John F. Cipollo

The Fine Structure of Caenorhabditis elegans N-Glycans ,

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematodeCaenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fuco-pausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholineN-glycans. Therefore, studies in C. elegansshould aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of itsN-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.

Unique Posttranslational Modifications of Chitin-Binding Lectins of Entamoeba invadens Cyst Walls

ABSTRACT Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an ∼30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.

Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica

N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man5GlcNAc2), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man5GlcNAc2, which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc1Man5GlcNAc2, which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive α-mannosidase trims some N-glycans to biantennary Man3GlcNAc2. Complex N-glycans of E. histolytica are made by the addition of α1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man5GlcNAc2, which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc.

The Caenorhabditis elegans bus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance

Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species.

The Fine Structure of Neisseria meningitidis Lipooligosaccharide from the M986 Strain and Three of Its Variants

Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose α-chain. The γ-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl α-chain even though previous reports suggested that the presence of an extended α-chain hinders C3 PEA substitution of Hep II. Approximately 50% of γ-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and γ-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.

Solid-Phase Chemical Modification for Sialic Acid Linkage Analysis: Application to Glycoproteins of Host Cells Used in Influenza Virus Propagation

Differentiation between the sialyl linkages is often critical to understanding biological consequence. Here we present a facile method for determining these linkages in glycans. Analysis of sialic acids is challenging due to their labile nature during sample preparation and ionization. Derivatization is often required via chemical reaction. Amidation derivatizes all sialic acids regardless of linkage, while esterification enables differentiation between α2,3-linked and α2,6-linked sialic acids. Reactions have been primarily performed on free glycans in solution but have been recently adapted to solid-phase providing unique advantages such as simplified sample preparation, improved yield, and high throughput applications. Here, we immobilized glycoproteins on resin via reductive amination, modified α2,6-linked sialic acids through ethyl esterification, and α2,3-linked sialic acids via amidation. N-glycans and O-glycans were released via enzyme and chemical reactions. The method was applied for analysis of three different MDCK cell lines used for influenza propagation and where distributions of α2,3 and α2,6 sialic acids are critical for cell performance. Linkage specific distribution of these sialic acids was quantitatively determined and unique for each cell line. Our study demonstrates that protein sialylation can be reliably and quantitatively characterized in terms of sialic acid linkage of each glycan using the solid-phase esterification/amidation strategy.