Glycopeptide Bonds Research Articles

Extraction, purification, characterization, and rheological properties of a glycoprotein from Cynomorium songaricum Rupr.

Cynomorium songaricum Rupr is widely known in China as a traditional herbal medicine. In this study, single-factor experiments and response surface methodology were used to optimize the extraction of Cynomorium songaricum Rupr glycoprotein (CSG). The results show that a maximum glycoprotein yield of 6.39±0.32% was achieved at a ratio of solid to liquid 32:1 for 4.2H at 52°C. Then, the IR, monosaccharide composition, amino acid composition, type of glycopeptide linkage, and average molecular weight of CSG-1 purified from CSG were characterized. The results indicate that CSG-1 presented the characteristic absorption peak of polysaccharide and protein, including four monosaccharides and 17 amino acids, had O-linked glycopeptide bonds, Mw , Wn , Mw /Mn , Mp , and the z-average were 5.343×106 , 3.203×106 , 1.668, 8.911×106 , and 6.948×106 , respectively. Besides, CSG-1 solution was described by the Herschel-Bulkley model and it behaved as a shear-thinning fluid. Also, under a frequency sweep the moduli G' and G″ both increased with increasing CSG-1 concentration and the CSG-1 dispersions had weak thermal stability over the temperature sweep. These results provide a scientific basis for the further study of Cynomorium songaricum Rupr.

Method development of glycoprotein biomarkers for cancers.

Method development of glycoprotein biomarkers for cancers.

Physicochemical property and structural characteristics of glycoprotein from Camellia oleifera Abel seed cake

The glycoprotein was extracted from defatted cake of Camellia oleifera Abel and purified by column chromatography. Physicochemical property and structural characteristics of the glycoprotein were investigated by color qualitative reaction, isoelectric point (PI) test, SDS-PAGE, β-elimination reaction, fourier transform infrared (FTIR) spectroscopy and automatic amino acid analyzer. The results showed that the glycoprotein was a kind of acidic polysaccharide, its relative molecular weight was 2436ku and PI value was 3.6. The type of glycopeptide bond oligosaccharide was O-linkage, and glycosidic bond was pyranose α-glycoside linkage type. The glycoprotein by IR analysis showed the typical absorption of polysaccharide and the presence of was indicated. The glycoprotein determined by automatic amino acid analyzer showed the glycoprotein also contained all human essential amino acids and the proportion was appropriate, which was of great value to develop. The physicochemical property and structural characteristics of glycoprotein from Camellia oleifera Abel seed cake was investigated in this paper, which provided theory basis for further research.

Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.

Formation of the sugar-amino acid linkage is a crucial event in the biosynthesis of the carbohydrate units of glycoproteins. It sets into motion a complex series of posttranslational enzymatic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biological functions. These reactions occur throughout the entire phylogenetic spectrum, ranging from archaea and eubacteria to eukaryotes. It is the aim of this review to describe the glycopeptide linkages that have been found to date and specify their presence on well-characterized glycoproteins. A survey is also made of the enzymes involved in the formation of the various glycopeptide bonds as well as the site of their intracellular action and their affinity for particular peptide domains is evaluated. This examination indicates that 13 different monosaccharides and 8 amino acids are involved in glycoprotein linkages leading to a total of at least 41 bonds, if the anomeric configurations, the phosphoglycosyl linkages, as well as the GPI (glycophosphatidylinositol) phosphoethanolamine bridge are also considered. These bonds represent the products of N- and O-glycosylation, C-mannosylation, phosphoglycation, and glypiation. Currently at least 16 enzymes involved in their formation have been identified and in many cases cloned. Their intracellular site of action varies and includes the endoplasmic reticulum, Golgi apparatus, cytosol, and nucleus. With the exception of the Asn-linked carbohydrate and the GPI anchor, which are transferred to the polypeptide en bloc, the sugar-amino acid linkages are formed by the enzymatic transfer of an activated monosaccharide directly to the protein. This review also deals briefly with glycosidases, which are involved in physiologically important cleavages of glycopeptide bonds in higher organisms, and with a number of human disease states in which defects in enzymatic transfer of saccharides to protein have been implicated.

NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.

Demonstration of a glycoprotein derived from the 24p3 gene in mouse uterine luminal fluid

A glycoprotein in mouse uterine luminal fluid was purified to homogeneity via a series of purification steps involving Sephadex G-100 chromatography, Sephadex G-50 chromatography and HPLC on a reverse-phase C18 column, in that order. Automated Edman degradation was unable to determine the N-terminal residue of the glycoprotein and the partial sequences determined from its trypsin digests were found to be identical with the protein sequence deduced from 24p3 cDNA. The core protein and the total amount of carbohydrate together gave a molecular mass of 25.8 kDa. Results from the characterization of the glycopeptide bond indicated the presence of N-linked carbohydrate but no O-linked carbohydrate in the protein, which has two potential sites for N-linked carbohydrate at Asn81 and Asn85, as deduced from analysis of the primary structure. The core protein was shown to have a molecular mass equal to that of the putative protein deduced from cDNA, suggesting that this protein may contain no signal peptide. Results of Northern-blot analysis for various tissues of adult mice revealed that the 24p3 gene was expressed in lung, spleen, uterus, vagina and epididymis.

A Strategy for a Convergent Synthesis of N-Linked Glycopeptides on a Solid Support

Oligosaccharides and glycopeptides are of considerable importance in molecular biology and pharmacology. However, their synthesis is complicated by the large number of different linking sites between each saccharide unit, the need for stereochemical control, the chemical sensitivity of the glycopeptide bonds, and the need to harmonize diverse protecting groups. Here, an efficient solid-phase synthesis of three N-linked glycopeptides based on glycal assembly is presented. The peptide domain can be extended while the ensemble remains bound to the polymer. The glycopeptides synthesized here are among the largest N-linked glycopeptides ever accessed by either solution- or solid-phase synthesis.

Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum.

The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo-beta-N-acetylglucosaminidase H, and particularly peptide N-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS)

Methods for the identification of N-asparaginyl and O-seryl/threonyl glycosidic linkages to aminosugars in glycoproteins

Methods are presented for the identification of certain glycopeptide bonds in glycoproteins. Mucin-type linkages are determined following treatment of glycoproteins with alkaline sodium [ 3H]borohydride. Such treatment cleaves O-glycosidic bonds to serine and threonine and simultaneously labels the sugar and amino acid components of the linkage. Following acid hydrolysis and dansylation, the sugar component of the linkage is identified as its corresponding dansyl-hexosaminitol by fluorographic techniques. A method is described for the separation of dansyl-galactosaminitol and dansyl-glucosaminitol by thin-layer electrophoresis in borate buffers. The amino acid component of the glycopeptide linkage is identified by fluorography following two-dimensional thin-layer chromatography of its dansyl derivative on polyamide plates. For the analysis of plasma-type glycoproteins, glycopeptides are prepared by exhaustive pronase digestion and purified by gel filtration chromatography. Final purification is effected by dansylation and thin-layer electrophoresis. The linkage compound 2-acetamido-1- N-β- l-aspartyl-2-deoxy-β- d-glucopyranosylamine is isolated from such glycopeptides as its dansyl derivative following partial acid hydrolysis. Its identity is confirmed by comparison of its properties with those of the synthetic compound. Thus the components of the glycosylamine linkage are identified following complete acid hydrolysis, redansylation, and separation by thin-layer electrophoresis.

Characterization of the glycopeptide bond in legumin from Pisum sativum L

Legumin and vicilin, the reserve storage globulins in pea cotyledons, are glycoproteins containing neutral and amino sugars. The amino sugar has been characterized as glucosamine and the neutral sugars are principally mannose [ 1 ] . In native glycoproteins, glucosamine usually occurs as an acetylated derivative (N-acetyl glucosamine) and is frequently involved in glycopeptide linkages [2]. Acetylated amino sugars are involved in glycopeptide linkages with the hydroxy amino acids serine or threonine or alternatively, with peptidyl asparagine [3]. The glycopeptide linkage in plant glucosamine-containing glycoproteins is considered in [4-j]. To extend our studies concerning the biosynthesis of glucosamine-containing glycoproteins [ 1,8-IO] , it is necessary to determine the nature of the glycopeptide linkage. Exogenously-supplied glucosamine is incorporated into legumin and vicillin in developing pea cotyledons [ 1 ] . We now report that some of this exogenously-supplied glucosamine may be isolated from legumin as a GlcNAc-Asn glycopeptide linkage.

Water-soluble glycoprotein from Urtica dioica leaves

A neutral and an acidic carbohydrate-protein polymer were isolated from leaves of Urtica dioica. The neutral fraction was a glycoprotein, containing the serine- O-galactoside glycopeptide bond. Methylation analysis revealed a highly branched structure, arabinose constituting the exterior and mainly galactose the interior part of the carbohydrate moiety. Galacturonic acid was the major component of the acidic fraction.

Studies on the Carbohydrate Units of Thyroglobulin: EVALUATION OF THEIR MICROHETEROGENEITY IN THE HUMAN AND CALF PROTEINS

Information has been obtained in regard to the carbohydrate units of human and calf thyroglobulin through a study of the glycopeptides obtained after extensive Pronase digestion of these proteins. Human thyroglobulin was shown to contain, as has been previously reported for the calf protein, carbohydrate units consisting only of mannose and N-acetylglucosamine (unit A) as well as more complex units consisting of mannose, Nacetylglucosamine, galactose, N-acetylneuraminic acid, and fucose (unit B). The units A of the human and calf thyroglobulin were similar, consisting on an average of 8 to 9 residues of mannose and 2 of N-acetylglucosamine. There were 7 to 8 units of this type in the human protein and 5 to 6 in the calf. Chromatography of human and calf unit A glycopeptides on Dowex 50-X2 revealed extensive microheterogeneity, with the mannose content varying from 5 to 11 residues per unit, but with the glucosamine content remaining constant at 2 residues in each. The average unit B of the human thyroglobulin was found to consist of 3 mannose, 4 N-acetylglucosamine, 2 to 3 galactose, 1 fucose, and 1 sialic acid residues. It differed from that previously reported for the calf in containing fewer residues of sialic acid, galactose, and glucosamine. There were found to be 22 units of the B type in the human thyroglobulin compared with 13 to 14 of these units in the calf protein. The human unit B was also found to exhibit pronounced microheterogeneity, as was revealed by fractionation of its glycopeptides on DEAE-cellulose. The variation was primarily evident in the sialic acid, galactose, and glucosamine residues, with the mannose of the core portion remaining constant at 3 residues. The galactosamine residues of the human thyroglobulin were shown, by density gradient centrifugation, to be an integral part of this protein and to occur in units distinct from carbohydrate units A and B, which have been designated as unit C. Alkaline sulfite treatment of glycopeptides containing galactosamine suggested that the C type of unit is linked to serine and threonine residues by O-glycosidic bonds, in contrast to the glycosylamine type of linkage to asparagine which attaches units A and B to the peptide chain. During Pronase digestion, some of the glycopeptide bonds linking unit A were split, yielding neutral oligosaccharides containing 7 to 8 mannose and 2 N-acetylglucosamine residues, with 1 of the latter in a reducing terminal position.