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.     

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.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

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.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

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.     

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.     

Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells.

The [3H]mannose-labelled glycopeptides from two lectin-resistant lines of Chinese-hamster ovary cells were fractionated by chromatography on lentil lectin-Sepharose and concanavalin A-agarose columns and subsequently analysed by gel filtration in comparison with the glycopeptides of the parental cell line. Essentially all of the [3H]mannose-labelled asparaginyl-oligosaccharides from the 'single-mutant' cells selected for resistance to phytohaemagglutinin and the 'double-mutant' cells selected for additional resistance to concanavalin A were not bound to lentil lectin, whereas approximately one-sixth of the parental-cell glycopeptides were bound and specifically eluted with alpha-methyl mannoside. These bound and eluted glycopeptides represented a specific subset of the complex acidic-type asparaginyl-oligosaccharides. The percentage of radiolabelled glycopeptides and oligosaccharides from each cell line that were specifically bound to concanavalin A was consistent with the relative sensitivities of the three cell lines to this lectin. The major radiolabelled species in the endoglycosidase digest of the 'double-mutant'-cell glycopeptides (Man4GlcNAc1-size neutral oligosaccharides) were not bound to concanavalin A, whereas essentially all of the other neutral-type oligosaccharides were bound. In addition, the larger neutral-type oligosaccharides (Man8--9GlcNAc1) were more strongly bound to concanavalin A than were either the smaller neutral-type or the di-antennary acidic-type structures.     

Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.     

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.     

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.     

Studies on the carbohydrate-protein linkage region in bovine corneal keratan sulfate. III. Evidence for a biantennary structure

Peptidokeratan sulfates were retained by a concanavalin A-Sepharose column and readily eluted with 20 mM methyl alpha-D-mannoside from the column. Linkage region-enriched glycopeptides, prepared by endo-beta-galactosidase digestion of the peptidokeratan sulfates, behaved similarly to the peptidokeratan sulfates on the same affinity chromatography. Further, behaviors of both the peptidokeratan sulfates and the linkage region-enriched glycopeptides on pea lectin-Agarose columns were consistent with the results of the chromatography on concanavalin A-Sepharose. These findings confirmed the typical biantennary structure previously suggested for the linkage region on the basis of structural studies of the linkage region-enriched glycopeptides.     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

Alterations in N-linked oligosaccharides of glycoproteins during rat liver regeneration

[3H]Mannose-labeled glycopeptides in the slices after partial hepatectomy were characterized by column chromatography using Sephadex G-50, DE-52 and Con A-Sepharose, and further by digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H. They contained both 'complex type' and 'high-mannose type' oligosaccharides. A higher proportion of 'complex type' oligosaccharides was contained in regenerating liver 24 h after partial hepatectomy than in control. This tendency was increased gradually with time and was most pronounced at 144 h. In our previous studies, the activities of microsomal N-acetylglucosaminyltransferase towards endogenous and exogenous acceptors at 144 h after partial hepatectomy were shown to exceed most prominently that in control. No differences in the oligosaccharides were observed at 240 h when the deficit of liver had been restored. The oligosaccharides of glycopeptides in the incubation media were mostly 'complex type' and the differences between regenerating liver and control were observed only at 144 h. These results suggest that oligosaccharide processing of glycoproteins is regulated at the transfer step of peripheral N-acetylglucosamine to core oligosaccharides 144 h after partial hepatectomy, and that these alterations in oligosaccharides of glycoproteins may be related to hypertrophy and hyperplasia of hepatic cells in liver regeneration.     

Sialylated oligosaccharides O-glycosidically linked to glycoprotein C from herpes simplex virus type 1.

Glycoprotein C (gC) was purified by immunoabsorbent from herpes simplex virus type-1-infected BHK cells labeled with [14C]glucosamine for 11 h and chased for 3 h. Glycopeptides obtained by pronase digestion of gC were fractionated by Bio-Gel filtration and concanavalin A-Sepharose chromatography. Each glycopeptide fraction was analyzed for amino sugar composition by thin-layer chromatography. The majority of radioactivity was recovered as N-acetylglucosamine, but a significant amount of labeled N-acetylgalactosamine was detected and recovered preferentially in some glycopeptide species. Mild alkaline borohydride treatment of the glycopeptides resulted in the release of small degradation products which contained N-acetylgalactosaminitol as the major labeled component and a drastic reduction of N-acetylgalactosamine in the residual glycopeptides. These results demonstrated that gC carries O-glycosidically linked oligosaccharides in addition to the N-linked di- and triantennary glycans previously described (F. Serafini-Cessi, F. Dall'Olio, L. Pereira, and G. Campadelli-Fiume, J. Virol. 51:838-844, 1984). Chromatographic behavior on DEAE-Sephacel chromatography and neuraminidase digestion of O-linked oligosaccharides indicated the presence of two major sialylated species carrying one and two sialic acid residues, respectively. The characterization of a peculiar glycopeptide species supported the notion that some of the O-linked oligosaccharides are bound to a cluster of hydroxyamino acids located near an N-glycosylation site which carries one N-linked diantennary oligosaccharide.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

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.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

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.