 1H-n.m.r. of these two oligosaccharides indicated that they have similar conformations in solution, and there is no evidence for the occurrence of inter-molecular interactions which might partially hinder the binding of anti-i to the octasaccharide methyl beta-glycoside. These results are consistent with the size of the i antigen being in the region of a hexasaccharide. It is proposed that the methyl aglycon group of the hexasaccharide methyl beta-glycoside confers an above normal activity by presenting a hydrophobic area for additional contact in the vicinity of the antibody-combining site.
Asunto(s)
Epítopos/inmunología , Glicoproteínas/inmunología , Sistema del Grupo Sanguíneo I/inmunología , Oligosacáridos/farmacología , Polisacáridos/farmacología , Secuencia de Carbohidratos , Humanos , Espectroscopía de Resonancia Magnética , Meconio/química , Datos de Secuencia MolecularRESUMEN
N-Phthaloylation of lactosamine gave various glycosyl donors (beta-chloride, beta-trichloroacetimidate) and glycosyl acceptors (3',4'-diol). Coupling of the chloride with a methyl beta-D-glycoside led to the tetrasaccharide fragment, beta-D-Galp-(1----4)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAcOMe. Acetolysis of the protected tetrasaccharide, followed by treatment with hydrogen chloride, gave a tetrasaccharide chloride which was coupled with the methyl beta-glycoside of lactosamine. A hexasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]2-beta-D-Galp-(1----4)-bet a- D-GlcpNAcOMe, was thus obtained by this ("n + 1") method. A more efficient ("n + n") method was applied for the synthesis of an octasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]3-beta-D-Galp- (1----4)-beta-D-GlcpNAcOMe (38), where di- and tetra-saccharide intermediates having a 3,4-O-isopropylidene-beta-D-galactopyranosyl nonreducing terminal group and a benzyl beta-D-glycoside group were precursors, either as glycosyl donors (beta-trichloroacetimidates) or glycosyl acceptors (3,4-diols as nonreducing terminal groups). Thus, doubling the length of the repetitive oligosaccharide sequence could be efficiently accomplished at each glycosylation step.
Asunto(s)
Sistema del Grupo Sanguíneo I , Oligosacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Humanos , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligosacáridos/química , Rotación Óptica , Relación Estructura-ActividadRESUMEN
1,5-Anhydro-3-O-benzyl-2,6-dideoxy-4-O-(3,4-di-O-benzyl-2,6-dideoxy-beta -D- arabino-hexopyranosyl)-D-arabino-hex-1-enitol (17), which corresponds to the B-C fragment of various orthosomycins, was prepared from phenyl 2,3-di-O-benzyl-6-deoxy-4-O-(3,4-di-O-benzyl-2,6-dideoxy- beta-D-arabino-hexopyranosyl)-1-thio-beta-D-glucopyranoside (16) by reductive lithiation. The synthesis of 16 involved a stereoselective coupling of phenyl 2,3-di-O-benzyl-6-deoxy-1-thio-beta-D-glucopyranoside (9) and 1,2-di-O-acetyl-3,4-di-O-benzyl-6-deoxy-beta-D-glucopyranose (14) followed by deoxygenation at C-2'. Glycosylation of methyl 2-O-benzyl-6- deoxy-4-O-methyl-beta-D-galactopyranoside (25) with 3,4,6-tri-O-acetyl-2-deoxy- 2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate, followed by deamination at C-2', led stereospecifically to methyl 2-O-benzyl-6-deoxy-4-O-methyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-beta-D-ara bino- hexopyranosyl)-beta-D-galactopyranoside (26). The 2-deoxy unit of 26 was then modified by consecutive axial introduction of a C-Me group at position 3', protection of HO-3', and deoxygenation at C-6', in order to obtain methyl 3-O-(3-O-benzoyl-2,6-dideoxy-3-C-methyl-beta-D-arabino-hexopyranosyl)-2- O- benzyl-6-deoxy-4-O-methyl-beta-D-galactopyranoside (39), which corresponds to the D-E fragment of orthosomycins. A glycosyloxyselenation-oxidation-elimination sequence was performed on 39 and either 1,5-anhydro-3,4-di-O-benzyl-2,6- dideoxy-D-arabino-hex-1-enitol (40) or 1,5-anhydro-3-O-benzyl-2,6-dideoxy-4-O-(3,4-di-O-benzyl-2,6- dideoxy-beta-D-arabino-hexopyranosyl)-D-arabino-hex-1-enitol (17) to give the C-D-E tri-and B-C-D-E tetrasaccharide fragments, respectively. Each fragment contained the spiro-ortholactone junction with an (R) configuration at the anomeric carbon atom of the C-unit.
Asunto(s)
Antibacterianos/síntesis química , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Fenómenos Químicos , Química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Trisacáridos/síntesis químicaRESUMEN
Methyl 4-O-(2-O-benzoyl-4,6-O-benzylidene-beta-D-galactopyranosyl)-2,3, 6-tri-O-benzoyl-beta-D-glucopyranoside (2) was prepared in four steps from methyl beta-lactoside. Crystalline 2 was a convenient substrate for the synthesis of branched oligosaccharides derived from lactose. High-yield glycosylations were performed first at the 3'-position and then, after removal of the benzylidene group, at the 6'-position, using trichloroacetimidates of N-phthaloylamino sugars. 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate was thus used in two consecutive glycosylations, and also in a simultaneous disubstitution of the triol 9, leading in each sequence to the branched tetrasaccharide, beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)- beta-D-GlcOMe. Similar glycosylations performed with 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl trichloroacetimidate afforded the branched hexasaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-beta-D-GlcOMe, which corresponds to the human-milk oligosaccharide lacto-N-neohexaose and has a strong blood-group I activity.
Asunto(s)
Antígenos de Grupos Sanguíneos , Glucolípidos/biosíntesis , Sistema del Grupo Sanguíneo I , Oligosacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Oligosacáridos/metabolismoRESUMEN
A beta 1-6N-acetylglucosaminyltransferase has been identified in microsomal preparations from hog gastric mucosa which is able to synthesize branch points in branched lactosaminoglycans (blood group I antigenic structures). The enzyme can be assayed specifically using the synthetic trisaccharide GlcNAc beta 1-3Gal beta 1-4Glc beta-OMe as acceptor. The product of the transferase reaction was isolated and identified by methylation analysis as, (Formula: see text) Into this tetrasaccharide two galactose residues were incorporated by the specific beta-N-acetylglucosaminide beta 1-4-galactosyltransferase from bovine milk. Thus a hexasaccharide was formed which was shown to inhibit strongly a murine monoclonal and a human anti-I antibody. Using a variety of oligosaccharides and glycolipids, which correspond to structures found in linear lactosaminoglycan chains, the acceptor substrate specificity of the branching enzyme was determined. From these results it is concluded that branching occurs only during the elongation process at the nonreducing end and follows a well-defined order. N-Acetylglucosamine is first transferred to position 3 of a terminal galactose followed immediately by the addition of a second N-acetylglucosamine to position 6; only then the 1-3 and the 1-6 branches are further elongated by galactose residues.
Asunto(s)
Antígenos de Grupos Sanguíneos , Mucosa Gástrica/enzimología , Sistema del Grupo Sanguíneo I , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Pruebas de Inhibición de Hemaglutinación , Cinética , Espectrometría de Masas , Microsomas/enzimología , Especificidad por Sustrato , PorcinosRESUMEN
The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.