The N-linked carbohydrate chains of porcine thyroglobulin were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F (PNGase-F). The resulting oligosaccharides were fractionated by a combination of fast protein liquid chromatography and high performance liquid chromatography and analyzed by 500-MHz 1H NMR spectroscopy. The major acidic compounds are mono- and disialylated, fucosylated diantennary compounds terminated with alpha(2-6)-linked sialic acid on the Man alpha(1-3) branch. The Man alpha(1-6) branch shows a large heterogeneity. It can be terminated with Man-4', GlcNAc-5', or Gal-6', whereas the Gal-6' residue may be extended with Gal alpha(1-3), NeuAc alpha(2-3), or Sia alpha (2-6). In the major structures 8% of alpha(2-6)-linked sialic acid was found as NeuGc instead of NeuAc. The main compounds have sulfated homologues bearing a sulfate group (6-20%) at C-3 of Gal-6' or at C-6 of GlcNAc-5 as follows. [formula: see text] 相似文献
The capsular polysaccharide of Neisseria meningitidis serogroup H is composed of the following repeating unit, Gro = glycerol; (formula; see text) Partial O-acetylation of the D-Galp moieties is found for C-2 (21%) and C-3 (57%). The structural elucidation of the biopolymer is based on sugar analysis, methylation analysis, partial acid hydrolysis, using gas-liquid chromatography/mass spectrometry studies, and NMR spectroscopy with 1H, 13C and 31P. 相似文献
The carbohydrate chains of the pathological human immunoglobulins M from two patients with Waldenström's macroglobulinemia were released by hydrazinolysis. The N-acetyllactosamine-type glycans were obtained by affinity chromatography on concanavalin A and fractionated by high-voltage paper electrophoresis. The primary structure of the major compounds was elucidated on the basis of carbohydrate analysis, methylation analysis, including mass-spectrometry, and 500 MHz 1H-NMR spectroscopy. For both patients, this appeared to be a monosialyl monofucosyl biantennary structure; the compounds differed by the presence of an intersecting N-acetylglucosamine residue. 相似文献
TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz1H-NMR spectroscopy to beAbbreviations hCG
human chorionic gonadotropin
- hCG-
-subunit
- hCG-
-subunit
- PNGase-F
peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52)
- endo-F
endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96)
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide gel electrophoresis
- CBB
coomassie brilliant blue R 250
- GlcNAc
N-acetylglucosamine
- NeuAc
N-acetylneuraminic acid
- Man
mannose
- Gal
galactose
- Fuc
fucose 相似文献
Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O2l-dehydroascorbic acid + H2O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz1H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be:
Abbreviations AAO
ascorbic acid oxidase
- PNGase-F
peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase-F
- GalNAc
N-acetylgalactosamine
- GlcNAc
N-acetylglucosamine
- Man
mannose
- Xyl
xylose
- GLC
gas-liquid chromatography
- FPLC
fast protein liquid chromatography
- NMR
nuclear magnetic resonance
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
The oligosaccharides of chick embryo type I procollagen were isolated from the carboxyl-terminal propeptide fragment by exhaustive digestion with papain and pronase, and then purified as a mixture of glycopeptides. The structures of the oligosaccharides were established by high-resolution 1H-NMR spectroscopy and found to be a mixture with respect to the non-reducing terminal residues as shown below: The percentages refer to the relative amount of those mannose residues present in the mixture. The data suggest that the oligosaccharides are a microheterogeneous mixture of high-mannose type glycans containing between six and nine mannose residues per carbohydrate unit. Such carbohydrate chains, although not uncommon for glycoproteins, had never been found before for collagen or collagen-related compounds. 相似文献
Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense
down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. 相似文献
New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied. 相似文献
Glycoproteins were isolated from the ovary of the starfish Asterias rubens (L.). After delipidation, sugar analysis revealed the presence of mannose, glucose and N-acetylglucosamine in a molar ratio of 9.0:1.3:2.3. Subsequently, hydrazinolysis, re-N-acetylation, reduction and high-voltage paper electrophoresis were carried out, resulting in a mixture of neutral oligosaccharide alditols which was fractionated on Bio-Gel P-4. The alditols, investigated by 500-MHz 1H-NMR spectroscopy, turned out to be of the oligomannose type or of the gluco-oligomannose type containing 9 mannose and 1-3 glucose residues. The most abundant compounds were established to be: (Formula: see text) and (Formula: see text). 相似文献