Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.      

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Carbohydrate components of lipovitellin of the sand crab Emerita asiatica

Lipovitellin II (Lv II), the major yolk protein of the anomuran crab Emerita asiatica, was purified using heparin-sepharose affinity column chromatography. The purified Lv II was a glycoprotein as it was stainable with periodic acid-Schiff's reagent. Quantitative analysis of sugars showed the presence of fucose, mannose, galactosamine, N-linked oligosaccharides, as well as O-linked oligosaccharides containing N-acetyl hexosamine as the terminal residue. The amount of N-linked oligosaccharides is higher than that of the O-linked oligosaccharides. Biogel P-4 column chromatographic separation of the radiolabeled oligosaccharides of Lv II showed the presence of five different O-linked oligosaccharides and four different N-linked oligosaccharide species. HPTLC separation of the neoglycolipids prepared from the O-linked oligosaccharides also showed the presence of five different O-linked oligosaccharide species. N-linked oligosaccharides contain significant quantities of mannose. Unisil column chromatographic purification in conjunction with HPTLC separation revealed three neutral glycolipid species such as monoglycosylceramide, diglycosylceramide, and triglycosylceramide in the Lv II. The functional significance of these carbohydrate components of the major yolk protein during embryogenesis of the sand crab is discussed.     

Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

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.     

Evidence for the presence of complex carbohydrates in Candida albicans cell wall glycoproteins

In order to test the hypothesis that cell wall glycoproteins of Candida albicans contained non-mannan oligosaccharides, the sugar composition of cell wall extracts and fractions of cell wall extracts was examined by means of fluorophore-assisted carbohydrate electrophoresis (FACE). In addition to the expected mannose, glucose, and N-acetyl-glucosamine, this analysis showed the presence of galactose, N-acetyl-galactosamine, fucose, and sialic acid and two unknown sugars. These sugars are also associated with complex oligosaccharides of mammalian glycoproteins. Presence of fucosylated cell wall components was further demonstrated by lectin-blotting analysis of cell wall extracts. Besides their structural role, complex carbohydrate structures on the surface of C. albicans may represent additional motifs through which interactions of this fungus with host cells and tissues could be established.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Differences of alpha3beta1 integrin glycans from different human bladder cell lines

Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.     

Human transferrin receptor contains O-linked oligosaccharides

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

Carbohydrate structure of glycoprotein 65 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus

The secondary envelope-gene product, glycoprotein 65 (gp65), of the polycythemia-inducing variant of Friend spleen focus-forming virus (F-SFFVp) was isolated from F-SFFVp-infected normal rat kidney cells cultivated in the presence or absence (-Glc) of glucose. Oligosaccharide side chains present were sequentially liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, by chromatographic comparison with oligosaccharide standards and by methylation analysis. The results demonstrate that gp65 contains oligomannosidic, hybrid and N-acetyllactosaminic glycans. The oligomannosidic glycans represent the same partially glucosylated species with six to nine mannose residues present in F-SFFVp gp52, the biosynthetic precursor of gp65 [Strube, K.-H. Schott, H.-H. and Geyer, R. (1988) J. Biol. Chem. 263, 3762-3771]. Oligosaccharides of the hybrid type were found to comprise one sialylated lactosamine unit and three or four alpha-linked mannose residues. Analysis of the N-acetyllactosaminic glycans revealed that gp65 carries fucosylated, partially sialylated bi-antennary, tri-antennary and tetra-antennary oligosaccharides, in addition to incomplete species. The glycosylation of gp65(-Glc) is characterized by the presence of oligomannosidic glycans with five to nine mannose residues, similar hybrid-type species and by increased amounts of incomplete N-acetyllactosaminic oligosaccharides, a decrease in sialylation and the lack of tetra-antennary species.     

Conformation analysis of carbohydrate chains of glycoconjugates

A problem of conformations of carbohydrate chains of glycoconjugates-glycoproteins and glycolipids--is reviewed. Experimental data (NMR, X-Ray) and theoretical conformational analysis data are discussed. Spatial structures of O-linked oligosaccharides from blood-group glycoproteins, N-linked oligosaccharides of different types (oligomannosidic, complex, hybrid, bisect) and carbohydrate chains of glycosphingolipids are considered.     

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.     

N-glycosylation of a baculovirus-expressed recombinant glycoprotein in three insect cell lines

Summary The capacity of two Trichoplusia ni (TN-368 and BTI-Tn-5bl-4) and a Spodoptera frugiperda (IPLB-SF-21A) cell lines to glycosylate recombinant, baculovirus-encoded, secreted, placental alkaline phosphatase was compared. The alkaline phosphatase from serum-containing, cell culture medium was purified by phosphate affinity column chromatography. The N-linked oligosaccharides were released from the purified protein with PNGase F and analyzed by fluorophore-assisted carbohydrate electrophoresis. The majority of oligosaccharide structures produced by the three cell lines contained two or three mannose residues, with and without core fucosylation, but there were structures containing up to seven mannose residues. The oligosaccharides that were qualitatively or quantitatively different between the cell lines were sequenced with glycosidase digestions. The S. frugiperda cells produced more fucosylated oligosaccharides than either of the T. ni cell lines. The smallest oligosaccharide produced by S. frugiperda cells was branched trimannose. In contrast, both T. ni cell lines produced predominantly dimannose and linear trimannose structures devoid of α 1–3-linked mannose.     

Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

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.