Studies on the carbohydrate-protein linkage region in bovine corneal keratan sulfate. I. Isolation of linkage-region glycopeptides under mild conditions

Isolation of linkage-region glycopeptides from corneal peptidokeratan sulfate was attempted under mild conditions. Peptidokeratan sulfate, which had been found in advance of the present study to contain three mannose residues per chain as a major component of the carbohydrate-protein linkage region, was digested with Pseudomonas endo-beta-galactosidase. The disaccharide-repeating chain was partially hydrolyzed, and almost all the galactose and N-acetylglucosamine residues were found in oligosaccharides of various sizes. The resulting linkage region-enriched glycopeptides were separated by gel filtration from these oligosaccharides and then fractionated by DEAE-cellulose and Dowex 50 chromatography with the guidance of the mannose content. The glycopeptides obtained were highly enriched in the linkage region and a large portion of them was free from sulfate groups, suggesting that they could be used to elucidate the structure of the linkage region.     

Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose.

Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.     

Heterogeneity of bovine corneal proteokeratan sulfate

Proteokeratan sulfate was extracted and purified from bovine corneal stroma and then characterized by chemical and biochemical analyses. It was fractionated into several fractions by affinity chromatography on a concanavalin A-Sepharose column or by hydrophobic chromatography on a phenyl-Sepharose column. These fractions differed widely from one another in carbohydrate content, though no significant differences of their amino acid compositions were observed. One fraction (ca. 25%, on a dry weight basis) tightly bound to a concanavalin A-Sepharose column, compared with another fraction (ca. 65%) weakly bound to the same column, was poor in galactose and N-acetylglucosamine, but contained mannose in a high proportion. Fractions (ca. 30%) tightly bound to a phenyl-Sepharose column, in contrast to the one (ca. 66%) weakly bound, had low carbohydrate contents, like the fraction tightly bound to a concanavalin A-Sepharose column. Additionally, the fractions tightly bound to these affinity columns exhibited strong inhibitory actions on erythrocyte-concanavalin A agglutination. To obtain further details of the carbohydrate moiety of the proteokeratan sulfate, an attempt was made to separate and characterize peptidokeratan sulfate and Asn-linked oligosaccharide derived from some proteokeratan sulfate fractions. The present work revealed that the proteokeratan sulfate contains keratan sulfate and high mannose-type oligosaccharide in an approximate chain number ratio of 3.5:1.0, the keratan sulfate content varies widely and the oligosaccharide content increases with decrease of the keratan sulfate content, and the protein core is homogeneous at least with respect to the amino acid composition.     

Structure of the oligosaccharide chains in human alpha 1-protease inhibitor.

Two glycopeptides were obtained from alpha 1-protease inhibitor after extensive pronase digestion and chromatography on Bio-Gel P-10 and concanavalin A-Sepharose. these glycopeptides were characterized by compositional analysis and sequential exoglycosidase digestion followed at each step by methylation analysis. The partially methylated alditol acetates obtained were resolved by gas chromatography and identified by mass spectrometry. The proposes structures of the oligosaccharide moieties of the glycopeptides are given below. (formula: see text) The relative amounts of the two glycopeptides isolated from concanavalin A-Sepharose suggest that each protein molecule contains four carbohydrate chains; one large chain (A) and three small chains (B).     

Separation of the integral membrane glycoproteins E1 and E2 of Semliki Forest virus by affinity chromatography on concanavalin A-Sepharose.

The membrane glycoproteins E1 and E2 of Semliki Forest virus are of about equal size but can be separated from each other by affinity chromatography on a concanavalin A-Sepharose column in the presence of sodium dodecyl sulfate. The E1 protein eluted like glycopeptides containing two peripheral sugar branches composed of N-acetylglucosamine, mannose, galactose and sialic acid. The E2 eluted like glycopeptides containing only N-acetylglucosamine and mannose.     

Alterations in fucosyl oligosaccharides of glycoproteins during rat liver regeneration.

[3H]Fucose-labelled glycopeptides in the slices of liver 24h after partial hepatectomy were fractionated on Sephadex G-50. Glycopeptides from regenerating liver contained a higher proportion of lower-Mr components than did controls. Regenerating liver contained a higher proportion of glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside than did controls. Concanavalin A-bound glycopeptides from each source were entirely bound to a lentil lectin-Sepharose column. Both the concanavalin A-bound and -unbound fractions from regenerating liver were indistinguishable from the respective controls by Bio-Gel P6 column chromatography and neuraminidase digestion. These results show that fucosyl glycopeptides from regenerating liver contain a higher proportion of biantennary species with core fucose residues than do controls. Glycopeptides from regenerating livers 12h, 72h and 144h after partial hepatectomy were also examined; however, the difference was not significant. These observations suggest that the alterations in fucosyl glycopeptides may be related to rapid growth of hepatocytes 24h after partial hepatectomy. No significant difference was found in either [3H]mannose- or [3H]fucose-labelled glycoproteins from regenerating liver and from controls by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, suggesting that the alteration in glycopeptides should depend on some differences in the late stage of oligosaccharide processing.     

Studies on haptoglobin binding to concanavalin A

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.     

Postnatal changes in N-linked oligosaccharides of glycoproteins in rat liver.

[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.     

Hybrid sialylated N-glycans are minor constituents of normal BHK-cell glycoproteins and a prominent feature in glycoproteins of some ricin-resistant cell lines.

Baby-hamster kidney (BHK) cells were labelled metabolically by growth in media containing radioactive sugars and the asparagine-linked glycopeptides (N-glycans) obtained by Pronase digestion of disrupted cells were fractionated by chromatography on concanavalin A-Sepharose. About 2-3% of the total [3H]galactose- or [3H]fucose-labelled glycopeptides were found to be bound tightly to the lectin column and were eluted with 500 mM-methyl alpha-mannoside. Further analysis of these minor components by chromatography on Bio-Gel P4, lentil-lectin-Sepharose and DEAE-Sephacel and sensitivity to alpha-mannosidase indicates the presence in BHK-cell glycopeptides of hybrid structures of the following form: (Formula: see text) Similar structures were identified as major features of the glycoproteins of ricin-resistant mutants RicR17 and RicR19 as described previously for RicR21 cells [Hughes, Mills & Stojanovic (1983) Carbohydr. Res. 120, 215-234]. The RicR15 cell line also produces significant amounts of hybrid N-glycans. The studies show that the novel N-glycans accumulating in ricin-resistant mutants are derived by a metabolic pathway that exists to a minor extent in normal BHK cells.     

Studies on the characterization of the linkage-region between polysaccharide chain and core protein in bovine corneal proteokeratan sulfate

1) A new method of enrichment of the linkage-region in corneal proteokeratan sulfate is described, which consists of desulfation of peptidokeratan sulfate, followed by chromatography on Con A-Sepharose 4B and enzymatic degradation with beta-D galactosidase and beta-N-acetyl-D-glucosaminidase. 2) After permethylation, hydrolysis, reduction with sodium borohydrid and acetylation gas chromatography/mass spectrometry analyses were performed. The followings products could be detected as their peracetates: 2,3,4-tri-O-methylfucitol; 2,3,4,6-tetra-O-methylmannitol; 3,4,6-tri-O-methylmannitol; 2,4-di-O-methylmannitol; 2,3,4,6-tetra-O-methylgalactitol; 2,4,6-tri-O-methylgalactitol; 2,4-di-O-methylgalactitol. 3) The results point to the presence of a branched linkage region in the proteokeratan sulfate molecule with one mannose as the branching point and two mannose residues as the starting point of two disaccharide chains.     

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

小鼠Lewis肺癌组织中糖肽的分离纯化及其对肿瘤细胞与层粘连蛋白基质粘着的影响

小鼠Lewis肺癌组织经氯仿甲醇去除脂类,用木瓜蛋白酶消化,再经Sephadex柱层析分离得到总糖肽。它有明显地抑制小鼠Lewis肺癌细胞,S180肉瘤细胞及人巨细胞肺癌细胞与层粘连蛋白(Laminin,LN)基质粘着的作用;对层粘连蛋白受体(LN-R)与其配体的识别及结合也具有同样明显的阻断效应。糖肽的上述作用均具有剂量依赖性。进一步经ConA-Sepharose CL-4B亲和层析将总糖肽分为三个部分。与ConA不结合的糖肽部分对Lewis肺癌细胞与LN基质的粘着也具有剂量性抑制作用。     

Isolation of mammalian tRNAAsp and tRNATyr by lectin-Sepharose affinity column chromatography.

tRNAAsp from rabbit liver, rat liver and rat ascites hepatoma was readily isolated by concanavalin A-Sepharose (Con A-Sepharose) affinity column chromatography. tRNATyr from these sources was extensively purified by Ricinus communis lectin-Sepharose column chromatography. These results, together with the chromatographic behaviour of four tRNAs (tRNATyr, tRNAHis, tRNAAsn and tRNAAsp) on acetylated DBAE-cellulose column chromatography suggested that tRNAAsp contains a Q nucleoside species having a mannose moiety while tRNATyr contains Q nucleoside with galactose. The sugars attached in 4-position of cyclopentene diol in the Q molecule are therefore not present at random in the four tRNAs, but present only in each specific tRNA. This is the first case which shows that plant agglutinin interacts with nucleic Acid as well as polysaccharide and glycoproteins.     

Endo-N-acetyl-beta-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification.

Endo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.96, endoglucosaminidase) has been partially purified (520-fold with respect to the cytoplasmic activity) by using concanavalin A-Sepharose, CM-Sephadex and Bio-Gel P-150 chromatography. From the influence of exogenous glycopeptides on the endoglucosaminidase activity it can be concluded that this activity consists of one enzyme hydrolysing both N-acetyl-lactosaminic-type and oligomannosidic-type substrates. Glycoproteins present in the homogenate inhibit the endoglucosaminidase activity. On re-examination of the subcellular distribution of endoglucosaminidase (after removal of inhibiting glycoproteins from the respective subcellular fractions), its cytoplasmic localization was confirmed.     

Changes in cell-surface fucose-containing glycopeptides and adhesion of cultured intestinal epithelial cells as a function of cell density.

Confluent cultured intestinal epithelial cells displayed greater adhesion to the substratum than did subconfluent cells. Subconfluent and confluent cells were labelled with [3H]fucose for 24h and the cell-surface components were released by mild Pronase treatment. After extensive Pronase digestion, cell-surface and cell-residue glycopeptides were fractionated on Bio-Gel P-6. The cell surface contained a higher proportion of lower-molecular-weight glycopeptides than the residue. No significant difference in elution pattern was found between total cell-surface glycopeptides of subconfluent and confluent cells. However, confluent cells contained almost twice as much [3H]-fucose-labelled glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside as subconfluent cells. When the bound glycopeptides were chromatographed on Bio-Gel P-6, it was found that confluent cells contained a larger proportion of lower-molecular-weight glycopeptides than subconfluent cells. This difference in size was eliminated after treatment of glycopeptides with sialidase. When growth of subconfluent cells was inhibited with a non-toxic concentration of retinoic acid, no significant effect on the elution pattern of [3H]fucose-labelled glycopeptides was observed on either Bio-Gel P-6 or concanavalin A-Sepharose. No significant difference was found in the total [3H]fucose-labelled glycoproteins from subconfluent and confluent cells by two-dimensional gel electrophoresis. It is suggested that the differences in [3H]fucose-labelled glycopeptides between subconfluent and confluent cells are cell-density-dependent rather than growth-dependent, and that these differences are likely to result from some changes in glycosylation mechanism(s). Furthermore, the differences in cell-surface glycopeptides may be related to the changes in the adhesion of the cells to the substratum.     

Carbohydrate heterogeneity of vesicular stomatitis virus G glycoprotein allows localization of the defect in a glycosylation mutant of CHO cells

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N-acetylglucosamine residue linked β1,6 to mannose.     

Temporal aspects of O-glycosylation of glycoprotein C from herpes simplex virus type-1.

Herpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.