Terminal Galactose Moieties Research Articles

Single-mutations at the galactose-binding site of enzymes GalK, GalU and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-D-galactose and azido-functionalized Gb3 analogs.

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson-Fabry disease and an elevated level of deacylated Gb3 (lyso-Gb3) is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1,4βGal1,4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of GalK, GalU and LgtC enzymes, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes GalK/GalU generate UDP-6-azido-6-deoxy-d-galactose (UDP-6AzGal), which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the three enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy-d-galactose-1-phosphate, UDP-6AzGal and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V and LgtC-Q187S, respectively, is 3- to 6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6AzGal with ~90 percent conversion yields and products AzGlobotriose and lyso-AzGb3 with substrate conversion of up to 70 percent. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series.

PH-Induced Conformational Changes of Human Bocavirus Capsids.

Human bocavirus 1 (HBoV1) and HBoV2-4 infect children and immunocompromised individuals, resulting in respiratory and gastrointestinal infections, respectively. Using cryo-electron microscopy and image reconstruction, the HBoV2 capsid structure was determined to 2.7 Å resolution at pH 7.4 and compared to the previously determined HBoV1, HBoV3, and HBoV4 structures. Consistent with previous findings, surface variable region (VR) III of the capsid protein VP3, proposed as a host tissue-tropism determinant, was structurally similar among the gastrointestinal strains HBoV2-4, but differed from HBoV1 with its tropism for the respiratory tract. Towards understanding the entry and trafficking properties of these viruses, HBoV1 and HBoV2 were further analyzed as species representatives of the two HBoV tropisms. Their cell surface glycan-binding characteristics were analyzed, and capsid structures determined to 2.5-2.7 Å resolution at pH 5.5 and 2.6, conditions normally encountered during infection. The data showed that glycans with terminal sialic acid, galactose, GlcNAc or heparan sulfate moieties do not facilitate HBoV1 or HBoV2 cellular attachment. With respect to trafficking, conformational changes common to both viruses were observed at low pH conditions localized to the VP N-terminus under the 5-fold channel, in the surface loops VR-I and VR-V and specific side-chain residues such as cysteines and histidines. The 5-fold conformational movements provide insight into the potential mechanism of VP N-terminal dynamics during HBoV infection and side-chain modifications highlight pH-sensitive regions of the capsid.IMPORTANCE Human bocaviruses (HBoVs) are associated with disease in humans. However, the lack of an animal model and a versatile cell culture system to study their life cycle limits the ability to develop specific treatments or vaccines. This study presents the structure of HBoV2, at 2.7 Å resolution, determined for comparison to the existing HBoV1, HBoV3, and HBoV4 structures, to enable the molecular characterization of strain and genus-specific capsid features contributing to tissue tropism and antigenicity. Furthermore, HBoV1 and HBoV2 structures determined under acidic conditions provide insight into capsid changes associated with endosomal and gastrointestinal acidification. Structural rearrangements of the capsid VP N-terminus, at the base of the 5-fold channel, demonstrate a disordering of a "basket" motif as pH decreases. These observations begin to unravel the molecular mechanism of HBoV infection and provide information for control strategies.

Lectin staining of the uterovaginal junction and sperm-storage tubule epithelia in broiler hens

Mammalian sperm bind to terminal carbohydrates associated with glycoconjugates on the apical surface of oviduct epithelial cells in the caudal region of the oviduct and undergo cellular and molecular modifications associated with capacitation prior to ovulation. In contrast, chicken sperm are stored for up to 23 d in sperm-storage tubules (SST) localized in the uterovaginal junction (UVJ). Little is known of the cellular and molecular mechanisms that regulate sperm storage in and release from the SST. The purpose of this study was to identify glycoconjugates associated with the SST epithelial cell surface using lectins. Virgin hens and hens of higher and lower fertility in egg production for 6 to 16 wk were used in this study. Sections of UVJ mucosa containing SST were stained with fluorescent conjugated lectins and examined by confocal microscopy. Carbohydrate moieties associated with the UVJ and SST epithelia differed in their lectin binding patterns. No differences in the lectin binding patterns within the 2 epithelia were discernible between the virgin and younger and older hens. Minor differences were observed between the higher and lower fertility hens. Only lectins specific for galactose and N-acetylgalactosamine moieties were localized to the luminal surface of the SST. While resident sperm may be closely apposed to the SST epithelial cell apical microvilli, it is unlikely that sperm binding to the microvilli via terminal carbohydrates associated with glycoconjugates is a requisite for prolonged storage. However, the possibility of SST epithelial cell communication with resident sperm via shedding microvillous vesicles characterized by surface glycoconjugates with terminal galactose and N-acetylgalactosamine moieties is currently being investigated.

Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

All currently identified primary receptors of adeno-associated virus (AAV) are glycans. Depending on the AAV serotype, these carbohydrates range from heparan sulfate proteoglycans (HSPG), through glycans with terminal α2-3 or α2-6 sialic acids, to terminal galactose moieties. Receptor identification has largely relied on binding to natural compounds, defined glycan-presenting cell lines, or enzyme-mediated glycan modifications. Here, we describe a comparative binding analysis of highly purified, fluorescent-dye-labeled AAV vectors of various serotypes on arrays displaying over 600 different glycans and on a specialized array with natural and synthetic heparins. Few glycans bind AAV specifically in a serotype-dependent manner. Differential glycan binding was detected for the described sialic acid-binding AAV serotypes 1, 6, 5, and 4. The natural heparin binding serotypes AAV2, -3, -6, and -13 displayed differential binding to selected synthetic heparins. AAV7, -8, -rh.10, and -12 did not bind to any of the glycans present on the arrays. For discrimination of AAV serotypes 1 to 6 and 13, minimal binding moieties are identified. This is the first study to differentiate the natural mixed heparin binding AAV serotypes 2, 3, 6, and 13 by differential binding to specific synthetic heparins. Also, sialic acid binding AAVs display differential glycan binding specificities. The findings are relevant for further dissection of AAV host cell interaction. Moreover, the definition of single AAV-discriminating glycan binders opens the possibility for glycan microarray-based discrimination of AAV serotypes in gene therapy.

Sialic Acid Loss Regulates Platelet Survival and Integrity

It becomes increasingly apparent that, besides the intrinsic apoptotic machinery, surface glycan modifications regulate platelet survival. Platelets with reduced α2,3-linked sialic acid during sepsis due to S. pneumoniae infection, after cold storage, or in mice lacking the sialyltransferase ST3GalIV are cleared by the hepatic Ashwell-Morell receptor (AMR, ASGPR1/2). Platelet survival in Asgr2-/- mice was increased by ∼35% when compared to that of WT mice, which results in a ∼50% increase in circulating platelet counts, despite a loss of surface sialic acid. We reasoned that sialidase activity increases on the surface of circulating platelets as they age, a process that would facilitate sialic acid hydrolysis and removal from the circulation. To test this hypothesis, we directly injected the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) into WT mice and determined endogenous platelet circulatory times. Platelet survival was prolonged by ∼30% (T1/2 of 62.0 ± 2.7 h) in DANA-treated mice, compared to that of mock-treated mice (T1/2 of 47.5 ± 4.3 h). DANA injections decreased terminal sialic acid loss on circulating platelets by ∼40% by day 2, compared to control platelets, as evidenced by binding of RCA-I lectin that specifically recognizes terminal β1-4 galactose moieties exposed by sialic acid removal. Freshly isolated, resting platelets from Asgr2-/- mice (AMR-platelets) were significantly smaller in size (22%) and had increased sialidase Neu1 (∼5 fold), but not Neu3 surface expression, when compared to WT platelets or St3gal4-/- platelets, as measured by flow cytometry. We next investigated if AMR-platelets age/deteriorate faster upon in vitro storage. Platelets were isolated from WT, Asgr2-/- and St3gal4-/- mice and stored for 24hrs at room temperature, and sialidase expression (Neu1 and Neu3) as well as microvesiculation were measured by flow cytometry. Although significant Neu1 and Neu3 surface expression increase was measured on platelets from all phenotype after storage, Neu1 and Neu3 surface expression was significantly higher in AMR-platelets (∼2 and 4 fold, respectively) when compared to WT and St3gal4-/- platelets. AMR-platelets, but not St3gal4-/- platelets microvesiculated upon storage, consistent with a faster deterioration of aged AMR-platelets. We next injected into WT and Asgr2-/- mice the BH3 mimetic, ABT-737, which binds and inhibits the pro-apoptotic Bcl-2, Bcl-xL and Bcl-w. After injection of ABT-737, platelets in the Asgr2-/- mouse were cleared more efficiently (∼20%) from the circulation when compared to those in WT mice. Collectively, our data show that blood borne sialidases contribute to loss of sialic acid during circulation to regulate platelet survival. Our data also suggest that platelet glycan degradation, i.e. sialic acid loss, may trigger the intrinsic apoptotic machinery in platelets, linking glycan degradation and intrinsic apoptotic machinery in the clearance mechanisms regulating platelet survival. Disclosures:No relevant conflicts of interest to declare.

Generation and characterization of recombinant feline β-galactosidase for preclinical enzyme replacement therapy studies in GM1 gangliosidosis

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

Kinetic and Mechanistic Analysis of Trypanosoma cruzi Trans-Sialidase Reveals a Classical Ping-Pong Mechanism with Acid/Base Catalysis

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.

Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system

The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that beta-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn(2+)), a cofactor for beta-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn(2+) addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn(2+) addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn(2+) only had this effect late in the culture process. These data indicate that the addition of Mn(2+) has complex effects on stressed batch fed cultures.

Saposin B binds and transfers phospholipids

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.

Identification of a Novel Sialic Acid Transporter in Haemophilus ducreyi

Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.

Expression of an α-Galactosidase Gene in Petunia is Upregulated during Low-temperature Deacclimation

Previous studies of plant tolerance to low temperature have focused primarily on the cold acclimation response, the process by which plants increase their tolerance to freezing in response to low nonfreezing temperatures, while studies on the deacclimation process have been largely neglected. In some plants, cold acclimation is accompanied by an increase in raffinose family oligosaccharides (RFO). The enzyme α-galactosidase (EC 3.2.1.22) breaks down RFO during deacclimation by hydrolyzing the terminal galactose moieties. Here we describe the isolation of PhGAL, an α-galactosidase cDNA clone from Petunia (Petunia ×hybrida `Mitchell'). The putative α-galactosidase cDNA has high nucleotide sequence homology (>80%) to other known plant α-galactosidases. PhGAL expression increased in response to increased temperature and there was no evidence of developmental regulation or tissue specific expression. Increases in α-galactosidase transcript 1 hour into deacclimation corresponded with increases in α-galactosidase activity and a concomitant decrease in raffinose content, suggesting that warm temperature may regulate RFO catabolism by increasing the transcription of the α-galactosidase gene. This information has potential practical applications whereby α-galactosidase may be targeted to modify endogenous raffinose accumulation in tissues needed for freezing stress tolerance.

Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library.

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.

Infectivity and attenuation of Leishmania donovani promastigotes. II: Association of the loss of parasite infectivity with the terminal galactosylation of precursor acceptors present in virulent parasites by the developmentally regulated galactosyltransferase.

The beta1-4 galactosyltransferase enzyme of the Leishmania donovani promastigotes, was found to be developmentally regulated and expressed only in the attenuated parasites. The enzymatic product of soluble determinants of virulent promastigotes and the galactosyltransferase enzyme was found to stimulate the macrophage burst activity but inhibit in vitro intracellular parasitism. In contrast, removal of terminal galactose moieties from soluble determinants of attenuated parasites resulted in the inhibition of macrophage respiratory burst activity but did not now inhibit intracellular parasitism. We propose that the terminal galactosylation of acceptor substrates present in virulent parasites by the developmentally regulated galactosyltransferase is associated with loss of parasite virulence.

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins.

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12–28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30–66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Adaptation of Haemophilus influenzae to acquired and innate humoral immunity based on phase variation of lipopolysaccharide.

Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non-typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of licl and lic2, which contain translational switches based on intragenic tandem repeats of 5'-CAAT-3'. Genetic analysis showed that opaque organisms have an out-of-frame number of repeats in both licl, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galalpha1-4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C-reactive protein (CRP). The addition of ChoP by licl was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galalpha1-4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host-like antigen blocks antibody-mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galalpha1-4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody.

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an alpha 2-3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman's incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.

Galactose-induced dimerization of blocked ricin at acidic pH: evidence for a third galactose-binding site in ricin B-chain.

Blocked ricin is a glycoconjugate formed by covalent modification of each of the two galactose-binding sites of ricin with affinity ligands derived by modification of glycopeptides containing galactose-terminated, triantennary, N-linked oligosaccharides. Blocked ricin undergoes a pH-dependent reversible self-association, being predominantly dimeric at neutral pH and monomeric at acidic pH. The shift in the monomer-dimer equilibrium towards the monomeric form at acidic pH (pH 4) is inhibited by lactose, as shown by size-exclusion chromatography. This behavior of blocked ricin can be reproduced in studies with isolated blocked B-chain. The effect, which is dependent on the concentration of the sugar, is specific for sugars having terminal galactose moieties, or sugars having the same orientation of hydroxyl groups at C2 and C4 as galactose. These results are interpreted as providing further support for the notion that ricin B-chain has a third galactose-binding site, which may be important for the intracellular trafficking of ricin during intoxication of cells.

Analysis and production of two exopolysaccharides fromLactococcus lactissubsp.cremorisLC330

SUMMARYTwo polysaccharides produced concurrently byLactococcus lactissubsp.cremorisstrain LC330 have been identified. One had a high molecular mass (> 1 × 106Da) and was neutral. The second was smaller (∼ 10000 Da), charged and had a high phosphorus content. Sugar composition also differed. In chemostat culture the neutral polysaccharide was influenced by temperature and by nitrogen limitation. This polysaccharide was branched with terminal galactose moieties and contained galactose, glucose and glucosamine. The phosphopolysaccharide was more complex with glucose, rhamnose, galactose and glucosamine in an approximate ratio of 6:5:4:1.