Purification and Preliminary Biochemical Characterisation of the Human and Rat Forms of the Central Nervous System-Specific Molecule, F3-87-8

The F3-87-8 monoclonal antibody recognises a phylogenetically conserved antigenic determinant found exclusively in the mammalian CNS. We used this monoclonal antibody as the major purification step for obtaining pure. F3-87-8-bearing molecules from rat and human brains for biochemical analysis. In both rat and man, the F3-87-8 molecule is a heavily glycosylated protein, consisting of 47.6 and 47.0% carbohydrate by weight, respectively. In both species, it occurs as a doublet on polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the Mr of the human form being 130,000 and 100,000. In the rat, the Mr of the doublet is slightly but consistently lower than in man, and the higher-Mr band is more pronounced. The amino acid composition of the rat and human forms is virtually identical, with a high content of serine and threonine. Significant differences are seen in carbohydrate composition, the rat form containing more sialic acid and neutral sugar and less hexosamine than the human form. beta-Elimination studies, in conjunction with carbohydrate analysis, suggest the presence of approximately 40 O-linked and 10-15 N-linked oligosaccharides per polypeptide chain of 500 amino acids, the N-linked chains predominantly of the high-mannose type. This makes it likely that the molecule adopts an extended rather than a coiled conformation on the membrane.     

Analysis of the structure of the human T cell surface antigen T8 by limited proteolysis and chemical cleavage

The structure of the human T lymphocyte surface antigen T8 (Leu 2) has been explored utilizing limited proteolysis on viable cells and cellular lysates. The positions of the cleavage sites of trypsin and papain were placed relative to the single CNBr cleavage point. Additional data allowed the location of the amino and carboxyl termini relative to the enzymatic and chemical cleavage sites. This information, together with earlier evidence concerning the position of a membrane binding site, allowed the construction of a model illustrating the vectorial orientation of the molecule on the cell. Within this model, the approximate positions of disulfide linkages were indicated based on the results of nonreduced/reduced two-dimensional sodium sulfide-polyacrylamide gel electrophoresis. Carbohydrate moieties were localized using cleavage with trifluoromethanesulfonic acid, a reagent which cleaves both N-linked and O-linked oligosaccharides. Finally, the implications of the proteolysis experiments in relation to the function of T8 were discussed.     

A murine cytotoxic T lymphocyte cell line resistant to Vicia villosa lectin is deficient in UDP-GalNAc:beta-galactose beta 1,4-N-acetylgalactosaminyltransferase

We have reported the presence of N-acetylgalactosamine linked beta 1,4 to galactose on O-linked oligosaccharides of a cloned murine cytotoxic T cell line and the absence of these residues from the O-linked structures of a Vicia villosa lectin-resistant mutant line, VV6, derived from parental B6.1.SF.1 cells (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12528-12535). This study shows that B6.1.SF.1 cells contain an enzyme which transfers N-acetylgalactosamine from UDP-GalNAc onto the O-linked tetrasaccharides of human glycophorin A, giving rise to pentasaccharides which contain beta-glycosidically linked N-acetylgalactosamine. Desialylated glycophorin was inactive as an acceptor. The enzyme also transfers N-acetylgalactosamine to the N-linked oligosaccharides of the Tamm-Horsfall glycoprotein. This glycoprotein is known to contain N-linked oligosaccharides with beta-linked N-acetylgalactosamine residues which constitute the Sda blood group determinant. This N-acetylgalactosaminyltransferase could not be detected in VV6 cells which can account for the lack of beta-linked N-acetylgalactosamine residues on its O-linked oligosaccharides. The two cell lines have comparable levels of UDP-GalNAc:apomucin N-acetylgalactosaminyltransferase, demonstrating that the enzyme deficiency in VV6 cells is selective. Both cell lines have a similar glycolipid content, with the major component being asialo-GM1. Since this glycolipid contains N-acetylgalactosamine linked beta 1,4 to galactose, it would appear that the N-acetylgalactosyltransferase involved in the biosynthesis of glycolipids is different from the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase. An independently derived murine CTL line also contains the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase, suggesting that the expression of this enzyme is a common characteristic of this type of cell line.     

Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains.

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.     

Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibian Pleurodeles waltl characterize by means of lectin histochemistry

The aim of this work was the characterization of the glycoconjugates of the premeiotic spermatogenetic cells of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. In the cytoplasm of the primordial germ cells, primary and secondary spermatogonia and primary spermatocytes, a granular structure can be observed close to the nucleus. These granules contain four types of sugar chains according to their appearance during the differentiation process: 1. some oligosaccharides that are identified in all the four cell types above mentioned, which include N-linked oligosaccharides with Fuc, Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc and O-linked oligosaccharides with Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc; 2. other glycan chains that are not present in the primary spermatocytes (N-linked oligosaccharides with DBA-positive GalNAc, GlcNAc, and a slight amount of Neu5Ac alpha2,6Gal/GalNAc and O-linked oligosaccharides with WGA-positive GlcNAc); 3. the sugar chains that are not in the earliest step of spermatogenesis (formed by both N-linked and O-linked oligosaccharides with Glc); and 4. other that appear at the earliest and latest stages, but not in the intermediate ones, (N-linked oligosaccharides with Man and O-linked oligosaccharides with SBA- and HPA-positive GalNAc and PNA-positive Gal beta1,3GalNAc). This structure could be related with the Drosophila spectrosome and fusome, unusual cytoplasmic organelles implicated in cystic germ cell development. Data from the present work, as compared with those from mammals and other vertebrates, suggest that, although no dramatic changes in the glycosylation pattern are observed, some cell glycoconjugates are modified in a predetermined way during the early steps of the spermatogenetic differentiation process.     

O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus

The O-linked oligosaccharides on mature forms of herpes simplex virus type 1 (HSV1) glycoproteins were characterized, and were found to account largely for the lower electrophoretic mobilities of these forms relative to the mobilities of immature forms. Other posttranslational modifications of HSV1 glycoproteins (designated gB, gC, gD and gE) were related temporally to the discrete shifts in electrophoretic mobilities that signal acquisition of the O-linked oligosaccharides. Fatty acid acylation (principally of gE) could be detected just prior to the shifts, whereas conversion of high-mannosetype N-linked oligosaccharides to the complex type occurred coincident with the shifts. The addition of O-linked oligosaccharides did not occur in cells treated with the ionophore monensin or in a ricinresistant cell line defective in the processing of N-linked oligosaccharides. We conclude that extension of O-linked oligosaccharide chains on HSV1 glycoproteins, and probably also attachment of the first O-linked sugars, occurs as a late posttranslational modification in the Golgi apparatus.     

Temporal aspects of O-glycosylation of glycoprotein C from herpes simplex virus type-1.

Herpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time.     

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.     

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.     

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.     

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.     

Evidence that leukosialin, CD43, is intensely sulfated in the murine T lymphoma line RDM-4.

Metabolic labeling of the murine T lymphoma cell line RDM-4 with [35S] sulfate results in intense incorporation into a cell-retained component of apparent Mr approximately 100,000. This macromolecule is identified as a glycoprotein by lectin chromatography. The sulfate is not incorporated as tyrosine sulfate. Release of the radiolabel by alkaline beta-elimination but not by endoglycosidase F is consistent with the sulfation of O- rather than N-linked oligosaccharides. The sulfated glycoprotein displays anomalous migration on SDS-PAGE in two respects: 1) the apparent Mr shifts from 115,000 to 87,000 on increasing the acrylamide concentration from 7 to 12%, and 2) on neuraminidase digestion migration is substantially reduced (apparent Mr 140,000). These properties indicate that the sulfated protein is both heavily glycosylated and extensively sialylated, and are characteristic of the lymphoid mucin, leukosialin (sialophorin, CD43). Specific labeling of the sialoglycoproteins of RDM-4 cells indicates that leukosialin, the most intensely labeled protein, comigrates with the sulfated protein on SDS-PAGE at varying acrylamide concentrations. Our data are therefore consistent with sulfation of at least some of the numerous O-linked oligosaccharides of this abundant glycoprotein in RDM-4 cells. No sulfation of CD43 in resting splenic T cells is observed.     

Unique anthranilic acid chemistry facilitates profiling and characterization of Ser/Thr-linked sugar chains following hydrazinolysis

A novel method for the analysis of Ser/Thr-linked sugar chains was made possible by the virtue of unique anthranilic acid (AA, 2-aminobenzoic acid [2AA]) chemistry for labeling carbohydrates in aqueous salt solutions (K. R. Anumula, Anal. Biochem. 350 (2006) 1-23). The protocol for profiling of Ser/Thr carbohydrates by hydrazinolysis was made simple by eliminating intermediary isolation steps involved in a sample preparation such as desalting and various chromatographic purification schemes. A 6-h hydrazinolysis was carried out at 60 degrees C for O-linked oligosaccharides and at 95 degrees C for total oligosaccharides (N-linked with some O-linked). Following evaporation of hydrazine (<10 min), the oligosaccharides were N-acetylated and derivatized with AA in the same reaction mixture containing salts. Presumably, the glycosyl-hydrazines/hydrazones present in the mixture did not interfere with AA labeling. Because AA is the most fluorescent and highly reactive tag for labeling carbohydrates, the procedures described are suitable for the analysis of a limited amount of samples ( approximately 5 microg) by the current high-resolution high-performance liquid chromatography (HPLC) methods. HPLC conditions developed for the separation of O-linked sugar chains based on size on an amide column were satisfactory for quantitative profiling and characterization. Common O-linked sugar chains found in fetuin, equine chorionic gonadotropin, and glycophorin can be analyzed in less than 50 min. In addition, these fast profiling methods were comparable to profiling by PNGase F (peptide N-glycosidase from Flavobacterium meningosepticum) digestion in terms of time, effort, and simplicity and also were highly reproducible for routine testing. The procedures for the release of sugar chains by hydrazinolysis at the microgram level, labeling with fluorescent tag AA, and profiling by HPLC should be useful in characterization of carbohydrates found in glycoproteins.     

Purification and characterization of a glycosylated proline-rich protein from human parotid saliva.

1. A glycosylated proline-rich protein (GPRP) was purified to homogeneity by subjecting parotid saliva to immunoaffinity, cation exchange, affinity and hydrophobic chromatography. 2. The purified GPRP had a molecular weight of 78 kDa as analyzed by SDS-PAGE. 3. The amino acid analysis revealed a preponderance of proline, glycine and glutamic acid/glutamine, which accounted for 77% of the total amino acids. 4. Cysteine, tyrosine or phenylalanine residues were not detected. 5. The glycoprotein contained 34% neutral sugars and the oligosaccharides were rich in mannose and N-acetylglucosamine, indicating that N-linked oligosaccharides were the predominant type of oligosaccharides in the molecule. 6. These observations were confirmed by treatment of the purified glycoprotein with specific N-glycosidase which removed the N-linked oligosaccharides leaving a core protein with an apparent molecular weight of 51 kDa. 7. The isoelectric point of GPRP was approx 7.0 and the molecule was not affected by reduction with 2-mercaptoethanol, indicating that no disulfide linkages were present. 8. The GPRP bound to hydroxyapatite and this binding could be partially inhibited by preincubation of the hydroxyapatite with parotid or submandibular saliva. 9. The purified GPRP also bound to a protein with an apparent molecular weight of 95 kDa present in submandibular saliva.     

Intrinsic glycosylation potentials of insect cell cultures and insect larvae

Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides. Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose.     

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.     

Specific cytotoxic T lymphocytes recognize the immediate-early transactivator Zta of Epstein-Barr virus.

We identified the immediate-early transactivator Zta of Epstein-Barr virus as a target for specific cytotoxic T lymphocytes (CTL). Cells pulsed with overlapping synthetic peptides representing the entire amino acid sequence of Zta proved to be efficient for the in vitro stimulation of Zta-specific CTL in several donors. With peptide-pulsed target cells, we found that CTL from several donors recognize a peptide comprising 15 amino acids. The immune response against this peptide exerted by CTL lines from different donors was found to be restricted by two different molecules of the major histocompatibility complex: HLA-B8 and HLA-Cw6. The latter molecule could for the first time be identified as a restricting element for a CTL response. The epitope of the HLA-B8-restricted CTL could be mapped to an octameric sequence between amino acid positions 190 and 197 of the Zta protein, whereas the minimal epitope of HLA-Cw6-restricted CTL consists of 11 to 15 residues between positions 187 and 201. Thus, the HLA-B8 and HLA-Cw6 epitopes widely overlap but are not completely identical. In vitro stimulation of blood lymphocytes from a panel of HLA-B8-positive or HLA-Cw6-positive virus carriers, using autologous cells pulsed with the Zta peptides comprising the HLA-B8 or HLA-Cw6 epitope, respectively, revealed in both cases that most of these donors developed a Zta-specific cytotoxic activity. These data, as well as the high spread of the major histocompatibility complex molecules HLA-B8 and HLA-Cw6 in most populations, suggest that an efficient CTL response directed against gene products of the immediate-early group of the lytic cycle exists in vivo in a considerable portion of virus carriers. A CTL response against proteins expressed immediately after the switch into the lytic cycle could eliminate lytically activated cells at an early stage and would thus efficiently prevent the production and release of progeny virions.     

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.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.