Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-->R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.     

Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures

This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.     

Reduction of metastatic properties of BL6 melanoma cells expressing terminal fucose(alpha)1-2-galactose after alpha1,2-fucosyltransferase cDNA transfection

We reported previously that transfection of BL6 melanoma cells that do not express the alpha1,3-galactosyltransferase (alpha1,3GT) gene with the alpha1,3GT cDNA resulted in synthesis and expression of alpha-galactosyl epitopes (Gal(alpha)1-3Gal(beta)1-4GlcNAc-R) and an impairment of their metastatic potentials. It was of interest to test whether inhibition of metastatic properties of BL6 melanoma cells is specifically associated with the appearance of the terminal alpha-Gal or whether capping N-acetyllactosamine with another oligosaccharide would also affect the metastatic properties of BL6 melanoma cells. For this purpose, BL6-2 clone isolated from B16BL6 melanoma was transfected with the alpha1,2-fucosyltransferase (alpha1,2FT) cDNA. The alpha1,2FT catalyzes a transglycosylation reaction, resulting in syntheses of the Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R structure, which is known as the H antigen of O blood group in humans and is also synthesized in some cells of mice. Transfection of BL6 melanoma cells with the alpha1,2FT cDNA resulted in the appearance of the terminal Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R epitopes reacting with the Ulex europaeus agglutinin lectin. In parallel, the transfected cells showed a decrease in N-acetyllactosamine sialylation. Decline in sialylation of the transfected cells is likely to be the result of competition between alphal,2FT and alpha2,3- or alpha2,6-sialyltransferases for the common substrate N-acetyllactosamine (Gal(beta)1-4GlcNAc-R) on N-linked carbohydrate chains of glycoproteins and glycolipids. The alpha1,2FT-transfected BL6-2 cells showed an increase in homotypic aggregation. In parallel, metastatic ability of the alpha1,2FT-transfected BL6-2 cells was reduced significantly in the immunocompetent as well as immunosuppressed (X-irradiated) mice. Thus, these data imply that capping N-acetyllactosamine with alphaGal or alphaFuc and the corresponding reduction in sialylation of BL6-2 melanoma cells were associated with reduction of their metastatic potential.     

An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.     

Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded homodimeric mucin-like glycoprotein on leukocytes that interacts with both P- and E-selectin. In this report we describe the structures of the Ser/Thr-linked O-glycans of PSGL-1 synthesized by HL-60 cells metabolically radiolabeled with 3H-sugar precursors. In control studies, the O-glycans on CD43 (leukosialin), a mucin-like glycoprotein also expressed by HL-60 cells, were analyzed and compared to those of PSGL-1. O-Glycans were released from Ser/Thr residues by mild base/borohydride treatment of purified glycoproteins, and glycan structures were determined by a combination of techniques. In contrast to expectations, PSGL-1 is not heavily fucosylated; a majority of the O-glycans are disialylated or neutral forms of the core-2 tetrasaccharide Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH++ +. A minority of the O-glycans are alpha-1,3-fucosylated that occur as two major species containing the sialyl Lewis x antigen; one species is a disialylated, monofucosylated glycan, and the other is a monosialylated, trifucosylated glycan having a polylactosamine backbone. CD43 lacks the fucosylated glycans found on PSGL-1 and is enriched for the nonfucosylated, disialylated core-2 hexasaccharide. These results demonstrate that PSGL-1 contains unique fucosylated O-glycans that are predicted to be critical for high affinity interactions between PSGL-1 and selectins.     

Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1-->6GalNAcalpha core structure as a tumor-associated antigen

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.     

Purification and characterization of an alpha1,2,-L-fucosyltransferase, which modifies the cytosolic protein FP21,from the cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.     

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells

During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans.     

Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland

The glycosylation of a number of constituents of human saliva is known to modify its biological roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a peridontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionisation (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labelled arabinose oligomers instead of the corresponding oligomers from partially hydrolysed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were additionally found substituted with sialic acid (both alpha2-3-linked and alpha2-6-linked) and sulphate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues respectively, containing fucose alpha1-3-linked to the outer-arm GlcNAc residues and alpha1-2-linked to the galactose. All fucosylated structures contained a core (alpha1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amounts of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addition of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.     

Expression of alpha-1,3-galactose and other type 2 oligosaccharide structures in a porcine endothelial cell line transfected with human alpha-1,2-fucosyltransferase cDNA

The binding of xenoreactive natural antibodies to the Galalpha1-3Galbeta1-4GlcNAc (alpha-galactose) oligosaccharide epitope on pig cells activates the recipient's complement system in pig to primate xenotransplantation. Expression of human alpha-1, 2-fucosyltransferase in pigs has been proposed as a strategy for reducing the expression level of the alpha-galactose epitope, thereby rendering the pig organs more suitable for transplantation into humans. The aim of this study was to examine how the cell surface expression of alpha-galactose, H, and related fucosylated and sialylated structures on a pig liver endothelial cell line is affected by transfection of human alpha-1,2-fucosyltransferase cDNA. Nontransfected and mock-transfected cells expressed alpha-galactose, alpha-2,3-sialylated, and alpha-2,6-sialylated epitopes strongly, with low level expression of type 2 H and LewisX. By contrast, expression of the H epitope was increased 5-8-fold in transfected cells with a 40% reduction in the expression of alpha-galactose epitope and a 50% decrease in sialylation, as measured by binding of Maackia amurensis and Sambuccus nigra agglutinins. LewisX expression was reduced to background levels, while the LewisY neoepitope was induced in human alpha-1,2-fucosyltransferase-expressing pig cells. The activities of endogenous alpha-1,3-galactosyltransferase, alpha-1,3-fucosyltransferases, and alpha-2,3- and alpha-2, 6-sialyltransferases acting on lactosamine were unaffected. Our results show that a reduction in alpha-galactose epitope expression in porcine endothelial cells transfected with human alpha-1, 2-fucosyltransferase cDNA may be achieved but at the expense of considerable distortion of the overall cell surface glycosylation profile, including the appearance of carbohydrate epitopes that are absent from the parent cells.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)-GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.     

Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.     

Alpha 1,6-linked fucose affects the expression and stability of polysialic acid-carrying glycoproteins in Chinese hamster ovary cells

To determine the effect of alpha1,6-linked fucose modification of N-glycans on the expression of polysialic acids (PSAs), the expression of PSAs in a fucose-lacking mutant of Chinese hamster ovary (CHO) cells, Lec13, was compared with that in CHO K1 cells. PSA synthase activity in these cells and the antennary structures of N-glycans associated with the neural adhesion molecule (NCAM), which is a major PSA-carrying glycoprotein, did not differ between the two types of cells. Metabolic labeling of cells with [3H]glucosamine for 48 h followed by immunoprecipitation with anti-PSA monoclonal antibodies revealed that the amount of labeled PSA-carrying glycoproteins obtained from Lec13 cells was 10-times less than that from K1 cells, although the incorporation of [3H]glucosamine into total extracts and NCAM was almost the same. In contrast, when cells were pulse labeled with [35S]methionine followed by a 1 h chase, there was not such a great difference in PSA-carrying protein synthesis between K1 and Lec13 cells. However, during a prolonged chase period, PSA-carrying proteins rapidly decreased in Lec13 cells, whereas those in K1 cells did not change. The degradation of PSA-carrying glycoproteins in Lec13 cells was partly prevented when the cells were grown in fucose-containing medium. Therefore, fucose modification of core N-glycans may affect the efficient expression of PSAs through the intracellular stability of PSA-carrying glycoproteins.     

GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.     

Stable expression of the Golgi form and secretory variants of human fucosyltransferase III from BHK-21 cells. Purification and characterization of an engineered truncated form from the culture medium

Stable BHK-21 cell lines were constructed expressing the Golgi membrane-bound form and two secretory forms of the human alpha1, 3/4-fucosyltransferase (amino acids 35-361 and 46-361). It was found that 40% of the enzyme activity synthesized by cells transfected with the Golgi form of the fucosyltransferase was constitutively secreted into the medium. The corresponding enzyme detected by Western blot had an apparent molecular mass similar to those of the truncated secretory forms. The secretory variant (amino acids 46-361) was purified by a single affinity-chromatography step on GDP-Fractogel resin with a 20% final recovery. The purified enzyme had a unique NH2 terminus and contained N-linked endo H sensitive carbohydrate chains at its two glycosylation sites. The fucosyltransferase transferred fucose to the O-4 position of GlcNAc in small oligosaccharides, glycolipids, glycopeptides, and glycoproteins containing the type I Galbeta1-3GlcNAc motif. The acceptor oligosaccharide in bovine asialofetuin was identified as the Man-3 branched triantennary isomer with one Galbeta1-3GlcNAc. The type II motif Galbeta1-4GlcNAc in bi-, tri-, or tetraantennary neutral or alpha2-3/alpha2-6 sialylated oligosaccharides with or without N-acetyllactosamine repeats and in native glycoproteins were not modified. The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.     

N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.     

Etiological and clinical characteristics of infectious optic neuritis

Infection by the tapeworm Echinococcus granulosus in the intermediate host results in the development of a hydatid cyst which contains the protoscoleces within a fluid-filled cavity enclosed by the bilayered cyst membrane. N-glycans were enzymatically released from crude extracts of homogenates of hydatid cyst membranes and protoscoleces and their structures were defined by high sensitivity fast atom bombardment mass spectrometry in conjunction with sequential exoglycosidase digestions. The major N-glycans from the cyst membrane were found to be non-charged structures having complex-type antennae and core fucosylation. The antennae are either truncated at the first N-acetylglucosamine or are extended with beta-galactose to form N-acetyllactosamine (lacNAc). A significant proportion of the lacNAc backbones are capped by alpha-galactose. The resulting Gal alpha-Gal beta-terminal structures may account for the earlier observation that antibodies against the blood group P1 epitope recognise components of hydatid cyst extracts. The complex-type N-glycans identified in the protoscoleces extracts were the same as the neutral structures found in the cyst membrane but a small proportion of high mannose structures and truncated di- and trimannosyl core structures were also identified. Sialylated N-glycans were identified as minor constituents of the cyst membrane preparation but were not observed in protoscoleces extracts. Whether the sialylated glycans are host derived or endogenously synthesized by the parasite remains to be established. This is the first reported structural analysis of N-glycans from cestodes and provides new insights into protein glycosylation in helminths.