Small Glycopeptides Research Articles

Protective mechanisms against the antitumor agent bleomycin: lessons from Saccharomyces cerevisiae.

Bleomycin is a small glycopeptide antibiotic used in combination therapy for the treatment of a few types of human cancer. The antitumor effect of bleomycin is most likely caused by its ability to bind to DNA and induce the formation of toxic DNA lesions via a free radical reactive (Fe.bleomycin) complex. However, the chemotherapeutic potential of bleomycin is limited, as it causes pulmonary fibrosis and tumor resistance at high doses. The chemical structure and modes of action of bleomycin have been extensively studied and these provide a foundation towards improving the therapeutic value of the drug. This review provides a first account of the current state of knowledge of the cellular processes that can allow the yeast Saccharomyces cerevisiae to evade the lethal effects of bleomycin. This model organism is likely to provide rapid clues in our understanding of bleomycin resistance in tumor cells.

Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein.

When glycoproteins formed in the endoplasmic reticulum (ER) are misfolded, they are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome. This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER. It has been established in yeast that several distinct proteins are involved in this translocation and degradation processes. Small glycopeptides formed in the ER are exported to the cytosol in a similar manner. This glycopeptide export system is conserved from yeast to mammalian cells, suggesting its basic biological significance for eukaryotic cells. These two export systems (for misfolded glycoproteins and glycopeptides) share some properties, such as a requirement for ATP and involvement of Sec61p, a central membrane protein presumably forming a dislocon channel for export of proteins. However, the machinery of glycopeptide export is poorly understood. In this study, various mutants known to have an effect on export/degradation of misfolded glycoproteins were examined for glycopeptide export activity with a newly established assay method. Surprisingly, most of the mutants were found not to exhibit a defect in glycopeptide export. The only gene that was found to be required on efficient export of both types of substrates was PMR1, the gene encoding the medial-Golgi Ca(2+)/Mn(2+)-ion pump. These results provide evidence that although the systems involved in export of misfolded glycoproteins and glycopeptides share some properties, they have exhibited distinct differences.

Iron Oxidation and Deposition in the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia)

The shell of the bivalve Montacuta ferruginosa is covered with a rust-colored biofilm. This biofilm includes filamentous bacteria and protozoa encrusted with a mineral, rich in ferric ion and phosphate. The aim of this research was to study two possible microbial iron precipitation pathways in the biofilm, namely, microbial iron oxidation and microbial degradation of organic Fe(III) complexes. The iron-oxidizing activity was assayed spectrophotometrically by monitoring the formation of the dye Wurster blue in biofilm extracts. Iron-oxidizing activity was effectively detected in extracts obtained by oxalic acid treatment of biofilm fragments. Extracts obtained without oxalic acid treatment, heated extracts, or extracts supplemented with HgCl 2 did not show any activity. This suggests that an iron-oxidizing factor (IOF), possibly an enzyme, coprecipitated with the mineral. Additional information gathered by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel-filtration chromatography, and UV spectrophotometry indicate that the IOF would be a small peptide or glycopeptide (1,350 Da). Microbial degradation of organic Fe(III) complexes was assayed with biofilm fragments incubated in a medium containing ferric citrate. Analysis of the supernatants after various intervals revealed that the complex was degraded by living microorganisms much faster than in the heat-killed negative controls. We conclude that ferric iron precipitation in the biofilm may proceed by way of microbial Fe(II) oxidation as well as microbial degradation of organic Fe(III) complexes.

In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains.

A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.

Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope.

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.

Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with immobiline membranes.

The purification of small glycopeptides (a Hepta-Tyr of the teicoplanin family, exhibiting broad activity against highly glycopeptide-resistant enterococci) by isoelectric focusing in multicompartment electrolyzers with buffering, isoelectric membranes, is described. The main obstacle to such a preparative technique, in common with all focusing methodologies, is the poor solubility of the analyte at the pI value with resultant precipitation and coprecipitation of all impurities with the main fraction. A good solubilizing power was obtained in hydro-organic solvents, particularly a mixture of 6 M urea and 20-25% trifluoroethanol. Best results, however, were obtained with mixtures of 8 M urea and zwitterionic detergents, notably the 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) family. A unique behavior of the peptide was found in concentration gradients of CHAPS: solubility increases up to 3.5% CHAPS, but the curve shows a maximum and then solubility decreases again at 5% CHAPS. In mixtures of 8 M urea and 3.5% CHAPS, sample loads of 500 up to 1000 mg Hepta-Tyr could be purified in a single run, with recoveries > 90% and purity in excess of 99%. The main glycopeptide fraction (pI 8.56) was collected into an isoelectric trap delimited by pI 8.46 and pI 8.65 membranes. Attempts at purifying the glycopeptide by most known reversed phase-high performance liquid chromatography (RP-HPLC) techniques failed completely.

Complexes of Fe(III) with amino sugars and small glycopeptides

The complexes of Fe(III) with small molecules (glycopeptides, amino sugars) related to peptidoglycan monomer (PGM) structure were prepared and characterized by chemical and physicochemical methods. A simple method for preparation using separation of the reaction products on a molecular sieve was introduced. Using this method, monomeric complexes of small molecular weight were obtained. The likely structures were proposed on the basis of analytical results.

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

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.

Glycopeptide Elicitors of Stress Responses in Tomato Cells

Induction of ethylene, an early symptom of the stress response in tomato (Lycopersicon esculentum [L.] Mill) cells, was used as a bioassay to purify elicitor activity from yeast extract. The purified elicitor preparation consisted of small glycopeptides (mean relative molecular weight of approximately 2500) and induced ethylene biosynthesis and phenylalanine ammonia-lyase activity half-maximally at 15 nanograms per milliliter. Elicitor activity was partially abolished by pronase and almost completely by endo-beta-N-acetylglucosaminidase H, alpha-mannosidase, or periodate. The oligosaccharides released upon treatment with endo-beta-N-acetylglucosaminidase H competitively inhibited the elicitor activity of the glycopeptides. This suppressor activity was abolished by periodate oxidation and alpha-mannosidase treatment. The suppressors were chromatographically separated into four active fractions with sizes corresponding to 7 to 10 monosaccharides. They consisted predominantly of mannose and contained also N-acetylglucosamine and glucose. The suppressors had no effect on the response of the tomato cells to a different elicitor, derived from cell walls of Phytophthora megasperma f. sp. glycinea. This strongly suggests that different recognition sites exist for different elicitors in tomato cells, and that the oligosaccharide suppressors act specifically on the perception of just one elicitor. The hypothesis is put forward that the suppressors bind to one of the elicitor recognition sites nonproductively, i.e. without producing a signal, thereby preventing induction of the stress responses by the corresponding elicitor.

Purification of the α- l-rhamnosidase of Penicillium decumbens and characterisation of two glycopeptide components

The α- l-rhamnosidase component of naringinase from Penicillium decumbens was purified and characterised as a monomeric enzyme with a mol. wt. of ∼90,000 and with a 50% content of carbohydrate. Digestion of the enzyme with pronase and fractionation of the products on Sephadex G-100 gave two glycopeptides. The larger glycopeptide contained galactofuranose and mannose residues together with lesser proportions of ethanolamine and glucose. Methylation analysis and 13C-n.m.r. spectroscopy indicated its structure to be similar to that of the cell-wall peptido-galactomannan of P. charlesii. The smaller glycopeptide was of the N-linked type with 10–12 mannose residues per asparagine. The 1H-n.m.r. spectrum indicated the structure to resemble those reported for Saccharomyces cerevisiae glycopeptides, with the addition of 1–2 galactofuranose residues and 3 ethanolamine substituents to the oligosaccharide unit.

Structural similarity of the type-specific group B streptococcal polysaccharides and the carbohydrate units of tissue glycoproteins: evaluation of possible cross-reactivity.

Type-specific capsular polysaccharides of group B streptococci show striking structural similarity with the terminal sugar sequences of tissue glycoconjugates. The polysaccharides have been put forward as vaccines against neonatal meningitis. A potential source of hazard in immunization of pregnant mothers may be the presence of the cross-reactive components in adult or fetal tissues. A radioactive ligand binding assay was used to test human immune sera to type Ia, II and III group B streptococcal polysaccharides for binding to tissue-derived glycopeptides showing structural similarities with the streptococcal polysaccharides. Of the 13 glycopeptides of human and rat tissues studied, representing a wide selection of structures known to occur in glycoproteins, only two showed some reactivity with the antisera. The reactivity with human small intestinal glycopeptides could be explained by the presence of natural blood group A antibodies, and was not related to the streptococcal group B antibodies. The basis of the reactivity of a high-molecular-weight glycopeptide from rat kidney with some of the sera was unknown, but was unrelated to the vaccination and clearly could not be inhibited with the streptococcal polysaccharides. Thus, no immunological cross-reactions of the tissue glycopeptides studied could be demonstrated with the group B streptococcal antisera.

Submaxillary mucins. Intermolecular interactions and gel-forming potential of concentrated solutions

The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport and processing of the glycosylated precursor of Concanavalin A in jack-bean

Concanavalin A (ConA) is a tetrameric lectin which is synthesized in the developing cotyledons of jack bean (Canavalia ensiformis L.) as a glycosylated precursor, pro-concanavalin A (pro-ConA). The processing of pro-ConA involves the excision of a small glycopeptide from the center of the pro-ConA molecule, and the ligation of the two polypeptides. In this paper, we show that pro-ConA is associated with the endoplasmic reticulum/Golgi fraction of the cells, and that the processing of pro-ConA occurs in the protein bodies. Processing is a complex process and different intermediate-sized polypeptides appear at different times during cotyledon development. The ConA-related polypeptides which accumulate during seed development may be the products of alternate processing events or breakdown products of ConA, rather than precursors of ConA. When glycosylation is prevented by tunicamycin, there is very little transport of pro-ConA out of the endoplasmic reticulum/Golgi system to the protein bodies; the unglycosylated pro-ConA which is transported is slowly processed. Tunicamycin does not prevent the transport of canavalin (a protein which is not glycosylated) or the transport and processing of the small amounts of glycosylated pro-ConA synthesized in the presence of the drug. This is, to our knowledge, the first demonstration that the transport of a glycoprotein in plant cells is dependent on the presence of the glycan.

Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. Partial amino acid sequence and lectin activity.

Concanavalin A (ConA), which is not a glycoprotein, is synthesized as a glycoprotein precursor (pro-ConA) which is post-translationally processed. This processing results in the loss of a small glycopeptide with a high mannose oligosaccharide. Carrington et al. (Carrington, D.M., Auffret, A., and Hanke, D.E. (1985) Nature 313, 64-66) determined the nucleotide sequence of a cDNA for pro-ConA, and in the derived amino acid sequence the only glycosylation site is in the middle of the molecule. Furthermore, the derived amino acid sequence of the putative precursor of ConA was found not to be colinear with that of ConA. Here we show that pro-ConA is located primarily in an endoplasmic reticulum-rich organelle fraction. Pro-ConA was purified from this fraction and subjected to amino acid sequencing. The first 12 amino acids at the N-terminal end of pro-ConA correspond to amino acids 119-130 of mature ConA, and to amino acids 30-41 of the putative pre-pro-ConA, the sequence of which was derived from the nucleotide sequence of a cDNA. Amino acid sequencing of a tryptic glycopeptide with the high mannose side chain showed that the first 17 amino acids of this peptide correspond to amino acids 154-170 of pre-pro-ConA. The last six amino acids in this series correspond to the first six amino acids of mature ConA. These data fully support the hypothesis of Carrington et al. that the biosynthesis of ConA involves a post-translational peptide cleavage, transposition, and ligation within the original polypeptide. Pro-ConA from the organelle fraction does not bind to Sephadex G-50, indicating that it has no lectin activity. The processing of pro-ConA apparently imparts biological activity to this lectin.

Thyroid cell surface glycoproteins. Nature and disposition of carbohydrate units and evaluation of their blood group I activity.

The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllactosamine sequences in the N-linked carbohydrate units of GP-1 and GP-3 was suggested by the composition and size of the oligosaccharides released by hydrazinolysis and was demonstrated by endo-beta-galactosidase treatment. The cleavage products from digestion with this enzyme were identified as NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----3Gal, Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal, Gal beta 1----4GlcNAc beta 1----3Gal, and GlcNAc beta 1----3Gal with the tetrasaccharides constituting the predominant species. The terminal alpha-D-Gal residues accounted for the binding of GP-1 and GP-3 glycopeptides to Bandeiraea simplicifolia I-agarose; concanavalin A-Sepharose affinity chromatography indicated that most of the N-linked carbohydrate units of both glycoproteins contained more than two branches. Difference in the branching on the poly-N-acetyllactosamine sequences of GP-1 and GP-3 was suggested by the finding that only the latter glycoprotein, as well as its glycopeptides, reacted with anti-blood group I antibodies; neither glycoprotein demonstrated blood group i antigenicity. Examination of cultured thyroid follicular cells revealed that both I and i determinants were present at the cell surface.

Increased urinary excretion of acid mucopolysaccharides and glycopeptides in hypothyroidism following thyroid hormone therapy

Urinary excretion of acid mucopolysaccharides and glycopeptides in the urine of an untreated patient with cretinism were measured before and after thyroid hormone replacement. Urinary uronic acid and hexose excretion in the CPC-precipitated fraction increased four to ten times after thyroid hormone administration. The maximum excretion was observed after 2 months of thyroid replacement. The excreted acid mucopolysaccharides consisted of chondroitin sulphate A/C and minor quantities of heparan sulphate. Urinary excretion of glycopeptides, particularly small molecular glycopeptides rose also four to five-fold with thyroid hormone administration. These data suggest that thyroid hormone markedly influences the metabolism of acid mucopolysaccharides and glycoproteins. Possibly, the lack of thyroid hormone caused a decreased activity of various lysosomal glycosidases and sulphatases.

O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity

Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of O-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.

Fucosylation of Membrane Proteins in Soybean Cultured Cells

Cultures of soybean cells incorporate [5,6-(3)H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose(15-17)-N-acetylglucosamine(2) (GlcNAc(2)) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose(12-13)-GlcNAc(2) structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.The membrane glycoproteins were doubly-labeled by incubating soybean cells in [(3)H]fucose and [(14)C]mannose. By repeated separation on Biogel P-4, six glycopeptides were purified that ranged in size from Hexose(8)GlcNAc(2) to Hexose(15-17)-GlcNAc(2). The three larger glycopeptides (I, II, III) were highly labeled with [(3)H]fucose and also contained [(14)C] mannose. Evidence that both isotopes were in the same glycopeptide was obtained by the finding that the mannose-labeled glycopeptides were shifted to smaller-sized structures when the [(3)H]fucose was removed by mild acid hydrolysis. Glycopeptide IV also contained [(3)H]fucose and [(14)C] mannose but only part of the [(3)H]fucose was released by mild hydrolysis. Glycopeptides V and VI contained only small amounts of tritium, but were labeled with [(14)C]mannose. None of the six glycopeptides was susceptible to the action of endo-beta-N-acetylglucosaminidase H, and none of these glycopeptides was bound to columns of concanavalin A-sepharose. The smaller glycopeptides (IV, V, VI) were partially susceptible to alpha-mannosidase digestion, but this enzyme did not release any radioactive mannose from the larger-sized glycopeptides. These data indicate that the fucosylated glycopeptides are N-linked structures containing fucose, mannose, GlcNAc, and probably other sugars, and that the mannose units are blocked by other sugars. Thus, these results indicate that plant membrane glycoproteins contain a significant amount of modified oligosaccharide side chains.

Increased sialylation of complex glycopeptides during differentiation of mouse embryonal carcinoma cells

High-molecular-weight, asparagine-linked glycopeptides – the lactosaminoglycans – are the major class of protein-bound carbohydrates synthesized by F9 cells; these cells synthesize only minor amounts of smaller glycopeptides. In contrast, F9ACC19, an endodermal cell line derived from F9 cells, synthesizes only minor amounts of lactosaminoglycans and a high proportion of smaller glycopeptides. Biochemical analysis of the small glycopeptides from F9ACC19 cells revealed that they are larger, bind less efficiently to concanavalin-A Sepharose and contain more sialic acid than their counterparts from F9 cells. Both cell types contain a small proportion of high-mannose glycopeptides. When synthesized by F9ACC19 cells, the glycopeptides of vesicular stomatitis virus show a high level of sialylation as compared to those synthesized by F9 cells, where few or no sialic-acid residues are present; this shows that the differences observed in total glycopeptides reflect differences in the glycosylation machinery of the cells. Consistent with this observation, sialyltransferase activity in vitro using a variety of acceptors was found to be markedly higher in F9ACC19 than in F9 cells, while galactosyltransferase activity was reduced several fold in F9ACC19 cells. These data support the hypothesis that the increased sialyltransferase activity in endodermal differentiated F9ACC19 cells may block the terminal galactose residue of glycopeptides, thereby inhibiting the synthesis of lactosaminoglycans in these cells.

Concanavalin A is synthesized as a glycoprotein precursor

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (Mr) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with Mr 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-Mr polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 Mr. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [(3)H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, α-mannosidase or β-N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (Mr=30000). The 4000 decrease in Mr is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.