Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [³H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (M_r > 5000) ³H-labeled glycopeptide. Digestion of these ³H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [³H]glucosamine-labeled cells with melittin caused ³H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total ³H-labeled glycoproteins from the cell and 3% of the ³H-labeled proteoglycans. The ³H-labeled glycoproteins were digested with Pronase and the resulting ³H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (M_r > 5000) now comprised about 30% of these released ³H-labeled glycopeptides. These high molecular weight ³H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released ³H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides.     

Differentiation-associated decrease in the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonic adenocarcinoma cells.

CaCo-2 cells are human colonic adenocarcinoma cells which can differentiate spontaneously into enterocytes when maintained confluent for extended periods of time. Cells kept in culture for 4 days (rapidly growing), 7-9 days (early confluence) and 19-22 days (late confluence) were incubated for 24 h with L-[5,6-3H]fucose or D-[6-3H]glucosamine in order to examine the changes in glycoprotein carbohydrate structure that occur during this differentiation. Labelled glycopeptides obtained by exhaustive Pronase digestion of the cell-surface and cell-pellet fractions were fractionated on Bio-Gel P-6. A high-Mr glycopeptide fraction which was excluded from Bio-Gel P-6 was present in all cases. These glycopeptides were then fractionated by affinity chromatography on Datura stramonium agglutinin-agarose. The glycopeptides which were specifically bound to the lectin column were largely degraded by endo-beta-galactosidase, thereby indicating that they consisted of fucosylated polylactosaminoglycans. The proportion of labelled polylactosaminoglycans decreased with increasing time in culture, whereas sucrase activity, which is characteristic of differentiated enterocytes, increased. These results demonstrate that a relatively large decrease in the proportion of fucosylated polylactosaminoglycans occurs with differentiation of CaCo-2 cells.     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with 35S-methionine-labeled cells showed that HPA recognized two glycoproteins of Mr 170,000 and 400,000 among the secreted products of fibroblasts and two major cellular glycoproteins of Mr 40,000 and Mr 180,000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulse-labeled shortly with 35S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

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.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins

The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis. The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide. The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase. These terminal sugars are linked to the alpha-mannose residues of the glycan cores. The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.     

The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones

Lysosome membrane glycoproteins, lamp-1 and lamp-2, have been shown to contain 18 and 16 N-glycans, some of which are modified by poly-N-acetyl-lactosamine. We have localized the polylactosaminoglycans to specific sites on lamp-1 and lamp-2 purified from human chronic myelogenous leukemia cells. Polylactosaminoglycan-containing glycopeptides, obtained by trypsin, pepsin, and V8 protease digestion of the glycoproteins, were isolated by Datura stramonium agglutinin affinity chromatography, gel filtration, and reverse phase high performance liquid chromatography. The poly-N-acetyllactosaminyl structures of isolated glycopeptides were confirmed by the susceptibility of their released oligosaccharides to endo-beta-galactosidase. Amino acid analysis and sequencing demonstrated that polylactosaminoglycans were located at Asn-34, Asn-93 and/or Asn-102, and Asn-195 and/or Asn-200 in lamp-1, and at Asn-4 and/or Asn-10, and Asn-279 in lamp-2. These results indicated that only certain glycosylation sites can be selectively modified by poly-N-acetyllactosamine, and those sites may confer the requirement by beta 1----3-N-acetylglucosaminyl transferase.     

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

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

Characterization of a high molecular weight glycoprotein secreted by the peri-implantation bovine conceptus

Cow conceptuses were flushed from uteri on Day 17 of pregnancy and cultured with [3H]glucosamine and [14C]leucine. A high molecular weight glycoprotein (HMWG) having an Mr = 765,000 was isolated by a combination of anion-exchange and gel-filtration chromatography. Selective chemical and enzymatic degradations were performed. The HMWG was resistant to Pronase and peptide: N-glycanase F. Only endo-beta-galactosidase and harsh alkaline reducing conditions were successful in dissociating carbohydrate from the protein core, suggesting that carbohydrate chains are N-linked to Asn and contain beta-galactosidic linkages. The intact molecule could bind to an affinity column of Datura stramoniom lectin, suggesting the presence of beta(1-4)-linked oligomers of N-acetylglucosamine. The susceptibility of HMWG to endo-beta-galactosidase suggests that at least some of these oligomers are substituted with galactose to form N-acetyllactosamine. Binding of HMWG to lectin could be inhibited partially with N-acetyllactosamine or completely with a mixture of N, N'-diacetylchitobiose and N, N', N"-triacetylchitotriose. In summary, properties of the HMWG suggest it contains lactosaminoglycan components and is almost identical to an HMWG secreted by the Day 16 ovine conceptus. Thus, embryos of these two ruminant species secrete similar molecules during early pregnancy.     

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.     

Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-³H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [³H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.     

Biosynthesis of proteodermatan sulfate in cultured human fibroblasts

Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.     

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.     

Hyaluronectin is produced by oligodendrocytes and Schwann cells in vitro

A hyaluronectin (HN)-like antigen was found in rat O-2A progenitors and oligodendrocytes, as well as in Schwann cells and in their culture medium. The HN-like antigen secreted in culture supernatants had a higher molecular mass than HN extracted from rat brain at acidic pH. In vitro the secreted HN-like antigen was spontaneously and slowly degraded into species whose Mr was close to that of HN found in acidic brain extract. In brain or nerve neutral pH extracts, both HN-like antigen and HN were present. The high Mr of the secreted antigen, the homology in amino acid sequences between HN and N-terminal domain of PG-M/versican, in addition to a positive hybridization between Schwann cell RNAs and a probe obtained with primers derived from HN sequences also found in versican suggested that HN is closely related to the large proteoglycan PG-M/versican. The presence in Schwann cell extract of a HN mRNA whose Mr was compatible with the size expected for HN showed that HN may be directly secreted by cells and not only the consequence of a proteolytic cleavage. The similarity of HN with PG-M (V3) suggested that HN found in vivo could be the result of an alternative splicing of a single gene. We conclude that HN as other members of the PG-M/versican family is a marker of oligodendrocytes and Schwann cells in culture.     

Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor

Mouse and hamster sperm receptors, called mZP3 (approximately 83,000 Mr) and hZP3 (approximately 56,000 Mr), respectively, are glycoproteins located in the ovulated egg zona pellucida. Certain of the glycoprotein O-linked oligosaccharides are essential for sperm receptor activity. Here, we transfected mouse embryonal carcinoma (EC) cells with mZP3 and hZP3 genes placed under control of a constitutive promoter. Transfected cells synthesized and secreted large amounts of the glycoproteins, called EC-mZP3 and EC-hZP3. Although the primary structures of mZP3 and hZP3 polypeptides (44,000 Mr) are very similar to one another, EC-mZP3 (approximately 83,000 Mr) and EC-hZP3 (approximately 49,000 Mr) were glycosylated to very different extents, such that they resembled their egg counterparts. Like egg mZP3, EC-mZP3 inhibited binding of sperm to ovulated eggs and induced sperm to acrosome-react in vitro. In addition, large numbers of sperm bound to aggregates of mZP3-transfected EC cells in vitro. On the other hand, unlike egg hZP3, EC-hZP3 did not exhibit either sperm receptor or acrosome reaction-inducing activity, and sperm failed to bind to aggregates of hZP3-transfected EC cells. Thus, transfected EC cells not only express sperm receptor genes, but also discriminate between very similar polypeptides with respect to glycosylation and, in the case of mZP3, add specific oligosaccharides essential for biological activity. In addition, the results demonstrate that EC cells can serve as a source for large amounts of functional mouse sperm receptor.     

Presence of erythroglycan on human K-562 chronic myelogenous leukemia-derived cells

Total glycopeptides from human K-562 cells, labeled metabolically with [3H]glucosamine or [3H]mannose, were prepared by extracting the cells with organic solvents to remove lipids and by digesting the residue with pronase. 3H-labeled glycopeptides were fractionated on Sephadex G-50 revealing a high molecular weight fraction (Mr = 7,000 to 11,000), comprising approximately 10% of the [3H]glucosamine and 25% of the [3H]mannose label. Digestion of this glycopeptide fraction with endo-beta-galactosidase from Escherichia freundii, specific for a repeating structure of Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3), results in the following four products as resolved by Bio-Gel P-2 gel filtration: 1) a disaccharide with the structure beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 2) a trisaccharide with the structure beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; 3) a tetrasaccharide with the sequence alpha-N-acetylneuraminyl leads to beta-galactosyl leads to beta-2-deoxy-2-acetamidoglucosyl leads to beta-galactose; and 4) a larger, complex fragment which contains mannose and beta-2-deoxy-2-acetamidoglucose and which is probably the protein linkage region. In addition, visualization of radiolabeled glycoproteins by fluorography on polyacrylamide gels revealed a 105,000-dalton "Band 3"-like glycoprotein and other bands that were sensitive to endo-beta-galactosidase. These results indicate that the K-562 cell line bears a glycopeptide, erythroglycan, which has been found on erythrocytes, and that this polymer is expressed mainly in the fetal form as a linear chain.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells.

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by 125I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce 125I-WGA binding more than 12%. In contrast, endo-beta-galactosidase as well as a combination of beta-galactosidase with beta-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by 3H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-3H-galactose. Analyses of the 3H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-beta-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine. Twenty-four hours later, 3H-glycopeptides in the axon and cell body were isolated and resolved by serial lectin affinity chromatography. Of the eight populations, the biantennary-type glycopeptides (GPbi) and those that bind to WGA (GPwga) were preferentially associated with rapidly transported glycoproteins. In contrast, the glycopeptide that consists of N-acetylglucosamine O-linked to ser/thr was mostly retained in the cell body. GPbi and GPwga were also preferentially transported to growth cones. Analyses of RUQ cells, exposed to 3H-glucosamine in vitro for 36 h showed an enrichment of GPbi and GPwga at the growth cone relative to the cell body. The disposition of the various glycopeptides in growing neurons was also examined using FITC lectins. FITC-coupled WGA, Vicia vellosa, and lentil lectin showed extensive staining of the cell body, but only WGA stained the growth cones. To investigate if GPwga interacts specifically with target cells, these glycopeptides were isolated from the neurons of 180 abdominal ganglia. GPwga, other Aplysia glycopeptides, and glycopeptides prepared from ovalbumin were coupled separately to fluorescent spheres. The spheres were then added to muscle cells isolated from the auricle of the heart, which is innervated by many neurons from the ganglion. While spheres coupled to GPwga bound to the muscle cell surface, the other glycopeptides did not. These results indicate that glycopeptides class GPwga, found among rapidly transported glycoproteins and on the growth cone surface, is able to bind to muscle cells and may therefore play some role in neuron-target interactions.