Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).     

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.     

Mucolipidosis II and III alpha/beta in Brazil: Analysis of the GNPTAB gene

Mucolipidosis II and III (MLII and MLIII) alpha/beta are rare autosomal recessive lysosomal storage diseases (LSDs) caused by pathogenic variations in the GNPTAB gene. GNPTAB gene codes for the α and β subunits of phosphotransferase, the enzyme responsible for synthesis of the mannose-6-phosphate (M₆P) marker that directs lysosomal enzymes to the lysosome.     

Carbohydrate phenotyping of human and animal milk glycoproteins

Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)^x, sialyl-Le^x, Le^a, sialyl-Le^a and Le^b carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Le^b and sialyl-Le^x), enteropathogenic (H type 1, Le^a and Le^x) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection. Published in 2005.     

Carbohydrate composition of serum low and high density lipoproteins of nonhuman primate species.

1. Carbohydrate composition of serum low and high density lipoproteins obtained from 5 nonhuman primate species (chimpanzee, patas, baboon, rhesus, and spider) and humans was studied. 2. Individual lipoproteins were isolated from pooled sera of each species by ultracentrifugal flotation between the densities 1.019-1.063 for LDL-2; 1.063-1.12 for HDL-2; and 1.12-1.21 for HDL-3. After delipidation, sialic acid, fucose, glucosamine, mannose, galactose, and glucose were determined on apo LDL-2, apo HDL-2, and apo HDL-3. 3. Glucosamine, galactose, and mannose constituted a major component of the sugars in apo LDL-2, with similar relative proportions in all species. Sialic acid, fucose, and glucose formed a minor component, the proportions of which varied greatly among the species. 4. Unlike apo LDL-2, sialic acid, fucose, and glucosamine constituted the bulk of the sugars in apo HDL-2 and apo HDL-3. Mannose, galactose, and glucose were minor components, with galactose predominating. 5. Qualitative differences were observed in electrophoretic mobilities of apo HDL-2 and apo HDL-3 on polyacrylamide gel. One faster moving band was unique to chimpanzee. 6. Intraspecies differences in the content of sialic acid and fucose of apolipoproteins may be related to lipoprotein metabolism and species susceptibility (or resistance) to either spontaneous or diet-induced atherosclerosis.     

Polypeptide composition of the purified Photosystem II pigment-protein complex from spinach

The Photosystem II pigment-protein complex, the chlorophyll α-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine.The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43 000 and 27 000. The chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100°C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43 000 and 27 000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Carbohydrate moieties of glycoproteins a re-evaluation of their function

Glycosylation-deficient mutants and inhibitors of glycosulation have been used to investigate the biological function of the carbohydrate moieties of glycoproteins. These and other experimental findings are reviewed and critically evaluated in the present treatise.An hypothesis is proposed to explain the biological significance of the covalent attachment of carbohydrate to protein. It is proposed that the carbohydrate acts as a chemical ‘tag’ which, upon interaction with a specific intracellular membrane receptor, directs glycoproteins to specific cellular organelles following synthesis on the rough endoplasmic reticulum. It is further proposed that secretion of extracellular products is a more general phenomenon which appears not to be absolutely carbohydrate dependent. Also, data are presented which support the view that the carbohydrate moeity is required for the proteolytic or conformational stabilization of the protein component of glycoproteins, but not for the mediation of protein specific biological activity. A model is presented which suggests, for the first time, that the localization function of carbohydrates is not restricted to lysosomal enzymes.     

Desmosomal glycoproteins II and III. Cadherin-like junctional molecules generated by alternative splicing

We have cloned the human genes coding for desmosomal glycoproteins DGII and DGIII, found in desmosomal cell junctions, and sequencing shows that they are related to the cadherin family of cell adhesion molecules. Thus a new super family of cadherin-like molecules exists which also includes the other major desmosomal glycoprotein, DGI (Wheeler, G. N., Parker, A. E., Thomas, C. L., Ataliotis, P., Poynter, D., Arnemann, J., Rutman, A. J., Pidsley, S. C., Watt, F. M., Rees, D. A., Buxton, R. S., and Magee, A. I. (1991) Proc. Natl. Acad. Sci. U.S.A., in press). DGIII differs from DGII by the addition of a 46-base pair exon containing an in-frame stop codon resulting in mature protein molecular weights of 84,633 for DGII and 78,447 for DGIII. The unique carboxyl-terminal region of DGII contains a potential serine phosphorylation site explaining why only DGII is phosphorylated on serine. The cadherin cell adhesion recognition sequence (His-Ala-Val) is replaced by Phe-Ala-Thr, suggesting that DGII/III may be adhesive molecules using a different mechanism.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Carbohydrate composition and taxonomy of the genusDipodascus

Carbohydrates released during acid hydrolysis of intact cells ofDipodascus were studied by gas-liquid chromatographic analysis as their trimethylsilyl derivatives. In addition, cells were characterized by pyrolysis gas-liquid chromatography and pyrolysis mass spectrometry. The data obtained support the classification ofDipodascus uninucleatus in a separate genusDipodascopsis. Glucuronic acid is present inD. uninucleatus and, therefore, a possible affinity to fungi classified in the Zygomycetes is considered.Dipodascus aggregatus andDipodascus australiensis were found to be rather different, but very close toGeotrichum candidum and related species.     

Polypeptide composition of the purified photosystem II pigment-protein complex from spinach.

The Photosystem II pigment-protein complex, the chlorophyll alpha-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine. The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43,000 and 27,000. the chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100 degrees C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43,000 and 27,000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Analysis of the peptide composition of purified beef-heart complex III by dodecylsulfate electrophoresis.

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex III and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex.