The structure of a trisialyl di-antennaryN-type glycopeptide obtained from rat plasma hemopexin

Rat hemopexin is a plasma glycoprotein that contains 18.3% carbohydrate consisting of onlyN-glycosidically-linked oligosaccharide chains. Glycopeptides obtained from hemopexin by Pronase^® digestion could be separated on Concanavalin A-Sepharose into three fractions. The lectin-binding fraction has been characterized as a mixture of monosialyl and disialyl di-antennary compounds ending inN-acetylneuraminic acid residues (2-6)-linked to galactose in the respective branches [Bernard N, Lombart C, Strecker G, Montreuil J, Van Halbeek H, Vliegenthart JFG (1983) Biochimie 65:185–92].The structures of the glycans in the Concanavalin A non-binding fractions were determined by a combination of methylation analysis and 500-MHz¹H-NMR spectroscopy. Some of them appeared to be tri-antennary glycans. However, the major component of these fractions possesses the following structure: This type of structure has been encountered before in some bovine blood coagulation factors as well as in rat -acid glycoprotein, but the¹H-NMR parameters for it are first reported here. Furthermore, by methylation analysis, the occurrence of the NeuAc2-8NeuAc disaccharide element was demonstrated in a minor part of the carbohydrate moiety of rat hemopexin. This element has also been reported previously for rat brain glycopeptides.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

The structure of nephritogenoside. A nephritogenic glycopeptide with alpha-N-glycosidic linkage

Nephritogenoside is a glycopeptide with the ability to induce chronic progressive glomerulonephritis (end stage kidney) in homologous animals, by a single footpad injection. This substance contains a novel carbohydrate-peptide linkage, i.e. the trisaccharide (3 glucose residues) chain is alpha-N-glycosidically linked to the polypeptide chain through the amido nitrogen of an asparagine residue at the N-terminal. It was found that the peptide portion of nephritogenoside is composed of twenty-one amino acids (Asn1-Pro-Leu-Phe-Gly5-Ile-Ala-Gly-Glu-Asp10-Gly-Pro-Thr-Gly-Pr o15-Ser-Gly-Ile- Val-Gly20-Gln21) and that the peptide portion has a repeated Gly-X-Y structure.     

The isolation and the structure of new glycopeptide from blood group substances

Three new glycopeptides with O-glycosidic and one glycopeptide with N-glycosylaminic carbohydrate-peptide linkages have been isolated after degradation of blood group substances (BGS). Their structure have been determined as O-(α-GalNAc)-Ser(I), O-(GalNAc)-(Pro)-Ser(II), O-(GalNAc 1 → 3 GalNAc)-(Thr-Ala)-Ser(III), N-(β-GlcNAc)-Asn(IV). The isolation of glycopeptide I confirmed α-configuration of O-glycosidic carbohydrate-peptide bonds. The structure of glycopeptide III with two galactosamine residues is in accordance with the data on the presence of hexosamine core of BGS carbohydrate chains.     

Carbohydrate structure of the major glycopeptide from human cold-insoluble globulin

Cold-insoluble globulin (CIg) is a member of a group of circulating and cell-associated, high-molecular-weight glycoproteins termed fibronectins. CIg was isolated from human plasma by affinity chromatography on gelatin-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified glycoprotein gave a double band that migrated near myosin. The CIg glycopeptides were released by pronase digestion and isolated by chromatography on Sephadex G-50. Affinity chromatography of the major G-50 peak on Con A-Sepharose resulted in two fractions: one-third of the glycopeptides were unbound and two-thirds were weakly bound (WB). Sugar composition analysis of the unbound glycopeptides by GLC of the trimethylsilyl methyl glycosides gave the following molar ratios: sialic acid, 2.5; galactose, 3.0; N-acetylglucosamine, 4.9; and mannose, 3.0. Sugar composition analysis of the WB glycopeptides gave the following molar ratios: sialic acid, 1.7; galactose, 2.0; N-acetylglucosamine, 4.1; and mannose, 3.0. The WB CIg glycopeptides cochromatographed on Sephadex G-50 with WB transferrin glycopeptides giving an estimated molecular weight of 2,800. After degradation with neuraminidase alone or sequentially with β-galactosidase the CIg and transferrin glycopeptides again cochromatographed. Methylation linkage analysis of the intact and the partially degraded glycopeptides indicated that the carbohydrate structure of the major human CIg glycopeptide resembles that of the major glycopeptide from transferrin.     

The structure of a glycopeptide (GP-II) isolated from Rhizopus saccharogenic amylase.

Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 3.2.1.24] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia, 2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II.     

A cytoskeletal structure with associated polyribosomes obtained from HeLa cells

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cell's ultrastructural features, such as 100Å filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100Å filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.     

Partial structure of a membrane glycopeptide from virus-transformed hamster cells.

The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.