Carbohydrate Composition Analysis Research Articles

Sensitive method for carbohydrate composition analysis of glycoproteins by high-performance liquid chromatography

A new high-perforrnance liquid chromatographic system has been developed for separating completely the neutral alditols and aminoalditols derived from sugars in complex carbohydrates. In combination with reduction by NaB 3H 4, this system affords a very sensitive and rapid method of monosaccharide composition analysis of glycoproteins and glycolipids.

On the macromolecular composition of the zona pellucida from porcine oocytes

The chemical and immunological relation between the glycoprotein components of the pig oocyte zona pellucida resolved by two-dimensional polyacrylamide gel electrophoresis was investigated. After disulfide bond reduction, four microheterogeneous glycoprotein components with apparent molecular weights of 25K, 55K, 65K, and 90K were resolved. When disulfide bonds were left unreduced, two microheterogeneous glycoprotein components were resolved with apparent molecular weights of 55K and 90K. Actin was present, but as a contaminant of the zona pellucida rather than as a true component. The structural relation of these components was investigated using deglycosylation with trifluoromethanesulfonic acid, limited proteolysis with Staphylococcus aureus V8 protease, amino acid and carbohydrate composition analyses, sequence analysis, and monoclonal antibodies. The 25K and 65K components comigrated with the 90K component when disulfide bonds were not reduced. When the intermolecular disulfide bonds crosslinking the two components were reduced, the 25K and 65K components behaved independently. The 25K and 65K components were derived from the 90K glycoprotein family by proteolysis. The 25K component originated from the C-terminal end, and the 65K component from the N-terminal end of the 90K glycoprotein. The 55K component was composed of two chemically and antigenically distinct glycoproteins, termed 55K α and 55K β, that electrophoretically comigrated. The N-terminal amino acid of the 55K α family was blocked. The 55K β family had an N-terminal amino acid sequence of Asp-Val-Pro-Thr-Ile-Gly-Leu-Ser-X-Ala-Pro-Thr. Thus, the two to four electrophoretic components of the zona pellucida observed on gel electrophoresis are derived from three glycoprotein families.

The carbohydrate structures ofβ-galactosidase from human liver

Acid β-galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (Mr63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz1H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the1H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure

Structural analysis of the major caprine β-mannosidosis urinary oligosaccharides

Urinary oligosaccharides were studied in β-mannosidosis, a newly identified, inherited glycoprotein catabolic disorder associated with severe neonatal neurological deficits, widespread lysosomal storage vacuoles and a deficiency of plasma and tissue β-mannosidase. A preliminary analysis of the oligosaccharides was obtained by gel-permeation chromatography and mass chromatography. The major urinary oligossacharides were then isolated by gel-permeation chromatography, DEAE-Sephadex column chromatography and preparative paper chromatography, and were analyzed by carbohydrate composition analysis, methylation studies, mass spectrometry and glycosidase digestion. As a result of these studies, β-mannosyl-(1 → 4)- N-acetylglucosamine and β-mannosyl-(1 → 4)- β- N-acetylglucosaminyl-(1 → 4)- N-acetylglucosamine were identified as the major abnormal oligosaccharides. Galactosaminyl-( α1 → 3)-[fucosyl-( α1 → 2)]-galactose was also found in affected goat urine, while lactose was present in the urine of both control and affected goats.

Purification and characterization of particulate guanylate cyclase from sea urchin spermatozoa.

The particulate form of guanylate cyclase from sea urchin spermatozoa was purified to apparent homogeneity by chromatography on GTP-Sepharose and DEAE-Sepharose and by preparative gel electrophoresis. The sedimentation coefficient (S20,w) was 6.8 and the Stokes radius was 5.1 nm, from which a native molecular weight of 157,000 was calculated. A single protein or periodic acid-Schiff staining band of 135,000 Da was observed after Na dodecyl SO4 gel electrophoresis. Antibody was produced to guanylate cyclase and was shown by electrophoretic transfer experiments (Western blot) to interact with only the Mr = 135,000 band in cases where all of the detergent-extracted protein from spermatozoa was added to the Na dodecyl SO4 gels. Although guanylate cyclase was normally bound to concanavalin A-Sepharose, after endoglycosidase H treatment it failed to bind. Treatment of the enzyme with endoglycosidase H did not alter guanylate cyclase activity, but the apparent size of the enzyme decreased to 72,000 Da on Na dodecyl SO4 gels. An analysis of carbohydrate composition indicated that the oligosaccharides contained N-acetylglucosamine, mannose, galactose, and 2-aminoerythritol in molar ratios (1:3:0.75:2); after endoglycosidase H treatment the enzyme contained essentially no carbohydrate. Major amino acids in the enzyme were aspartic (Asn) and glutamic (Gln) which accounted for approximately 25 mol % of the enzyme amino acid composition. The purified enzyme displayed linear kinetics on double reciprocal plots and had a KMnGTP = 133 microM, KM2+ = 138 microM, KiMnGTP = 122 microM, KiMn2+ = 127 microM, and a V max in excess of 15 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C. Sodium nitroprusside did not stimulate the enzyme in either the presence or absence of added hemeproteins. These results indicate that the particulate form of guanylate cyclase from sea urchin spermatozoa is a glycoprotein which is distinctly different than the soluble form of the enzyme found in mammalian tissues.

Characterization of axonally transported glycoproteins in regenerating garfish olfactory nerve.

This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [3H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180-200K, 105-115K, and 80-90K components, while a 150-160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50-140K range.

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.

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal ( M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184 ). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.

Isolation and structural characterization of human lymphocyte and neutrophil gangliosides.

Gangliosides were isolated from purified preparations of human peripheral blood lymphocytes and neutrophils. Structural analyses and comparisons were performed by direct probe mass spectrometry and by degradation studies with the following enzymes: Escherichia freundii endo-beta-galactosidase; Clostridium perfringens and Arthrobacter ureafaciens neuraminidase; and jack bean beta-N-acetylhexosaminidase and beta-galactosidase. This combination of techniques allowed us to obtain carbohydrate composition and sequence information without the aid of methylation or carbohydrate compositional analyses using only 1-2 mg of purified gangliosides. On the basis of these studies we propose that human lymphocytes and neutrophils have gangliosides with the following structures. NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure A NeuAc alpha 2 leads to ? GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure B NeuAc alpha 2 leads to ? Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure C All three compounds were isolated from both cell types with structure A being the major lymphocyte ganglioside and structure C the major neutrophil ganglioside. Structure B is a novel ganglioside and may represent a leukocyte-specific glycosphingolipid. Neuraminidase degradation studies demonstrated that only one ganglioside species of each cell type contains an internally linked sialic acid residue, and on the basis of thin layer chromatographic analysis this component is the same as the major brain ganglioside, GM1 (II3-N-acetylneuraminosyl-gangliotetraosylceramide). In addition, large gangliosides with the general structure NeuAc alpha 2 leads to ?(Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3)n Gal beta 1 leads to 4Glc beta 1 leads to 1Cer were isolated. These results are discussed as they relate to blood group antigens and specific cell surface markers in human leukocytes.

Purification and properties of invertases extractable from Phytophthora megasperma var. sojae mycelia

Suspensions of washed mycelia of the fungus Phytophthora megasperma Dreschs. var. sojae A. Hildbrand (Pms) catalyze the hydrolysis of sucrose. This is β-fructofuranosidase (invertase) since sucrose and raffinose (Gal p α1-6-Glc p α1-β2Fru f) serve as substrates, whereas maltose and 4-nitrophenyl-α- d-glucopyranoside do not. The invertase was extracted from frozen mycelia of Pms race 1 and from race 3 by a combination of mechanical disintegration and treatment with 10 m m dithiothreitol at 40 °C for 1 h. Each of the invertases was purified 156- to 192-fold in yields of 21 to 35% by a combination of procedures which included binding the enzyme to and releasing it from an activated-thiol Sepharose gel. The purified enzymes from both sources give similar isoelectric focusing gel patterns with at least five discrete protein bands in the p I range of 3.6 to 4.2. All of the bands stain positively for glycoprotein and sucrose-hydrolyzing activity. The purified enzymes have protein to carbohydrate ratios of 3.2. Analysis of carbohydrate composition by gas chromatography of the alditol acetates revealed the presence of mannose and glucose as the major components along with smaller amounts of galactose. The proportions of mannose and glucose differ significantly in Pms-1 and Pms-3 preparations. No amino sugars were detected. The pH optimum for the purified enzymes is 6.2 and the K m for sucrose is 1.8 ± 0.3 m m. Although several lines of evidence indicated that the extracted mycelial invertase contains sulfhydryl groups, the free sulfhydryl groups are not essential for activity. Iodoacetamide is not inhibitory and p-chloromercuribenzoate is not inhibitory at 10 μ m or lower concentrations; Cu(NO 3) 2 in the range of 1 μ m to 1 m m actually stimulated the activity. The properties of these enzymes are significantly different from those of the invertases purified from the culture fluids of these same races ( E. Ziegler and P. Albersheim, 1977, Plant Physiol., 59, 1104–1110).

Carbohydrate composition and sequence analysis of a derivative of brain disialoganglioside by mass spectrometry, with molecular weight ions at m-e 2245. Potential use in the specific microanalysis of cell surface components.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCarbohydrate composition and sequence analysis of a derivative of brain disialoganglioside by mass spectrometry, with molecular weight ions at m/e 2245. Potential use in the specific microanalysis of cell surface componentsKarl A. KarlssonCite this: Biochemistry 1974, 13, 18, 3643–3647Publication Date (Print):August 1, 1974Publication History Published online1 May 2002Published inissue 1 August 1974https://doi.org/10.1021/bi00715a003RIGHTS & PERMISSIONSArticle Views67Altmetric-Citations111LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (459 KB) Get e-Alerts Get e-Alerts

Use of mass spectrometry for the carbohydrate composition and sequence analysis of glycosphingolipids.

Mass spectrometry has been applied for a detailed structural characterization of glycosphingolipids. Methylated and methylated plus reduced glycolipids (amide groups of ceramide and amino sugars were reduced to the corresponding amines) were found the most useful derivatives. Molecular weight ions [M – 1] were not obtained for methylated lipids with more than four sugar units, but were easily obtained for reduced derivatives, so far with six sugars. Spectra of reduced derivatives showed very abundant ions containing the complete carbohydrate and the fatty acid, allowing a conclusion to be made concerning the carbohydrate composition (ratio of hexose and hexosamine and sialic acid). In addition, conclusive information was obtained for the carbohydrate sequence, including branching of the chain. By a combined use of both derivatives the long chain base and fatty acid composition was also evident. Spectra are discussed for a synthetic monoglycosylceramide, the Forssman glycolipid hapten (pentaglycosylceramide) and two gangliosides (a triglycosylceramide and a pentaglycosylceramide). The mass spectrum of the Forssman glycolipid provides the first conclusive evidence for two amino sugars in the terminal disaccharide of the molecule.

Carbohydrate composition and sequence analysis of cell surface components by mass spectrometry. Characterization of the major monosialoganglioside of brain

FEBS LettersVolume 32, Issue 2 p. 317-320 Full-length articleFree Access Carbohydrate composition and sequence analysis of cell surface components by mass spectrometry. Characterization of the major monosialoganglioside of brain Karl-Anders Karlsson, Karl-Anders Karlsson Department of Medical Biochemistry, University of Göteborg, Fack, S-400 33 Göteborg 33, SwedenSearch for more papers by this author Karl-Anders Karlsson, Karl-Anders Karlsson Department of Medical Biochemistry, University of Göteborg, Fack, S-400 33 Göteborg 33, SwedenSearch for more papers by this author First published: June 01, 1973 https://doi.org/10.1016/0014-5793(73)80863-4Citations: 58AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 S.-I. Hakomori, G.D. Strycharz, Biochemistry, 7, (1968), 1279– 2 B. Siddiqui, S.-I. Hakomori, J. Biol. Chem., 246, (1971), 5766– 3 H.R. Herschman, C.F. Fox A.D. Keith Membrane molecular biology (1972), Sinauer Ass. Publishers Stamford, Conn. USA 471– 4 S.-I. Hakomori, Chem. Phys. Lipids, 5, (1970), 96– 5 S. Roseman, Chem. Phys. Lipids, 5, (1970), 270– 6 S. Roth, E.J. McGuire, S. Roseman, J. Cell Biol., 51, (1971), 536– 7 K.-A. Karlsson, I. Pascher, B.E. Samuelsson, G.O. Steen, Chem. Phys. Lipids, 9, (1972), 230– 8 G. Dawson, C.C. Sweeley, J. Lipid Res., 12, (1971), 56– 9 S. Hammarström, B. Samuelsson, J. Biol. Chem., 247, (1972), 1001– 10 H. Björndal, C.G. Hellerquist, B. Lindberg, S. Svensson, Angew. Chem., 9, (1970), 610– 11 S.-I. Hakomori, J. Biochem., 44, (1964), 205– 12 H. Wiegandt, Adv. Lipid Res., 9, (1971), 249– 13 S.-I. Hakomori, K. Stellner, K. Watanabe, Biochem. Biophys. Res. Commun., 49, (1972), 1061– Citing Literature Volume32, Issue2June 01, 1973Pages 317-320 ReferencesRelatedInformation