A third glycopeptide (nephritogenoside) isolated from the glomerular basement membrane

A new glycopeptide was isolated from the glomerular basement membrane (GBM) of normal rats. Unlike already known glycopeptides, this glycopeptide has biological activity (nephritogenic activity) to induce glomerulonephritis when injected once into the footpads of homologous animals. A close relationship was found between the nephritogenic activity and the non-dialyzable glucose content of this glycopeptide. Thus the nephritogenic activity can be assessed quantitatively by estimating the content of "non-dialyzable glucose." Chemical purification of the nephritogenic glycopeptide involved the selective removal of inactive glycopeptide containing galactose, mannose, and N-acetyl-glucosamine (but no glucose). Trichloroacetic acid (TCA) treatment was a simple but highly effective procedure for selective removal of this inactive glycopeptide. The non-reducing terminus of the nephritogenic glycopeptide is alpha-D-glucopyranoside, and the glycopeptide reacts specifically with concanavalin A, even in the crude state. We propose that the nephritogenic glycopeptide is not an artifact produced during exhaustive proteolytic digestion, but a natural substance having a fixed molecular shape, even in the crude state, and whose union with GBM-proper can be easily broken by proteolytic digestion.     

Effect of trypsin on the cell surface proteins of hepatoma tissue culture cells. Characterization of a carbohydrate-rich glycopeptide released from a calcium binding membrane glycoprotein.

Concentrations of trypsin that bring about aggregation of hepatoma tissue culture (HTC) cells also release from the cell surface an Mr = 55,000 glycopeptide fragment. This glycopeptide fragment also accumulates in the medium, including serum-free medium, as a normal consequence of membrane protein turnover. The trypsin-released glycopeptide is labeled when cells are grown in the presence of fucose or leucine before treatment of the cells with the protease. Similarly, the glycopeptide fragment can be labeled by reacting cells in situ by lactoperoxidase-catalyzed radioiodination or by tritiated borohydride reduction of cells treated first with neuraminidase and galactose oxidase. The tryptic glycopeptide fragment was purified by concanavalin A-Sepharose chromatography, and hydroxyapatite chromatography in the presence of dodecyl sulfate. The amino acid and carbohydrate composition was determined, as was the sensitivity of the purified glycopeptide to a variety of endo- and exoglycosidases. The purified glycopeptide contains an average of 17 sialic acid residues and hence, shows charge heterogeneity after electrophoresis in isoelectric focusing gels. The charge heterogeneity can be eliminated completely by treatment with neuraminidase. The glycopeptide after this treatment is homogeneous. The trypsin-sensitive membrane glycoprotein which is the source of the Mr = 55,000 glycopeptide was identified by two-dimensional gel electrophoretic analysis of labeled cells, treated or not treated with trypsin. This glycoprotein, which has an apparent molecular weight of 85,000 and forms a homodimer in the presence of calcium ions, was purified and its identity as the parent of the Mr = 55,000 glycopeptide was confirmed by showing that the same Mr = 55,000 fragment was released by trypsin from the purified glycoprotein as was released from the intact cells.     

Chemoenzymatic synthesis of a MUC1 glycopeptide carrying non-natural sialyl TF-beta O-glycan

A MUC1 type of glycopeptide was synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol using benzyl and benzylidene-protected beta-D-Gal-(1-->3)-beta-D-GalNAc-Ser/Thr (TF-beta: a stereoisomer of the Thomsen-Friedenreich antigen). The synthetic glycopeptide was released from the resin with reagent K, and the resulting benzylated glycopeptide was deprotected under conditions of low-acidity trifluoromethanesulfonic acid (TfOH). The glycopeptide carrying duplicate non-natural O-glycans was dominant in the products, but was accompanied by a considerable amount of the glycopeptide missing one of the O-glycans. In contrast, the native alpha-glycoside was relatively stable under the acidic debenzylation conditions as shown by a parallel experiment with the glycopeptide involving alpha-D-GalNAc-Ser/Thr linkage. Enzymatic glycosylation with CMP-NeuAc was successful with both natural and non-natural O-glycans of the synthetic glycopeptide.     

Hydrogen-bonded structure of the complex N-linked fetuin glycopeptide

The conformation of the N-linked complex glycopeptide of fetuin was examined with hydrogen-exchange techniques. The glycopeptide molecule contains eight acetamido hydrogens stemming from five N-acetylglucosamine residues and three N-acetylneuraminic acid residues and also one from the remaining sugar-peptide linkage. The hydrogen-exchange rates of these secondary amides were compared with small molecule model compounds having identical primary structures at their exchangeable hydrogen sites. Differences between the model rates and glycopeptide rates therefore cannot be accounted for by primary structure effects but reflect conformational features of the glycopeptide. Two glycopeptide hydrogens exhibit significantly hindered exchange; the rest exchange at the model rates. Removal of the three N-acetylneuraminic acid residues from terminal positions on the three branches of the glycopeptide removes the slowed hydrogens. The remaining ones continue to exchange at the model rate. These results indicate that two of the eight sugar acetamido hydrogens are involved in intramolecular hydrogen bonds. A likely structure includes two hydrogen bonds between the three N-acetylneuraminic acid residues. These two hydrogens, slowed to a moderate degree, reflect a preferred conformation stabilized by about 1 kcal/mol in free energy. The solution conformation of the glycopeptide suggested by these results is one that is partially ordered and can be easily modulated, owing to the relatively small amount of energy stabilizing the preferred conformation.     

The comparison of an acidic glycopeptide from the ascitic tumor fluid and of the glycopeptide from Ehrlich cells

An acidic glycopeptide of molecular weight of 12000 daltons and of pI 5.6 from the ascitic tumor fluid of Ehrlich cells was isolated and characterized with regard to its physico-chemical properties and compared with an acidic glycopeptide of Ehrlich cells. Both glycopeptides have the same carbohydrate and amino acid components but differ in respect of quantity. The origin of an acidic glycopeptide from the ascitic tumor fluid of Ehrlich cells is briefly discussed.     

Studies of the diphtheria toxin receptor on chinese hamster cells

Concanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β-N-acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell-surface receptor analogue. Diphtheria toxin-resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10-to 15-fold more resistant to diphtheria toxin than wild-type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild-type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell-surface receptor.     

Comparison of glycopeptide in lung cancer tissues of different histological types

The glycopeptide was studied in the pulmonary cancer tissues of three different histological types including squamous cell carcinoma, undifferentiated cell carcinoma, and adenocarcinoma. The glycopeptide preparation was made by digesting the lung cancer tissues with proteolytic enzymes followed by removal of enzymes and undigested protein. The comparison of the relative proportion of the glycopeptide, which migrated toward the anode slower than hyaluronic acid, and was stained with both alcian blue and PAS, and contained higher sialic acid, was carried out among three different histologic types of lung cancer. This glycopeptide was highest in adenocarcinoma, followed by undifferentiated cell carcinoma, and was lowest in squamous cell carcinoma.     

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

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.     

A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.     

放线菌来源的糖肽类抗生素的药学机制研究进展

糖肽类抗生素具有较好的抑制革兰氏阳性细菌生长的活性,临床上广泛用于治疗革兰氏阳性细菌导致的严重感染性疾病,也被认为是对抗这类顽固性病原菌的最后一道防线。随着耐药菌的不断涌现,糖肽类抗生素的应用越来越受到限制。本文针对糖肽类抗生素的结构特征与药效关系、生物学活性和病原菌对于它们的耐药机制,以及糖肽类抗生素的生物合成机制及其结构的合成生物学改造等方面进行了概述。最后,对糖肽类抗生素在应用中面临的问题进行了展望。     

Enzymatic sulfation of exogenous high molecular weight glycopeptides by microsomal fraction of the rabbit uterine endometrium

Incorporation of radioactive sulfate into exogenous glycopeptides and glycoproteins from adenosine 3'-phosphate 5'-phospho[35S]sulfate was studied with the microsomal fraction of the uterine endometrium of rabbits. A high molecular weight (Mr greater than 750,000) glycopeptide fraction from rat adenocarcinoma was found to be active as substrate, while several other glycoproteins were not. The rate of incorporation of sulfate was almost proportional to the concentration of glycopeptide substrate, the quantity of microsomal fraction, and the length of the incubation period. Cellulose acetate membrane electrophoresis demonstrated that radioactivity was incorporated into the glycopeptide fraction. The radioactive glycopeptide was excluded from a Sephadex G-50 column, but the 35S radioactivity and oligosaccharides were found in the retarded fractions after treatment with alkali in the absence of sodium borohydride. These observations indicated the presence of an enzyme which catalyzes the transfer of sulfate residue from adenosine 3'-phosphate 5'-phosphosulfate into the carbohydrate units attached to the polypeptide via O-glycosidic linkage. The sulfotransferase activity was measured with the microsomal fractions from the animals which had been treated with female sex hormones. As a result, it was found that estrogen enhances the activity and that progesterone suppresses the effect of estrogen.     

Structure of the tryptic glycopeptide isolated from rabbit transferrin.

The structure of the tryptic glycopeptide isolated from rabbit transferrin was elucidated by use of sequential Edman degradations, specific exoglycosidases, endo-beta-N-acetylglucosaminidases, methylation analyses, and periodate oxidation studies. The glycopeptide consists of a heteropolysaccharide, AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3[AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6]-Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc, attached to a peptide, Asn-Ser-Ser-Leu-Cys, via a linkage involving N-acetyl-glucosamine and asparagine. The stoichiometry of this glycopeptide is 2 mol/mol of protein, indicating that rabbit transferrin contains two structurally identical glycopeptide segments.     

Cell surface glycoproteins I: Accumulation of a glycoprotein on the outer surface of mouse LS cells during mitosis

Surface glycopeptides derived from vertebrate cells have been separated into 4 classes by chromatography on DEAE cellulose columns. Among different cell types tested, significant differences were observed in the relative amounts of these 4 glycopeptide classes present on the cell surface. This type of heterogeneity is consistent with the expected biological role of cell surface glycoproteins. One glycopeptide, as revealed by the DEAE column analysis, was found to have a characteristic metabolic pattern in mouse LS cells. New accumulation of this structure, called glycopeptide 4, on the cell surface was detected only around the period of cell division (M phase) and not at other times during the cell cycle.     

Solid-phase synthesis of core 8 O-glycan-linked MUC5AC glycopeptide

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.     

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.     

Structure of the carbohydrate chain of free secretory component from human milk.

Secretory component from human milk was found to contain 23.4% carbohydrate, which includes galactose, mannose, fucose, glucosamine, and sialic acid. Secretory component could be degraded by pronase or base-borohydride to yield the same, single type of carbohydrate chain. In the glycopeptide produced by pronase digestion, aspartic acid was the only amino acid present in molar quantities after amino acid analysis, which suggests that the carbohydrate moiety is linked to the polypeptide chain at asparagine residues. The positions of links between the various sugar units were studied by methylation analyses of: secretory component, periodate-oxidized and reduced secretory component, the fragment produced by base-borohydride treatment, and the pronase glycopeptide after treatment with specific glycosidases. Sugars released from the glycopeptide by various glycosidases were also quantitated. From the results of these studies a branched chain structure was assigned to the carbohydrate chain of secretory component.