Sulfated N-linked oligosaccharides in mammalian cells. II. Identification of glycosaminoglycan-like chains attached to complex-type glycans

In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

The spectrum of incomplete N-linked oligosaccharides synthesized by endothelial cells in the presence of brefeldin A.

Previous studies in many cell lines have shown that Brefeldin A (BFA) inhibits the forward movement of newly synthesized glycoconjugates by fusing the cis-, medial-, and trans-Golgi compartments with the rough endoplasmic reticulum. Studies on the oligosaccharide processing of individual glycoproteins have yielded confusing and incomplete results regarding the location of the block. Assuming that all glycoproteins with N-linked oligosaccharides follow the same endoplasmic reticulum to the Golgi pathway, a more complete picture on the location and nature of the block can be determined by analyzing N-linked oligosaccharides synthesized in the presence of BFA. In bovine pulmonary artery endothelial cells, BFA (0.1 microgram/ml) reversibly inhibits the secretion of greater than 95% of Tran35S and [3H]Man-labeled glycoproteins without affecting protein synthesis or N-linked glycosylation. In addition, BFA inhibits the synthesis and secretion of 35SO4-labeled oligosaccharides. Initial oligosaccharide trimming is uninhibited, but further processing is affected since the majority (65%) of the chains terminate only in beta-GlcNAc residues. Concomitantly, the proportion of [3H]Man-labeled N-linked anionic oligosaccharides is reduced from 60 to 20%, and the great majority of the charge is due to one sialic acid. The rate-limiting step for sialylation appears to be the branch selective addition of beta-Gal residues. The remaining charge is due to sulfate esters (0.6%) which normally account for greater than 10% of the anionic substituents. BFA also reduces the amount of phosphorylated chains by 80% and greatly diminishes further phosphodiester processing since the majority of these oligosaccharides (60%) contain a Man-6-PO4 residue in an acid-sensitive diester linkage. The addition of all polylactosamine chains, outer-branch fucose and terminal alpha-Gal residues are completely inhibited by BFA. Secretion, fucosylation, and sialylation are completely restored when BFA is removed, but the other modification steps are only partially restored. Our results indicate that addition of sulfate esters, terminal alpha-Gal residues, polylactosamine chains, outer-branch fucose residues, some initial phosphorylation, and most phosphodiester processing may occur beyond a compartment where some beta-Gal and sialic acid residues can be added. Essentially, all of the effects on oligosaccharide processing are partially or completely reversible.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.     

The effect of swainsonine and castanospermine on the sulfation of the oligosaccharide chains of N-linked glycoproteins

MDCK (Madin-Darby canine kidney) cells infected with the NWS strain of influenza virus incorporate 35SO4 into complex types of oligosaccharides of the N-linked glycoproteins. On the other hand, when these virus-infected MDCK cells are incubated in the presence of swainsonine, an inhibitor of the processing mannosidase II, approximately 40-80% of the total [35S]glycopeptides were of the hybrid types of structures. Thus, these sulfated, hybrid types of glycopeptides were completely susceptible to digestion by endoglucosaminidase H, whereas the sulfated glycopeptides from infected cells incubated without swainsonine were completely resistant to endo-beta-N-acetylglucosaminidase H. When virus-infected MDCK cells were incubated in the presence of castanospermine, an inhibitor of the processing glucosidase I, the N-linked glycopeptides contained mostly oligosaccharide chains of the Glc3Man7-9GlcNAc2 types of structures, and these oligosaccharides were devoid of sulfate. Structural analysis of these abnormally processed oligosaccharides produced in the presence of swainsonine or castanospermine indicated that they differed principally in the processing of one oligosaccharide branch as indicated by the structures shown below. They also differed in that only the swainsonine-induced structures were sulfated. These data indicate that removal of glucose units and perhaps other processing steps are necessary before sulfate residues can be added. (Formula: see text).     

Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine.

The major envelope glycoproteins gp120 and gp41 of human immunodeficiency virus type 1, the causative agent for human AIDS, contain numerous N-linked oligosaccharides. We report here our discovery that N-acetylglucosamine residues within the complex-type N-linked oligosaccharides of both gp120 and its precursor, gp160, are sulfated. When human Molt-3 cells persistently infected with human T-cell leukemia virus IIIB were metabolically radiolabeled with 35SO4, gp160, gp120, and to some extent gp41 were radiolabeled. The 35SO4-labeled oligosaccharides were quantitatively released by N-glycanase treatment and were bound by immobilized Ricinus communis agglutinin I, a lectin that binds to terminal beta-galactosyl residues. The kinetics of release of sulfate upon acid hydrolysis from 35SO4-labeled gp120 indicate that sulfation occurs in a primary sulfate ester linkage. Methylation analysis of total glycopeptides from Molt-3 cells metabolically radiolabeled with [3H]glucosamine demonstrates that sulfation occurs at the C-6 position of N-acetylglucosamine. Fragmentation of the gp120-derived 35SO4-labeled glycopeptides by treatment with hydrazine and nitrous acid and subsequent reduction generated galactosyl-anhydromannitol-6-35SO4, which is the expected reaction product from GlcNAc-6-sulfate within a sulfated lactosamine moiety. Charge analysis of the [3H]galactose- and [3H]glucosamine-labeled glycopeptides from gp120 and gp160 indicates that approximately 14% of the complex-type N-linked oligosaccharides are sulfated.     

Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase

After adipocytes were labeled with Na2[35SO4], immunoadsorbed with immobilized antilipoprotein lipase, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, a labeled band was identified at 59,700 daltons, the molecular mass of chicken lipoprotein lipase (LPL). Excess unlabeled LPL prevented the immunoadsorption of this labeled species, hence the labeled species was determined to be LPL. Digestion of LPL with endo-beta-N-acetylglucosaminidase H (Endo H) caused a shift in mobility of LPL in SDS-PAGE with no loss of radioactivity, whereas digestion with glycopeptidase F resulted in removal of 99% of the radioactivity. Adipocytes cultured with Trans35S-label and tunicamycin produced an LPL species of 52,000 daltons, but tunicamycin abolished the incorporation of 35SO4 into LPL. This established that 35SO4 was incorporated into an N-linked oligosaccharide of LPL. Endo H digestion of pulse-chase labeled LPL revealed the presence of two complex and one high mannose-type N-linked oligosaccharides. A single 35SO4-labeled tryptic peptide was isolated by reverse phase chromatography. The amino acid sequence of the peptide established that the 35SO4 oligosaccharide is conjugated at Asn-45. Behavior of the 35SO4-labeled oligosaccharide on concanavalin A-agarose, sequential exoglycosidase digestion, and chemical analysis of the 35SO4 oligosaccharide confirms that this moiety is of the complex type. Sequential exoglycosidase digestion, thin layer chromatography of the released monosaccharides, and the use of glycosylation inhibitors established that the sulfated sugar is a core N-acetylglucosamine (GlcNAc). The data show that chicken LPL contains two complex and one high mannose N-linked oligosaccharides and that 35SO4 is incorporated into LPL on a GlcNAc residue of a complex oligosaccharide located at Asn-45.     

A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein

The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP.     

O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.     

Characterization of oligosaccharide chains on mouse mammary tumor virus envelope proteins and the implications for the mechanism of their glucocorticoid regulated processing

Glucocorticoid hormone is required for complete posttranslational processing of the glycosylated mouse mammary tumor virus envelope precursor, Pr74env in the murine T-lymphosarcoma cell line, W7MG1. Metabolic labeling studies with [35S]methionine, [3H]galactose, and [3H]mannose, combined with enzymatic digestion analyses with a variety of endoglycosidases, demonstrated that both proteolytic processing and N-linked oligosaccharide maturation depended, either directly or indirectly, on glucocorticoid action. Pr74 is found in both control and hormone-treated cells. In both cases Pr74 molecules carry high mannose and/or hybrid, but not complex, oligosaccharide chains with very little or no sialic acid. When cells are grown with glucocorticoid, Pr74 is converted to gp52 and gp33 with greatly increased efficiency, and these mature glycoproteins carry complex oligosaccharides containing sialic acid. No O-linked carbohydrate was detected on any of these species. According to this evidence, the glucocorticoid-regulated step in this pathway must occur at or before the final mannose trimming step in the Golgi that is required for formation of complex carbohydrate chains.     

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

We have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[3H]4. The 3H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The anionic oligosaccharides were further purified as well as structurally characterized using a variety of preparative and analytical techniques, including HPLC, endo- and exoglycosidase digestions, and lectin affinity chromatography. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. The sulfated oligosaccharides consisted of hybrid and complex type oligosaccharides with one or two branches terminating in SO4-4GalNAc beta 1,4. In contrast, the sialylated oligosaccharides consisted of a wide array of differing structures containing two or three peripheral branches as well as one, two, or three sialic acid moieties. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, we describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species. In the accompanying paper (Green, E.D., and Baenziger, J.U.(1987) J. Biol. Chem. 262, 36-44) we demonstrate the marked quantitative differences among the pituitary glycoprotein hormones in terms of sulfation, sialylation, and underlying oligosaccharide structures, as well as provide evidence for site-specific synthesis of oligosaccharides on individual hormones.     

Depletion of divalent cations within the secretory pathway inhibits the terminal glycosylation of complex carbohydrates of thyroglobulin.

Newly synthesized thyroglobulin transiting the secretory pathway is posttranslationally modified by addition of oligosaccharides to asparagine N-linked residues. The effect of divalent cation depletion on oligosaccharide processing of Tg was studied in FRTL-5 cells. Treatment with an ionophore, A23187, or thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum ATPases delayed Tg secretion. These effects were accompanied by a normal distribution of the marker of the endoplasmic reticulum protein disulfide isomerase. Analysis of the thyroglobulin oligosaccharides by Bio-gel P4 chromatography showed that in the presence of A23187 and thapsigargin the addition of peripheral sialic acid and possibly galactose is inhibited. These findings were strengthened by experiments of exoglycosidase digestion and SDS-PAGE analysis of the resulting products. These results reveal a cellular mechanism of production of thyroglobulin with incompletely processed complex chains, i.e., the ligand of the recently described GlcNAc and asialoglycoprotein receptors of the thyroid. Since A23187 and thapsigargin inhibit biosynthetically the addition of peripheral sugars on N-linked oligosaccharides chains, the thyroglobulin molecules secreted in the presence of A23187 and thapsigargin should greatly facilitate studies on the function of the GlcNAc and asialoglycoprotein receptors of the thyroid.     

Mannose 6-sulfate is present in the N-linked oligosaccharides of lysosomal enzymes of Dictyostelium

The major N-linked, anionic oligosaccharide found on several lysosomal enzymes of Dictyostelium discoideum contains five charges, composed of three sulfate esters and two residues of Man-6-P in phosphodiester linkage. Most of the SO4 was found as Man-6-SO4. This novel sulfated sugar was detected and quantitated by measuring the appearance of 3,6-anhydromannitol following acid hydrolysis and reduction of base-treated, reduced oligosaccharides. If SO4 is removed by solvolysis prior to the base treatment, the anhydrosugar is not formed, indicating that its presence is not an artifact of the procedure. That these oligosaccharides are derived from standard high-mannose-type oligosaccharides indicates that only one or, at most, two Man residues are unsubstituted at the 6-position.     

Immunoglobulin carbohydrate requirement for formation of an IgG-IgG complex.

In addition to their Fc oligosaccharides, some immunoglobulin molecules have oligosaccharides linked to variable segments of H or L chains. These Fab oligosaccharides are potential determinants of antibody specificity. This possibility was considered in a study of the IgG antiglobulin from a patient with IgG-IgG complexes. F(ab')2 fragments of the antiglobulin retained the ability to form complexes with normal IgG as detected by analytical ultracentrifugation. Removal of F(ab')2 sialic acids by neuraminidase abolished complex formation. Recombination experiments further localized antiglobulin activity to the L chains. Antiglobulin activity of the recombinant molecules was shown by analytical ultracentrifugation and by column chromatography with molecules containing 125I-labeled L chains. L chains from the subject's IgG were enriched in sialic acids. Thus, a sialic acid-containing oligosaccharide on the L chain of this antiglobulin is required for its binding action.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

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.     

Lymphocyte function-associated antigen 1 (LFA-1) contains sulfated N-linked oligosaccharides

The murine lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein heterodimer consisting of an Mr 180,000 alpha-chain and an Mr 95,000 beta-chain. Although LFA-1 has been studied extensively in the past few years due to its involvement in various antigen-specific T lymphocyte responses, virtually nothing is known about its glycosylation. In this report, we have analyzed the oligosaccharide moieties of the murine LFA-1 molecule. Utilizing a T lymphoma cell line, EL-4, it was found that [35S] sulfate, [3H]glucosamine, [3H]mannose, and [3H]fucose were incorporated into both the alpha- and beta-chains of LFA-1. Isolated alpha- and beta-chains from anti-LFA-1 immunoprecipitates of [3H]glucosamine-labeled NP-40 lysates were subjected to tryptic-chymotryptic digestion, and the resulting glycopeptides were fractionated by reverse-phase high performance liquid chromatography. Five major [3H]glucosamine-labeled glycopeptides were generated by this procedure from each of the two polypeptide chains. Treatment of the individual glycopeptides with almond emulsin peptide:N-glycosidase or Endo F demonstrated that the [3H]glucosamine label existed almost entirely in N-linked oligosaccharide structures (Mr 5000 to 10,000). By using similar techniques, the majority of the [35S]sulfate moieties were also found covalently bound to N-linked oligosaccharides. In addition, both [35S]sulfate-labeled alpha- and beta-chains were susceptible to Keratanase and endo-beta-galactosidase digestions, indicating the presence of sulfated N-acetyllactosamine sequences. The expression of [35S]sulfate-labeled LFA-1 on various cell types was also examined. LFA-1 was found to be sulfated only on thymocytes and splenic T cells, but not on macrophages, splenic B, or bone marrow cells.     

Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line

The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type molecules, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha 2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the major sulfated protein of mouse pancreatic acinar cells: A high molecular weight peripheral membrane glycoprotein of zymogen granules

The major sulfated protein of the mouse pancreatic acinar cell, gp300, hsa been identified and characterized with monoclonal and polyclonal antibidies. gp300 is a glycoprotein of M_r = 300,000 which contains ～40% of metabolically incroporated [³⁵S] sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis. demonstrating that the sulfate is carbohydrate-linked rather than tyrosine-linked. gp300 metabolically labeled with [^3H]glucosamine and [³⁵H]sulfate was chemically and enzymaticlly treated followed by Bio-Gel P-10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycan. Treatment of dual-labeled gp300 with PNGase F to cleave N-linked oligosaccharides released ～17% of [₃S]. Mild alkaline borohydride treatment after removal of N-linked sugar relased the remainder of both labels, indicating the presence of sulfated O-linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicating the presence of sulfated O-linked oligosaccharides. Biosunynthetic studies and PNGase digestion F digestion indicate that the core protein is ～210 KDa, with apparent contrinution of ～35 KDa N-linked sugar, and ～55 KDa O-linked sugar. Lectin blotting and glycosidase digestion demonstrated the presece of Galβ(1–3)GalNAc and sialic acid α(2–3)Gal in O-linked oligosaccharide, and Galβ(1–4)GLcNAc in N-linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral memberane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in reponse to hormone stimulation ofacini, so it is not a secertroy product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule bigeneses.