Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.
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Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand. (+info)
Gangliosides of human kidney.
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Five gangliosides isolated from human kidney have been characterized. The two main fractions were shown to be typical extraneural gangliosides in having lactose as their neutral carbohydrate moiety. Their structures were identified as: AcNeu(alpha2-3)Gal(beta1-4)Glc(beta1-1)Cer and AcNeu(alpha2-8)AcNeu(alpha2-3)Gal(beta1-4)Glc(beta1-1)Cer. The two main hexosamine-containing gangliosides are structurally related to human blood group substances of glycosphingolipid nature. The following structures are postulated: AcNeu(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(beta1-1)Cer and AcNeu(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc(beta1-1) Cer. The third hexosamine-containing ganglioside belongs to a different series of glycolipids and was shown to have the structure of a major ganglioside of human brain: AcNeu(alpha2-3)Gal(beta1-3)GalNAc(beta1-4)[AcNeu(alpha2-3)]Gal(beta1-4)Glc(beta1- 1)Cer. The fatty acid structure of different gangliosides was shown to resemble that of neutral glycolipids of human kidney with the nonhydroxy acids C16:0, C22:0, and C24:0 as major components. (+info)
Participation of a trisaccharide-lipid in glycosylation of oviduct membrane glycoproteins.
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Preincubation of a hen oviduct membrane preparation with UDP-Nactyl[14C]glucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (2/1)-extractable glycolipid. Treatment of the lipid with mild acid results in the release of a trisaccharide shown to have the structure beta-mannosyl-N-acetylglucosamineyl-N-acetylglucosamine. Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000... (+info)
Lectin receptor sites on rat liver cell nuclear membranes.
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The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells. (+info)
Role of surface proteins in Vibrio cholerae attachment to chitin.
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The role of surface proteins in Vibrio cholerae attachment to chitin particles in vitro was studied. Treatment of V. cholerae O1 ATCC 14034 and ATCC 14035 with pronase E reduced the attachment of bacteria to chitin particles by 57 to 77%. A statistically significant reduction was also observed when the attachment to chitin was evaluated in the presence of homologous Sarkosyl-insoluble membrane proteins (MPs) (67 to 84%), N-acetylglucosamine (GlcNAc) (62%), the sugar that makes up chitin, and wheat germ agglutinin (40 to 56%), a lectin that binds GlcNAc. The soluble oligomers N,N'-diacetylchitobiose or N,N', N"-triacetylchitotriose caused an inhibition of 14 to 23%. Sarkosyl-insoluble MPs able to bind chitin particles were isolated and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; two of these peptides (molecular sizes, 36 and 53 kDa) specifically bind GlcNAc. (+info)
Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids.
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The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I-LDL by up to 80%. The transfer of pyrene (py)-labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I-LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three-fold. WGA also specifically augmented the transfer of py-SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N-acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I-LDL to the platelets by up to 2.8-fold. On the other hand, the import of LDL-derived py-phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins. (+info)
Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus.
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The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo. (+info)
Quantitative determination of N-acetylglucosamine residues at the non-reducing ends of peptidoglycan chains by enzymic attachment of [14C]-D-galactose.
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The ability of human milk galactosyltransferase to attach D-galactose residues quantitatively to the C-4 of N-acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rod-shaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical 'mini' cells had chain lengths similar to those of the isogenic rod-like cells. (+info)