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1.
The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.  相似文献   

2.
Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.  相似文献   

3.
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.  相似文献   

4.
Structures of the asparagine-linked sugar chains of laminin   总被引:13,自引:0,他引:13  
This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.  相似文献   

5.
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.  相似文献   

6.
The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.  相似文献   

7.
The structures of the sugar chains present in two human monoclonal IgM molecules purified from the serum of a patient with Waldenstr?m's macroglobulinemia have been determined. The asparagine-linked sugar chains were liberated as oligosaccharides by hydrazinolysis and labeled by reduction with NaB3H4 after N-acetylation. Their structures were studied by serial lectin column chromatography and sequential exoglycosidase digestion in combination with methylation analysis. These two IgM's were shown to contain almost the same sugar chains. The sugar chains were a mixture of a series of high-mannose-type and biantennary complex-type oligosaccharides. The complex-type oligosaccharides contain Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc as their core and GlcNAc beta 1----, Gal beta 1----4GlcNAc beta 1---- and Neu5Ac alpha 2----6Gal beta 1----4GlcNAc beta 1---- groups in their outer chain moieties.  相似文献   

8.
Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.  相似文献   

9.
Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.  相似文献   

10.
The susceptibility of a variety of oligosaccharides to endo-beta-N-acetylglucosaminidase D was investigated. The oligosaccharides having the structures of Man alpha 1----6 (GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, derived from complex type triantennary sugar chains, released +/- Fuc alpha 1----6GlcNAcOT upon incubation with the enzyme at almost the same rate as Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. When the reaction products were reduced with NaB3H4 and analyzed by Bio-Gel P-4 column chromatography, a new radioactive peak was detected in both cases. This new radioactive oligosaccharide was confirmed to be Man alpha 1----6(GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAcOT in the former case and Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAcOT in the latter. These results indicated that endo-beta-N-acetylglucosaminidase D does not require the presence of a free hydroxyl group at the C-4 position of the alpha-mannosyl residue of the trisaccharide glycon: Man alpha 1----3Man beta 1----4GlcNAc beta 1----.  相似文献   

11.
This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.  相似文献   

12.
The fusion glycoprotein (F0) was isolated from Newcastle disease virus (NDV) particles metabolically labelled with [2-3H]mannose; it was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. In this manner, the majority of the oligosaccharides in NDV F0 could be liberated. After reduction with NaBH4, they were separated by high-performance liquid chromatography, and were subjected to structural analysis. Using micromethylation/capillary gas chromatography/mass fragmentography, alpha-mannosidase digestion, and acetolysis, it was found that the enzymatically released NDV F0 oligosaccharides are common oligomannosidic glycoprotein glycans of size classes (Man)8GlcNAc, Man)7GlcNAc, (Man)6GlcNAc, (Man)9GlcNAc, and (Man)5GlcNAc (in order of prevalence). The major structural isomers present in the NDV F0 (Man)8GlcNAc to (Man)5GlcNAc fractions were shown to lack mannose residues D2, D1D2 or D2D3, D1D2D3, and CD1D2D3, respectively, of (Man)9GlcNAc.  相似文献   

13.
Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.  相似文献   

14.
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.  相似文献   

15.
Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.  相似文献   

16.
T Endo  M Kasahara  A Kobata 《Biochemistry》1990,29(39):9126-9134
The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.  相似文献   

17.
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.  相似文献   

18.
The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.  相似文献   

19.
Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.  相似文献   

20.
The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.  相似文献   

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