Publications

It was recently demonstrated that complex-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni contain the unusual terminal sequence GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-2R. This structure suggests that the parasite might contain a novel glycosyltransferase that can add GalNAc to terminal GlcNAc residues in N-linked oligosaccharides. This report describes an assay for this enzyme, designated UDPGalNAc:GlcNAc beta 1-4 N-acetylgalactosaminyltransferase (beta 1-4GalNAcT), and demonstrates the presence of the enzyme activity in schistosome extracts. As an acceptor for the beta 1-4GalNAcT, we prepared from human fibrinogen a truncated biantennary glycopeptide that contained terminal GlcNAc residues. When this acceptor was incubated with schistosome extracts in the presence of UDP-[3H]GalNAc, Mn2+, and detergent, [3H]GalNAc was transferred to the glycopeptide acceptor. Approximately 75% of the radioactivity in the product isolated by lectin affinity chromatography was recovered as [3H]GalNAc following hydrolysis; likewise, a majority of the isolated product was bound by immobilized Wisteria floribunda agglutinin, a lectin that binds to schistosome-derived oligosaccharides containing terminal beta 1-4-linked GalNAc residues. The activity of the beta 1-4GalNAcT in schistosome extracts was dependent on time, protein, and UDPGalNAc.

We report the development of a solid-phase assay for the activity of the enzyme GDPFuc:Gal beta 1-4GlcNAc-R (Fuc to GlcNAc) alpha 1,3 fucosyltransferase (alpha 1,3FT). This enzyme generates the blood group antigen Lewis x (Lex)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-R from the acceptor Gal beta 1-4GlcNAc-R. In our method, the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (lacto-N-neotetraose, LNnT) from human milk was chemically conjugated to bovine serum albumin (BSA) to generate LNnT-BSA. As a source of alpha 1,3FT to develop the assay, we used extracts of COS7 cells created to stably express the human FucTIII and FucTIV genes, both of which have alpha 1,3FT activity. LNnT-BSA was immobilized in microtiter wells and incubated with GDPFuc and cell extracts. The Lex antigen generated by alpha 1,3FT was detected with a monoclonal IgM antibody (anti-CD15). Binding of this IgM-type antibody to product was detected by one of two methods. Method 1 was based on the binding of alkaline phosphatase-conjugated goat anti-mouse IgM. Method 2 was based on the binding of a streptavidin conjugate of the recombinant bioluminescent protein aequorin to biotinylated goat anti-mouse IgM. The alpha 1,3FT assay was linear with respect to time (0-3 h), extract added (0-40 micrograms), and was dependent on GDPFuc (20 microM optimal) and LNnT-BSA. Both methods 1 and 2 allowed measurement of alpha 1,3FT in extracts of the human cell line HL-60.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-carbohydrate recognition between the selectins and their ligands are among the earliest events in leukocyte recirculation, leukocyte recruitment into inflamed areas, and abnormal egress of leukocytes in diseases. Previously, we have described a dimeric sialoglycoprotein from myeloid cells with subunits of molecular mass = 120 kDa, which is selectively recognized by P-selectin (Moore, K.L., Stults, N.L., Diaz, S., Smith, D.F., Cummings, R.D., Varki, A., and McEver, R.P. (1992) J. Cell Biol. 188, 445-456). Here, we demonstrate that this P-selectin ligand carries alpha 2-3-linked sialic acids and the sialyl-Lewisx (SLex) tetrasaccharide motif. This glycoprotein contains 1% of the total membrane-bound sialic acids and a very small fraction of the total SLex on neutrophil membranes. In spite of a relative resistance to sialidase digestion, the predominant form of sialic acid on the ligand is N-acetylneuraminic acid. Selective periodate oxidation of the side chain of sialic acids does not affect P-selectin binding and allows the introduction of tritium label into the truncated sialic acids. beta-Elimination with alkaline borohydride releases labeled O-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [3H]glucosamine. The ligand from both neutrophils and HL-60 cells is also susceptible to cleavage by the enzyme O-sialoglycoprotease from Pasteurella hemolytica. Analysis of the specificity of this enzyme suggests that the P-selectin ligand carries large numbers of closely spaced sialylated O-linked oligosaccharides. O-Sialoglycoprotease abolishes both direct binding of P-selectin to HL-60 cells and the adhesion of HL-60 cells to immobilized P-selectin, without significantly decreasing overall cell surface SLex expression. This indicates that the 120-kDa ligand may be the major determinant of P-selectin:myeloid cell interaction in vivo. Finally, based on the current and previous data, we hypothesize that the high affinity recognition site(s) of this P-selectin ligand may be derived from a "clustered saccharide patch" of sialylated fucosylated O-linked oligosaccharide sequences.

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.

The S-type lactose-binding lectins are found in a variety of animal tissues and their primary sequences are highly conserved between species. Little is known, however, about their functions and endogenous ligands. We report here our studies on the carbohydrate binding specificity of one S-type lectin designated L-14, using glycopeptides and glycoproteins from Chinese hamster ovary cells and mouse teratocarcinoma F9 cells. Our studies demonstrate that L-14 binds with high affinity to oligosaccharides containing multiple repeating units (approximately 4) of the disaccharide [3Gal beta 1-4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. Interestingly, terminal beta-galactosyl residues are not necessary for high affinity binding of long PL-containing oligosaccharides to L-14. To characterize the glycoprotein ligands for L-14, we applied total [3H]galactose-labeled glycoproteins from differentiated F9 cells to L-14-Sepharose. Laminin was one of the major glycoproteins in both the cells and the media bound by the lectin. The possible functional significance of this interaction is suggested by the fact that in the absence of Ca2+ L-14 can mediate the binding in vitro of both CHO and F9 cells to immobilized laminin. Taken together, these studies demonstrate that L-14 binds with high affinity to laminin and to relatively long PL chains and indicate that L-14 can promote cell adhesion to laminin.

In this report, we describe studies on the structures of the N-linked oligosaccharides contained in glycoproteins synthesized by microfilariae of the canine heartworm, Dirofilaria immitis. Microfilariae were incubated in media containing either 2-[3H]mannose, 6-[3H]glucosamine, or 6-[3H]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were radiolabeled by both the mannose and glucosamine, whereas glycoproteins were not radiolabeled by the galactose. Glycopeptides from these total glycoproteins were fractionated and purified by serial lectin affinity chromatography, and the structures of the oligosaccharides in the isolated glycopeptides were analyzed by a variety of techniques. The N-linked oligosaccharides were shown to contain mannose (Man), fucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). However, they lacked sialic acid and galactose, which are commonly found in mammalian glycoproteins. GalNAc was shown to be in an unusual terminal position and beta-linked in the sequence GalNAc beta GlcNAc beta Man-R, where R is the typical branch of complex-type N-linked oligosaccharides. Similar structures were recently found by us to be synthesized by the helminthic parasite Schistosoma mansoni. These results demonstrate that glycoproteins synthesized by microfilariae of D. immitis have unusual carbohydrate moieties and may lead to a better understanding of the specific roles of glycoprotein oligosaccharides in host-parasite interactions.

We report our discovery that many glycoproteins synthesized by Chinese hamster ovary (CHO) cells contain fucose in O-glycosidic linkage to polypeptide. To enrich for the possible presence of O-linked fucose, we studied the lectin-resistant mutant of CHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferase I and are therefore unable to synthesize complex-type N-linked oligosaccharides. Lec1 cells were metabolically radiolabelled with [6-3H]fucose and total glycoproteins were isolated. Glycopeptides were prepared by proteolysis and fractionated by chromatography on a column of concanavalin A (Con A)-Sepharose. The sets of fractionated glycopeptides were treated with mild base/borohydride to effect the beta-elimination reaction and release potential O-linked fucosyl residues. The beta-elimination produced [3H]fucitol quantitatively from [3H]fucose-labelled glycopeptides not bound by Con A-Sepharose, whereas none was generated by treatment of glycopeptides bound by the lectin. The total [3H]fucose-labelled glycoproteins from Lec1 cells were separated by SDS-PAGE and detected by fluorography. Treatment of selected bands of detectable glycoproteins with mild base/borohydride quantitatively generated [3H]fucitol. Pretreatment of the glycoproteins with N-glycanase prior to the SDS-PAGE method of analysis caused an enrichment in the percentage of radioactivity recovered as [3H]fucitol. Trypsin treatment of [3H]fucose-labelled intact CHO cells released glycopeptides that contained O-linked fucose, indicating that it is present in surface glycoproteins. These findings demonstrate that many glycoproteins from CHO cells contain O-linked fucosyl residues and raise new questions about its biosynthesis and possible function.

We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.

Poly-N-acetyllactosamine (PL) sequences (repeating (3Gal beta 1-4GlcNAc beta 1)n) in complex-type N-linked oligosaccharides often occur in branched tri- and tetraantennary chains containing alpha-linked mannosyl residues disubstituted by N-acetyllactosaminyl units at C-2 and C-6 (2,6-branched mannose). We report here our studies on the factors affecting PL biosynthesis and the branching of N-linked oligosaccharides in glycoproteins from Chinese hamster ovary (CHO) cells. For our studies, we utilized a mutant CHO cell line designated Lec8 CHO, which lacks the ability to galactosylate its glycoproteins and consequently synthesizes glycoproteins containing terminal GlcNAc residues and lacking poly-N-acetyl-lactosamine and sialic acid. Glycoproteins in extracts of [3H]glucosamine- or [3H]mannose-labeled Lec8 CHO cells were galactosylated by exogenous beta 1-4-galactosyltransferase and analyzed by chromatography on leukoagglutinating phytohemagglutinin-Sepharose, a lectin reactive with glycoproteins containing 2,6-branched mannosyl residues. Approximately 10% of the radiolabeled glycoproteins were bound, and these were primarily of high molecular mass. Structural analyses of the bound glycoproteins demonstrated that they quantitatively contained 2,6-branched mannose. We then determined whether the "small i" N-acetylglucosaminyl-transferase (iGNT), which initiates PL biosynthesis, could specifically recognize glycoproteins in vitro and whether recognition was dependent on the presence of 2,6-branched mannose. When the galactosylated glycoproteins in extracts of Lec8 CHO cells were incubated with UDP-[3H]GlcNAc, the endogenous iGNT quantitatively added GlcNAc in beta 1-3-linkage to terminal galactosyl residues in the leukoagglutinating phytohemagglutinin-bound glycoproteins. These results demonstrate for the first time that 2,6-branched mannosyl residues are restricted to a subset of CHO glycoproteins and that the iGNT in vitro preferentially recognizes glycoproteins containing the 2,6-branched mannose determinant.