Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Schistosome glycans and innate immunity.

Schistosome glycans induce characteristic innate immune responses in the infected host. The molecular aspects of these responses, the pathways and receptors as well as the schistosome glycans and glycoconjugates involved, form an area of intense research. The relevant schistosome glycan elements and the possible mechanisms through which they act on the innate immune system are discussed in this review.

Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

Immunodiagnostically applicable monoclonal antibodies to the circulating anodic antigen of Schistosoma mansoni bind to small, defined oligosaccharide epitopes.

Gut-associated glycoproteins constitute a major group of the circulating excretory antigens produced by human Schistosoma species. The O-glycans of the relatively abundant circulating anodic antigen (CAA) from S. mansoni carry long stretches of unique -->6(GlcA beta 1-->3)GalNAc beta 1--> repeats. Specific anti-carbohydrate monoclonal antibodies (mAbs) are essential tools for the immunodiagnostic detection of CAA in the serum or urine of Schistosoma-infected subjects. In order to define the epitopes recognised by these anti-CAA mAbs, we screened a series of protein-coupled synthetic di- to pentasaccharide building blocks of the CAA polysaccharide for immunoreactivity, using ELISA and surface plasmon resonance spectroscopy. It was shown that anti-CAA IgM mAbs preferentially recognise -->6(GlcA beta 1-->3)GalNAc beta 1--> disaccharide units. Interestingly, no mouse anti-CAA mAbs of the IgG class were found that bind to the synthetic epitopes, although many of the IgG mAbs tested do recognise native CAA in a carbohydrate-dependent manner. In addition, both IgM and IgG class antibodies could be detected in human infection sera using the synthetic CAA fragments. These synthetic schistosome glycan epitopes and their matching set of specific mAbs are useful tools that further the development of diagnostic methods and are helpful in defining the immunological responses of the mammalian hosts to schistosome glycoconjugates.

Dominant antibody responses to Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc containing carbohydrate epitopes in Pan troglodytes vaccinated and infected with Schistosoma mansoni.

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)). In addition, high levels of IgM antibodies were found against the trimeric Lewis(x) epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals.

Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by surface plasmon resonance.

We report here that sera of children and adults infected with Schistosoma mansoni, S. haematobium, or S. japonicum contain antibodies against GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) and to a lesser extent to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)) and GalNAcbeta1-4GlcNAc (LDN). Surface plasmon resonance (SPR) spectroscopy was used to monitor the presence of serum antibodies to neoglycoconjugates containing these carbohydrate epitopes and to define the immunoglobulin M (IgM) and IgG subclass distribution of the antibodies. The serum levels of antibodies to LDN-DF are high related to LDN and Lewis(x) for all examined groups of Schistosoma-infected individuals. A higher antibody response to the LDN-DF epitope was found in sera of infected children than in sera of infected adults regardless of the schistosome species. With respect to the subclasses, we found surprisingly that individuals infected with S. japonicum have predominantly IgG antibodies, while individuals infected with S. mansoni mainly show an IgM response; high levels of both isotypes were measured in sera of individuals infected with S. haematobium. These data provide new insights in the human humoral immune response to schistosome-derived glycans.

Schistosome glycoconjugates in host-parasite interplay.

Schistosomes are digenetic trematodes which cause schistosomiasis, also known as bilharzia, one of the main parasitic infections in man. In tropical and subtropical areas an estimated 200 million people are infected and suffer from the debilitating effects of this chronic disease. Schistosomes live in the blood vessels and strongly modulate the immune response of their host to be able to survive the hostile environment that they are exposed to. It has become increasingly clear that glycoconjugates of schistosome larvae, adult worms and eggs play an important role in the evasion mechanisms that schistosomes utilise to withstand the immunological measures of the host. Upon infection, the host mounts innate as well as adaptive immune responses to antigenic glycan elements, setting the immunological scene characteristic for schistosomiasis. In this review we summarise the structural data now available on schistosome glycans and provide data and ideas regarding the role that these glycans play in the various aspects of the glycobiology and immunology of schistosomiasis.

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins.

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains.

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.

Phase variation in Helicobacter pylori lipopolysaccharide due to changes in the lengths of poly(C) tracts in alpha3-fucosyltransferase genes.

The lipopolysaccharide (LPS) of Helicobacter pylori expresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two alpha3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of alpha3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the alpha3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of alpha3-fucosyltransferase knockout mutants. The data also show that the two alpha3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 alpha3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the alpha3-fucosylation precedes alpha2-fucosylation [corrected], an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

Identification of an alpha3-fucosyltransferase and a novel alpha2-fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-->3[Gal(NAc)beta1-->4]GlcNAc sequence.

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-->4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-->2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1-->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3-FucT resembles human FucT V and VI rather than other known FucTs. N-ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-->2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-->3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2-FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.

Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand. A second site on sperm binds a nonfucosylated, beta-galactosyl-capped oligosaccharide.

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively, a terminal alpha-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc,was inactive (1-72 microM range). In contrast, a beta 4-galactosyl-capped trisaccharide (Gal beta 1,4GlcNAc beta 1, 4GlcNAc) and an alpha 3-galactosyl-capped trisaccharide (Gal alpha 1,3Gal beta 1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 microM and 5.3 microM, respectively). The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Gal beta 1,4[Fuc alpha 1,3] GlcNAc beta 1,4GlcNAc or Gal alpha 1,3Gal beta 1, 4[Fuc alpha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nM). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the alpha 3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta-galactosyl-capped trisaccharide.

Phase variation in Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

Enzyme-assisted synthesis of Asn-linked diantennary oligosaccharides occurring on glycodelin A.

The preparation of a series of sialylated and fucosylated N,N'-diacetyllactosediamine-type diantennary glycopeptides is reported. By sequential enzymatic action of jack bean beta-galactosidase, snail beta 4-N-acetyl-galactosaminyltransferase, bovine colostrum alpha 6-sialyltransferase and human milk alpha 3-fucosyltransferase, a diantennary glycopeptide obtained from asialo fibrinogen was converted at a 5-mumol scale to a series of structures occurring on the glycoprotein glycodelin A, which potentially inhibit human sperm-egg binding.

One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration.

The trisaccharide Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was enzymatically synthesized, with in situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4'-epimerase, UDP-Gal:GlcNAc beta 4-galactosyltransferase and UDP-Gal:Gal beta 1-->4GlcNAc alpha 3-galactosyltransferase, Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was formed in a 2.2 mumol ml-1 yield starting from the acceptor GlcNAc beta 1-->O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc epitope which pays an important role in various biological and immunological processes.

Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Sialylation patterns and branch location of dimeric N-acetyllactosamine units.

The N-linked carbohydrate chains of recombinant human erythropoietin expressed in CHO cells were quantitatively released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, separated from the remaining O-glycoprotein by gel-permeation chromatography, and subsequently fractionated via FPLC on Mono Q, HPLC on Lichrosorb-NH2 and high-pH anion-exchange chromatography on CarboPac PA1. The purified sialylated oligosaccharides were analyzed by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. When necessary, oligosaccharides were treated with endo-beta-galactosidase (and N-acetyl-beta-glucosaminidase) followed by 1H-NMR analysis of the incubation products, to obtain additional structural information. Di-, tri-, tri'- and tetraantennary N-acetyllactosamine-type oligosaccharides occur which can be completely (major) or partially (minor) sialylated. Three different types of alpha 2-3-linked sialic acids are present, namely, N-acetylneuraminic acid (95%), N-glycolylneuraminic acid (2%) and N-acetyl-9-O-acetylneuraminic acid (3%). In the case of partial sialylation, a non-random distribution of the sialic acids over the branches is observed. One or two extra N-acetyllactosamine units, being exclusively located in the branches attached to the alpha 1-6-linked Man residue, can be present in completely or partially sialylated di-, tri'-, and tetraantennary oligosaccharides. Tetraantennary oligosaccharides with N-acetyllactosamine repeats could be digested quantitatively with endo-beta-galactosidase from Bacteroides fragilis, whereas under the same conditions tri' antennary oligosaccharides hardly reacted (< 15%). Using endo-beta-galactosidase from Escherichia freundii, these tri'antennary oligosaccharides could be digested more extensively (> 75%). The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment, and purified via FPLC on Mono Q and HPLC on Lichrosorb-NH2. Two O-glycans were found, namely, Neu5Ac alpha 2-3Gal beta 1-3GalNAc-ol and Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol.

A strategy for the mapping of N-glycans by high-performance capillary electrophoresis.

We have evaluated high-performance capillary electrophoresis (HPCE) with respect to its suitability for use in establishing a carbohydrate-mapping database that would enable a carbohydrate structural analysis by mere comparison of migration times. The suitability of HPCE for carbohydrate structural assignments was ascertained by validation experiments. The migration times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation of usually less than 0.20%, requiring only femtomoles of N-glycan per injection for reliable measurements. By including mesityl oxide and sialic acid as internal standards and a triple-correction method, HPCE fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan-mapping database was established using a newly developed and optimized buffer system containing 1,5-diaminopentane as an organic modifier. Approximately 80 different sialylated N-glycans of known structure, which have thus far been measured and characterized, have been entered into our Lotus 1-2-3 mapping database. The database for structural determinations was tested using the N-linked carbohydrates released from recombinant human urinary erythropoietin (baby hamster kidney) by PNGase F treatment and from bovine serum fetuin and alpha 1-acid glycoprotein by automated and manual (large-scale) hydrazinolysis, respectively. The efficiency of the database and of the triple-correction method was further confirmed by HPCE measurements performed in a different laboratory and by a different analyst who used the HPCE system of a different manufacturer.

Structure of the O-linked carbohydrate chains of porcine zona pellucida glycoproteins.

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and subsequently separated from the O-glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two-dimensional 1H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal beta 1-4GlcNAc beta 1-3Gal beta 1-3GalNAc-ol, Gal beta 1-4(6SO4-)GlcNAc, and alpha 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac alpha2-3]0-1[Gal beta 1-4(6SO4-)GlcNAc beta 1-3]nGal beta 1-4GlcNAc beta 1-3Gal Beta 1-3GalNAc-ol. In addition, smaller compounds were identified in which the Gal beta 1-3GalNAc-ol core is substituted by Neu5Gc/Ac alpha 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc beta 1-3GalNAc-ol core structure. Analysis of the endo-beta-galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O-linked carbohydrate chains.

Human beta 1,4 galactosyltransferase and alpha 2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum.

Biosynthesis and intracellular transport of recombinant human full-length beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113-120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H-NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [35S]Met-labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of beta-galactosidase-GT or beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin-treated [35S]Met-labelled transformed yeast cells or labelling with radio-active sugars both indicated that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi-specific man-alpha 1,6-man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.

Structure determination of the disialylated poly-(N-acetyllactosamine)-containing O-linked carbohydrate chains of equine chorionic gonadotropin.

The disialylated poly-(N-acetyllactosamine)-containing O-linked oligosaccharide alditols, released by alkaline borohydride treatment of the enzymically N-deglycosylated beta-subunit of equine chorionic gonadotropin, were purified by fast protein liquid chromatography (FPLC) on Mono Q and analysed by fast ion bombardment mass spectrometry (FAB-MS) and 1H-NMR spectroscopy. The identified oligosaccharide alditols have the following structure: [Formula: see text]