Type Glycopeptides Research Articles

Interactions of concanavalin a with a trimannosyl oligosaccharide fragment of complex and high mannose type glycopeptides

It has previously been reported that the binding interactions of concanavalin A with a purified high mannose type glycopeptide from ovalbumin differs from that with simple mono- and oligosaccharides (Brewer, C.F. (1979) Biochem. Biophys. Res. Commun. 90, 117-122). We now report studies with a synthetic analog of complex type glycopeptides, and a synthetic trimannosyl oligosaccharide fragment that is common to both complex and high mannose type glycopeptides. We find that both synthetic oligosacchardes undergo similar interactions with concanavalin A which mimic the effects of binding corresponding larger glycopeptides. Furthermore, the relative affinity of the trimannosyl oligosaccharide is 130-fold greater than the binding of methyl-alpha-D-mannopyranoside. The results indicate that the trimannosyl oligosaccharide is a unique structural element recognized by the lectin.

Structural basis for the affinity of four insolubilized lectins, with a specificity for α-D-mannose, towards various glycopeptides with the N-glycosylamine linkage and related oligosaccharides

Precisions are given on the fine specificity and on the usefulness of im- mobilized concanavalin A, Lens culinaris, Vicia faba and Pisum sativum agglutinins for fractionation of glycopeptides with the N-glycosylamine linkage. While insolubilized concanavalin A represents a very useful tool for the fractionation of both N-acetyllactosaminic and oligomannosidic type glycopeptides or related oligo- saccharides,immobilized Lens culinaris, as well as Vicia faba or Pisum sativum agglutinins allow the subfractionation of some N-acetyllactosaminic glycopeptide populations on the basis of the presence of an α-L-fucose residue substituting in C-6 position the N- acetylglucosamine residue involved in the N-glycosylamine bond.

Studies on the Plasma Membrane of Normal and Psoriatic Keratinocytes. 6. Cell Surface and Shed Glycoproteins

Quantification and characterization of [3H]-fucose-labeled cell surface glycoproteins are reported. Two approaches have been compared; first the analysis of glycoprotein shed spontaneously into the medium during incubation of keratinocytes in vitro, and second the study of material released by exposure of the cell to proteolytic enzymes. It is shown that psoriatic keratinocytes "shed" glycoprotein more rapidly than normal, although the material is of similar molecular weight (mainly "biantennary" transferrin type glycopeptides). By contrast, the percentage of glycoprotein released by proteolysis of psoriatic keratinocytes is normal, but the molecular weight distribution of the labeled glycopeptides is markedly altered. The abnormal turnover and composition of fucose-labeled glycoproteins from the cell surface may be related to the loss of growth control in psoriatic epidermis.

The applicability of 500-MHz high-resolution 1H-NMR spectroscopy for the structure determination of carbohydrates derived from glycoproteins

The applicability of 500-MHz high-resolution 1H-NMR spectroscopy for the structure determination of carbohydrates derived from glycoproteins

Mannose containing glycopeptides of cells derived from human teratocarcinoma (line PA 1)

Human teratocarcinoma derived cells, line PA 1, maintained in the undifferentiated state, yielded upon exhaustive pronase digestion unusually large glycopeptides (fraction A), which showed on gel filtration an apparent molecular weight larger than 7400. These glycopeptides derived from whole cell proteins carried large-sized oligosaccharides as evidenced by repeated pronase treatments, hydrazinolysis, and beta-elimination experiments. The oligosaccharides consisted of mannose, fucose, galactose, and N-acetylglucosamine. The PA 1 cells contained also oligomannosyl type glycans, presumably linked to asparagine (fraction C glycopeptides). These glycopeptides were strongly bound to Con A--Sepharose and their oligosaccharides were released by endo-beta-N-acetylglucosaminidase H. The liberated glycans ranged from Man5GlcNAc to Man9GlcNAc as analyzed by paper chromatography. "Pulse-chase" experiments suggest that there is a precursor-product relationship between the mannose label in the fraction C (oligomannosyl type) glycopeptides and the fraction A glycopeptides.

Semliki Forest virus glycans analyzed by affinity chromatography, hydrazinolysis and paper chromatography.

N-acetyl-lactosamine type glycopeptides of Semliki Forest virus were fractionated on concanavalin A-Sepharose, and their oligosaccharides were liberated by hydrazinolysis and analyzed by paper chromatography. Comparison with labeled glycans from reference glycopeptides suggests that the viral N-acetyl-lactosamine type glycans are bi-, tri- and tetra-antennary.

Sequence analysis of lactosamine type glycans of individual membrane proteins of Semliki Forest virus.

3H-fucose and 14C-glucosamine labelled glycopeptides of the individual membrane proteins E1, E2 and E3 of Semliki Forest virus could be sequentially digested with alpha-neuraminidase, beta-galactosidase, N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, N-acetyl-beta-hexosaminidase and finally with alpha-fucosidase. The degradations of the virus glycopeptides proceeded in the same way as stepwise digestions of reference glycopeptides of the lactosamine type obtained from IgG and alpha 1-acid glycoprotein. This suggests that all three membrane glycoproteins of Semliki Forest virus contained glycans with a monosaccharide sequence characteristic for lactosamine type oligosaccharides. The number of both distal and proximal N-acetyl-glucosamine residues was estimated to be usually two. According to exo- and endo-glycosidase digestions, fucose seemed to be attached to the innermost N-acetyl-glucosamine unit.

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.

Isolation and characterization of surface glycopeptides from adult rat hepatocytes in an established line

The fractionation of glycopeptides isolated from the surface of an established line of adult rat hepatocytes, after 48 h metabolic labeling with [14C] glucose leads to the demonstration of the presence of two types of glycopeptides: i) O-glycopeptides containing N-acetylneuraminic acid, galactose and N-acetylgalactosamine, ii) N-glycopeptides with mannose, galactose, N-acetylgluosamine, fucose and N-acetylneuraminic acid. The former yield, by alkaline reductive degradation, a trisaccharide: sialyl-galactosyl-N-acetyl-galactosaminitol, a tetrasaccharide : di-sialyl-galactosyl-N-acetyl-galactosaminitol and a small amount of the disaccharide: galactosyl-N-acetyl-galactosaminitol. The latter are alkali-stable and can be divided by ion-exchange chromatography on DEAE-cellulose into i) acidic glycopeptides, not retained on a Concanavalin A-Sepharose column and belonging to the N-acetyllactosaminic type glycopedtides and ii) neutral glycopeptides which can be further fractionated on the Con-A-Sepharose column into non-retained glycopeptides belonging to the N-acetyllactosaminic type, and glycopeptides retained on the Sepharose-Con A column, containing only N-acetylglucosamine and mannose residues and related to the oligomannosidic type glycopeptides.

Circular dichroism of glycopeptide fractions from α1-acid glycoprotein, thyroglobulin, and ovalbumin

Using sequential pronase digestions, glycopeptide fractions were prepared from human α 1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from α 1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sialic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of —6400 deg·cm 2·dmol −1 to —7200 deg·cm 2·dmol −1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of —29 200 deg·cm 2·dmol −1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in a particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetylglucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.

Characterization of the fragments obtained by enzymic and alkaline degradation of rice-bran proteoglycans

Repeated digestion of the proteoglycan B from rice bran with Pronase and hemicellulase yielded two types of glycopeptides. The first group, obtained from the third enzymic digest, was a type of low molecular-weight glycopeptide and was fractionated into two subtractions by paper electrophoresis. The second group, obtained from the fourth enzymic digest, was a mixture of glycopeptides of relatively high molecular-weight and was fractionated into three subfractions by gel filtration on Sephadex G-75. Alkaline degradation of these glycopeptides or the intact proteoglycan A yielded a sugar-amino acid compound, whose structure was established as O-α- L-arabinofuranosyl-hydroxyproline. These results indicate that the carbohydrate-protein linkage of the rice-bran proteoglycan is an O-glycosyl linkage between L-arabinose and hydroxyproline.

Immunological investigations of penicillium—II. Primary binding of glycopeptides and glycopeptide derivatives to specific antibodies

An exocellular glycopeptide from Penicillium charlesii has been shown previously to contain a high mol. wt polysaccharide and several oligosaccharides attached to the peptide through seryl and threonyl residues. Guinea pigs immunized with the glycopeptide conjugated to bovine gamma globulin, produced nonprecipitating antibodies to the polysaccharide portion of the glycopeptide. However, a technique was developed for measuring the primary binding of the glycopeptide by the guinea pig antibodies. It was found that the antigen-antibody complex could be precipitated by 40% ammonium sulfate, whereas negligible quantities of the glycopeptide alone or glycopeptide incubated with normal guinea pig sera were precipitated under these conditions. Equations predicting the influence of the ratio of a weighted dissociation constant representing the various antigen-antibody complexes/antibody concentrations, β/[ Ab t] , on antigen binding and the influence of this ratio on inhibition of antigen binding by hapten were derived. The data obtained with the fungal glycopeptide as an antigen were interpreted in terms of this model. The experiments showed that antibodies in the guinea pig sera were directed toward the polysaccharide portion of the glycopeptide, and not toward the peptide portion or the oligosaccharides. Furthermore, a glycopeptide deficient in galactofuranosyl and phosphorus moieties from a uridine diphosphate galactose 4-epimerase negative mutant of P. charlesii competed with wild type glycopeptide for antibody. It appeared that mannopyranosyl residues associated with an, as yet unidentified, acid labile material were the major immunodeterminant groups of the glycopeptide which bound to guinea pig sera. In contrast to its behavior in guinea pig antisera, the polysaccharide portion of the glycopeptide did not compete with the glycopeptide for antibodies in rabbit antisera. In addition the peptide portion of the glycopeptide did not compete with the glycopeptide in the rabbit antisera. Further, dilute acid treatment of the glycopeptide did not destroy its ability to bind to rabbit antibodies. These data suggest that the oligosaccharides attached to the peptide serve as the antigenic determinats which bind to rabbit antibodies.

Glycopeptides from plasma membranes and microsomes of rat liver

Previous work in this laboratory [I] has demonstrated that the structural protein fraction prepared from rat liver microsomes consists of glycoproteins with 1.5% of the carbohydrate moieties made up of mannose, galactose, glucose, hexosamine and sialic acid. For chemical characterization of the carbohydrate moieties we attempted to isolate glycopeptides by proteolytic digestion. Two types of glycopeptides, i.e. one with acidic and another with neutral oligosaccharides, could be isolated. They apparently resembled those isolated by Li et al. [2 ] from an unfractionated membranous fraction of microsomes but differed significantly in carbohydrate composition from the latter. The microsomal structural protein fraction is derived largely from proteins of endoplasmic reticulum and partly from those of plasma membranes, Although they could not be separated from each other due to close similarity in gross chemical and physical properties [l] , it seemed interesting to see if each type of oligosaccharide mentioned above might correspond to each membrane structure. There are a few methods available for isolation of plasma membranes with a high degree of purity directly from rat liver homoge-

Composition of glycopeptides obtained by proteolytic digestion of the media of porcine aorta

Glycopeptides were isolated from the pronase-collagenase digested polymeric fibrous stroma of porcine aortic media. The glycopeptides were separated by gel filtration and high voltage electrophoresis and their sugar and aminoacid composition was determined. Two major types of glycopeptides could be isolated: one (type I) similar to the heteropolysaccharides isolated from structural glycoproteins of other connective tissues containing galactose, glucose, mannose, glucosamine, galactosamine, fucose and sialic acid. The other (type II) of low molecular weight corresponding to the hydroxylysylgalactoside and hydroxylysylgalactosido-glucoside, was previously isolated from several purified collagen preparations. Type I glycopeptides turned out to be very heterogenous and the seven fractions separated by high voltage electrophoresis all had different carbohydrate compositions. One fraction was devoid of sialic acid and fucose. The sialic acid/fucose ratio increased regularly with the anodic mobility of these fractions. The following chemical parameters were estimated from these results and are considered to be characteristic of the morphological pattern of aortic media: hexose content of the polymeric stroma of aorta, 6.6%; ratio of hydroxylysylglycopeptide/ heteropolysaccharides (ratio of glycopeptides of type II/type I), ∼ 1; ratio of hydroxylysyl mono- and disaccharide ( Hyl-Gal Hyl-GaIGIc ), ∼1. These chemical parameters were previously shown to be closely related to the morphological parameters (as fiber diameter) of the fibrous stroma of different connective tissues.

Glycans linked to the insoluble fraction of the dogfish cornea (Seyliorhinus canicula L.)

The distribution of the heteropolysaccharide chains in the insoluble tissular network of the dogfish cornea was investigated. For this purpose, the corneas were extracted with a buffer solution composed of 1 M CaCl2, 0.05 M tris and 0.02 M citric acid to remove soluble proteins. The residual insoluble stroma was successively hydrolysed by collagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. Two types of sugar chains were detected:(a) polyosides composed of galactose, glucose, mannose, glucosamine, fucose, xylose and sialic acid, some of them at least linked to aspartic acid (type 1);(b) galactose or glucosidogalactose linked to hydroxylysine (type 2).For one heteropolysaccharide chain of type 1, 6 galactosidohydroxylysines and 4 glucosidogalactosidohydroxylysines were found.The insoluble stroma was submitted to a fractionated extraction procedure by trichloroacetic acid and water in order to remove all collagenous material. The residual structural glycoprotein exempt from hydroxyproline was hydrolysed by pronase and the glycopeptide fraction was isolated by exclusion chromatography and electrophoresis. A single major glycopeptide fraction of the type 1 containing galactose, glucose, mannose, xylose and glucosamine was obtained.This clear‐cut separation of the two types of glycopeptides by the tricholoroacetic acid treatment indicates that they are bound to different peptide chains: glycane of type 1 is bound to the peptide chain of structural glycoproteins (insoluble in hot trichloroacetic acid) and the glycane of type 2 is bound to the peptide chains of collagen (soluble in hot trichloroacetic acid).

The Tryptic Glycopeptides of Bovine Thyrotropin: THEIR COMPOSITION AND SIMILARITIES TO THOSE OF LUTEINIZING HORMONE

The glycopeptides of purified bovine thyrotropic and luteinizing (interstitial cell-stimulating) hormones have been obtained by tryptic digestion of their reduced and S-carboxymethylated derivatives. A combination of gel filtration and countercurrent distribution yielded, in each case, fractions which were almost exclusively glycopeptides. Three different types of glycopeptides were in this fraction from thyroid-stimulating hormone, two of which were easily isolated. Their amino acid compositions and partial sequences are: Val-Glx-Asx-(Ser, Thr2, Glx, Cys3, His2)-(Tyr2, His)-Lys and Asx-Ile-(Ser, Thr2, Glx, Cys2, Ala2, Val)-Lys. These glycopeptides have amino acid compositions identical or nearly identical with those of the two tryptic glycopeptides from 1 of the 2 subunits of bovine-luteinizing hormone and are also very similar in compositions and partial sequences to the same peptides from ovine luteinizing hormone previously reported by Ward and his colleagues (Proceedings of the Third International Congress of Endocrinology, 1968). Considerable homology thus may exist between thyroid-stimulating hormone and one chain of luteinizing hormone in regions adjacent to two oligosaccharides, and peptide maps suggest that the similarity extends to other regions. A third glycopeptide from thyroid-stimulating hormone was more difficult to purify completely. Its probable amino acid composition is: Asx, Thr4, Gly, Ala, Cys3, Val, Met, Ile, Leu, Tyr, Arg. This peptide does not seem related to the third tryptic glycopeptide of luteinizing hormone, which originates from its other subunit. Each type of thyrotropin glycopeptide contains glucosamine, galactosamine, and mannose. A fraction of a residue of fucose is found in preparations of each type and, in one type, a fraction of a residue of galactose. The yields of these glycopeptides are consistent with attachment of 3 oligosaccharide units to each molecule.

Studies on the sugar fractions of the insoluble fibrous network of the corneal stroma

Calf corneas were extracted with a buffer containing M CaCl2, 0.05 M Tris and 0.02 M citric acid, at pH 7.5. The residual stroma was digested successively by collagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. The sugar and amino acid content of the isolated glycopeptides was determined. Two types of sugar chains were detected: (a) chains with a more complicated structure, containing galactose, glucose, mannose and glucosamine; (b) disaccharidic and monosaccharidic side chains containing galactose and glucose or galactose alone and linked O‐glycosidically to the OH groups of hydroxylysine. The Hyl‐Gal‐Glc compound was isolated and identified with the compound isolated by Butler and Cunningham from calf skin collagen, by the study of its composition, thin layer chromatography and high voltage electrophoresis.The stroma of the calf cornea contains about 4 times more hexoses in the small glycopeptides of type b (di‐ and monosaccharides) than in the higher molecular weight mannose‐containing chains.