Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.     

Glycoengineering of alphaGal xenoantigen on recombinant peptide bearing the J28 pancreatic oncofetal glycotope

In human pancreatic adenocarcinoma, alterations of glycosylation processes leads to the expression of tumor-associated carbohydrate antigens, representing potential targets for cancer immunotherapy. Among these pancreatic tumor-associated carbohydrate antigens, the J28 glycotope located within the O-glycosylated mucin-like C-terminal domain of the fetoacinar pancreatic protein (FAPP) and expressed at the surface of human tumoral tissues, can be a good target for anticancer therapeutic vaccines. However, the oncodevelopmental self character of the J28 glycotope associated with the low immunogenicity of tumor-associated carbohydrate antigens may be a major obstacle to effective anti-tumor vaccine therapy. In this study, we have investigated a method to increase the immunogenicity of the recombinant pancreatic oncofetal J28 glycotope by glycoengineering Galalpha1,3Galss1,4GlcNAc-R (alphaGal epitope) which may be recognized by natural anti-alphaGal antibody present in humans. For this purpose, we have developed a stable Chinese hamster ovary cell clone expressing the alphaGal epitope by transfecting the cDNA encoding the alpha1,3galactosyltransferase. These cells have been previously equipped to produce the recombinant O-glycosylated C-terminal domain of FAPP carrying the J28 glycotope. As a consequence, the C-terminal domain of FAPP produced by these cells carries the alphaGal epitope on oligosaccharide structures associated with the J28 glycotope. Furthermore, we show that this recombinant "alpha1,3galactosyl and J28 glycotope" may not only be targeted by human natural anti-alphaGal antibodies but also by the mAbJ28, suggesting that the J28 glycotope remains accessible to the immune system as vaccinating agent. This approach may be used for many identified tumor-associated carbohydrate antigens which can be glycoengineered to carry a alphaGal epitope to increase their immunogenicity and to develop therapeutic vaccines.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

Anti-alpha-galactosyl antibodies recognizing epitopes terminating with alpha1,4-linked galactose: human natural and mouse monoclonal anti-NOR and anti-P1 antibodies

The rare NOR erythrocytes, which are agglutinated by most human sera, contain unique glycosphingolipids (globoside elongation products) terminating with the sequence Galalpha1-4GalNAcbeta1-3Gal- recognized by common natural human antibodies. Anti-NOR antibodies were isolated from several human sera by affinity procedures, and their specificity was tested by inhibition of antibody binding to NOR-tri-polyacrylamide (PAA) conjugate (ELISA) by the synthetic oligosaccharides, Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), Galalpha1-4GalNAc (NOR-di), Galalpha1-4Galbeta1-3Galbeta1-4Glc ((Gal)3Glc), and Galalpha1-4Gal (P1-di). Two major types of subspecificity of anti-NOR antibodies were found. Type 1 antibodies were found to react strongly with (Gal)3Glc and NOR-tri and weakly with P1-di and NOR-di, which indicated specificity for the trisaccharide epitope Galalpha1-4Gal/GalNAcbeta1-3Gal. Type 2 antibodies were specific to Galalpha1-4GalNAc, because they were inhibited most strongly by NOR-tri and NOR-di and were not (or very weakly) inhibited by (Gal)3Glc and P1-di. Monoclonal anti-NOR antibodies were obtained by immunizing mice with NOR-tri-human serum albumin (HSA) conjugate and were found to have type 2 specificity. All anti-NOR antibodies reacted specifically with NOR glycolipids on thin-layer plates. The cross-reactivity of type 1 anti-NOR antibodies with Galalpha1-4Gal drew attention to a possible antigenic relationship between NOR and blood group P system glycolipids. The latter glycolipids include Pk (Galalpha1-4Galbeta1-4Glc-Cer) present in all normal erythrocytes and P1 (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) present only in P1 erythrocytes. Sera of some P2 (P1-negative) persons contain natural anti-P1 antibodies. This prompted us to test the specificity of anti-P1 antibodies. Natural human anti-P1 isolated from serum of P2 individual and mouse monoclonal anti-P1 were best inhibited by Galalpha1-4Galbeta1-4GlcNAc (P1-tri) and did not react with NOR-tri and NOR-di. Monoclonal anti-P1 bound to Pk and P1 glycolipids and not to NOR glycolipids. These results indicated an entirely different specificity of anti-NOR and anti-P1 antibodies. Human serum samples differed in the content of anti-alpha-galactosyl antibodies, including both types of anti-NOR. In the sera of some individuals, type 1 or type 2 anti-NOR antibodies dominated, and other samples contained mixtures of both types of anti-NOR. The biological significance of these new abundant anti-alpha-galactosyl antibodies still awaits elucidation.     

Recent intron gain in elongation factor-1alpha of colletid bees (Hymenoptera: Colletidae)

We discovered the presence of a unique spliceosomal intron in the F1 copy of elongation factor-1alpha (EF-1alpha) restricted to the bee family Colletidae (Hymenoptera: Apoidae). The intron ranges in size from 101 to 1044 bp and shows no positional sliding. Our data also demonstrate the complete absence of this intron from exemplars representing all other bee families, as well as from close hymenopteran relatives. A review of the literature finds that this intron is likewise absent from all other arthropods for which data are available. This provides unambiguous evidence for a relatively recent intron insertion event in the colletid common ancestor and, at least in this specific instance, lends support to the introns-late hypothesis. The comparative distribution of this novel intron also supports the monophyly of Colletidae and the exclusion of the Stenotritidae from this family, providing an example of the potential of some introns to act as robust markers of shared descent.     

Molecular evolution and structure of alpha-actinin

The N-terminal actin-binding domain of alpha-actinin is connected to the C-terminal EF-hands by a rod domain. Because of its ability to form dimers, alpha-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells. In the prototypic alpha-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats. We have found some atypical alpha-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one and two spectrin repeats, respectively. This implies that the four repeats present in modern alpha-actinins arose after two consecutive intragenic duplications from an alpha-actinin with a single repeat. Further, the evolutionary gene tree of alpha-actinins shows that the appearance of four distinct alpha-actinin isoforms may have occurred after the vertebrate-invertebrate split. The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early in the vertebrate radiation. The phylogeny also considers these atypical isoforms as the most basal to alpha-actinins of vertebrates and other eukaryotes. The analysis also positioned alpha-actinin of the fungi Encephalitozoo cuniculi close to the protozoa, supporting the suggestion that microsporidia are early eukaryotes. Because alpha-actinin is considered the basal member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily.     

Characterization of the alpha-2,8-polysialyltransferase from Neisseria meningitidis with synthetic acceptors, and the development of a self-priming polysialyltransferase fusion enzyme

Glycoconjugates containing polysialic acid have many biological activities and represent target molecules for therapeutic interventions. Enzymatic synthesis of these glycoconjugates should give access to these important molecules to evaluate their potential. The polysialyltransferases from both Neisseria meningitidis and Escherichia coli were cloned and expressed as recombinant proteins in E. coli. We have used synthetic acceptors to probe the acceptor requirement of these enzymes and to examine the basic enzymology. The minimum number of sialic acid residues (Neu5Ac) on the acceptor for activity in vitro was shown to be 2 for both enzymes, but a large increase in activity was seen if the acceptor had three Neu5Ac residues. The polysialyltransferase from N. meningitidis generated longer reaction products than the enzyme from E. coli on FCHASE acceptors. Examination of the products showed them to be a heterogeneous mixture, but products with >50 Neu5Ac residues could be seen using capillary zone electrophoresis analyses. In addition we made fusion proteins of these polysialyltransferase enzymes with the bifunctional alpha-2,3/alpha-2,8-sialyltransferase from Campylobacter jejuni to create self priming polysialyltransferases. These bifunctional sialyltransferases utilized various synthetic disaccharide acceptors with a terminal galactose, and we demonstrate here that the PST enzyme from N. meningitidis and its fusion protein with the C. jejuni sialyltransferase can be used to create polysialic acid on O-linked glycopeptides.     

Genome phylogenies indicate a meaningful alpha-proteobacterial phylogeny and support a grouping of the mitochondria with the Rickettsiales

Placement of the mitochondrial branch on the tree of life has been problematic. Sparse sampling, the uncertainty of how lateral gene transfer might overwrite phylogenetic signals, and the uncertainty of phylogenetic inference have all contributed to the issue. Here we address this issue using a supertree approach and completed genomic sequences. We first determine that a sensible alpha-proteobacterial phylogenetic tree exists and that it can confidently be inferred using orthologous genes. We show that congruence across these orthologous gene trees is significantly better than might be expected by random chance. There is some evidence of horizontal gene transfer within the alpha-proteobacteria, but it appears to be restricted to a minority of genes ( approximately 23%) most of whom ( approximately 74%) can be categorized as operational. This means that placement of the mitochondrion should not be excessively hampered by interspecies gene transfer. We then show that there is a consistently strong signal for placement of the mitochondrion on this tree and that this placement is relatively insensitive to methodological approach or data set. A concatenated alignment was created consisting of 15 mitochondrion-encoded proteins that are unlikely to have undergone any lateral gene transfer in the timeline under consideration. This alignment infers that the sister group of the mitochondria, for the taxa that have been sampled, is the order Rickettsiales.     

Aberrant O-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion

Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.     

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man(7)GlcNAc(2) and probably Man(6)GlcNAc(2). These two products were also observed when Man(8)GlcNAc(2) isomer B instead of Man(9)GlcNAc(2) was used as substrate. Other oligosaccharides, such as Man(6)GlcNAc(2)-Asn, Man(5)GlcNAc(2)-Asn, and the alpha1,3- and alpha1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an alpha1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.     

GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.     

Evidence of positively selected sites in mammalian alpha-defensins

Alpha-defensins are a family of mammalian antimicrobial peptides that exhibit variable activity against a panel of microbes, including bacteria, fungi, and enveloped viruses. We have employed a maximum-likelihood approach to detect evidence of positive selection (adaptive evolution) in the evolution of these important molecules of the innate immune response. We have identified 14 amino acid sites that are predicted to be subject to positive selection. Furthermore, we show that all these sites are located in the mature antimicrobial peptide and not in the prepropeptide region of the molecule, implying that they are of functional importance. These results suggest that mammalian alpha-defensins have been under selective pressure to evolve in response to potentially infectious challenges by fast-evolving microbes.     

A specific detection of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins based on the specificity of N-acetylglucosaminyltransferase VI

Malignant transformation is often accompanied by an aberrant glycosylation profile of the cell surface-in particular, the production of GlcNAcbeta1-6Manalpha1 branches in N-linked glycoproteins. To identify the target glycoproteins, we show a method using recombinant chicken N-acetylglucosaminyltransferase VI (GnT VI) and radiolabeled uridine (5'-)diphosphate-GlcNAc. The assay exploits the fact that GnT VI has a strict requirement for the GlcNAcbeta1-6Manalpha1 structure for activity, when a pyridylaminated free N-glycan is used as the acceptor substrate. Human asialo-agalacto alpha1-acid glycoprotein (AGP), which is known to contain GlcNAcbeta1-6Manalpha1 branches in its N-linked glycan chains, was radiolabeled when reacted with GnT VI, whereas human asialo-agalacto transferrin and bovine fetuin, neither of which contains a GlcNAcbeta1-6Manalpha1 structure were not, thus corroborating the specificity of the assay. Several proteins from human serum after pretreatment with sialidase and beta-galactosidase could be detected using the assay. One was identified as AGP from its mobility on SDS-PAGE, demonstrating the potential of this assay even with crude materials. Furthermore, this method could detect a protein that was also positively stained with leukoagglutinating phytohemagglutinin (L(4)-PHA) using glycoproteins prepared from WiDr human colon cancer cells. This method should provide a useful complement to the current method, which relies on the specificity of L(4)-PHA.     

The binding of human glial cell line-derived neurotrophic factor to heparin and heparan sulfate: importance of 2-O-sulfate groups and effect on its interaction with its receptor,GFRalpha1

We report ELISA studies of the glycosaminoglycan binding properties of recombinant human glial cell line-derived neurotrophic factor (GDNF). We demonstrate relatively high affinity binding as soluble heparin competes with an IC50 of 0.1 micro g/ml. The binding of GDNF to heparin is particularly dependent on the presence of 2-O-sulfate groups. Highly sulfated heparan sulfate is also an effective competitor for GDNF binding. We also show that heparin at low concentrations protects GDNF from proteolytic modification by an endoprotease and also promotes the binding of GDNF to its receptor polypeptide, GFRalpha1. In both of these actions, 2-O-desulfated heparin is less effective. Considered overall, these findings provide strong support for a hypothesis that the bioactivity of GDNF during prenatal development is essentially dependent on the binding of this growth factor to 2-O-sulfate-rich heparin-related glycosaminoglycan.     

The binding of VIP36 and alpha-amylase in the secretory vesicles via high-mannose type glycans

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.     

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.     

Specificity of human anti-NOR antibodies,a distinct species of "natural" anti-alpha-galactosyl antibodies

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.     

The structure of cell wall alpha-glucan from fission yeast

Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis.     

Contribution of alpha-gustducin to taste-guided licking responses of mice

We examined the necessity of alpha-gustducin, a G protein alpha-subunit expressed in taste cells, to taste-mediated licking responses of mice to sapid stimuli. To this end, we measured licking responses of alpha-gustducin knock-out (Gus-/-) mice and heterozygotic littermate controls (Gus+/-) to a variety of 'bitter', 'umami', 'sweet', 'salty' and 'sour' taste stimuli. All previous studies of how Gus-/- mice ingest taste stimuli have used long-term (i.e. 48 h) preference tests, which may be confounded by post-ingestive and/or experiential effects of the taste stimuli. We minimized these confounds by using a brief-access taste test, which quantifies immediate lick responses to extremely small volumes of sapid solutions. We found that deleting alpha-gustducin (i) dramatically reduced the aversiveness of a diverse range of 'bitter' taste stimuli; (ii) moderately decreased appetitive licking to low and intermediate concentrations of an 'umami' taste stimulus (monosodium glutamate in the presence of 100 microM amiloride), but virtually eliminated the normal aversion to high concentrations of the same taste stimulus; (iii) slightly decreased appetitive licking to 'sweet' taste stimuli; and (iv) modestly reduced the aversiveness of high, but not low or intermediate, concentrations of NaCl. There was no significant effect of deleting alpha-gustducin on licking responses to NH4Cl or HCl.