Biochemical characterization of a glycosyltransferase homolog from an oral pathogen Fusobacterium nucleatum as a human glycan-modifying enzyme

Bacterial glycosyltransferases have drawn growing attention as economical enzymes for oligosaccharide synthesis, with their easy expression and relatively broad substrate specificity. Here, we characterized a glycosyltransferase homolog (Fnu_GT) from a human oral pathogen, Fusobacterium nucleatum. Bioinformatic analysis showed that Fnu_GT belongs to the glycosyltransferases family II. The recombinant Fnu_GT (rFnu_GT) expressed in Escherichia coli displayed the highest glycosylation activity when UDP-galactose (Gal) was used as a donor nucleotide-sugar with heptose or Nacetylglucosamine (GlcNAc) as an acceptor sugar. Interestingly, rFnu_GT transferred the galactose moiety of UDP-Gal to a nonreducing terminal GlcNAc attached to the trimannosyl core glycan, indicating its potential as an enzyme for humantype N-glycan synthesis.     

Comprehensive analysis of glycosyltransferases in eukaryotic genomes for structural and functional characterization of glycans

Glycosyltransferases comprise highly divergent groups of enzymes, which play a central role in the synthesis of complex glycans. Because the repertoire of glycosyltransferases in the genome determines the range of synthesizable glycans, and because the increasing amount of genome sequence data is now available, it is essential to examine these enzymes across organisms to explore possible structures and functions of the glycoconjugates. In this study, we systematically investigated 36 eukaryotic genomes and obtained 3426 glycosyltransferase homologs for biosynthesis of major glycans, classified into 53 families based on sequence similarity. The families were further grouped into six functional categories based on the biosynthetic pathways, which revealed characteristic patterns among organism groups in the degree of conservation and in the number of paralogs. The results also revealed a strong correlation between the number of glycosyltransferases and the number of coding genes in each genome. We then predicted the ability to synthesize major glycan structures including N-glycan precursors and GPI-anchors in each organism from the combination of the glycosyltransferase families. This indicates that not only parasitic protists but also some algae are likely to synthesize smaller structures than the structures known to be conserved among a wide range of eukaryotes. Finally we discuss the functions of two large families, sialyltransferases and β4-glycosyltransferases, by performing finer classifications into subfamilies. Our findings suggest that universality and diversity of glycans originate from two types of evolution of glycosyltransferase families, namely conserved families with few paralogs and diverged families with many paralogs.     

On the origin of family 1 plant glycosyltransferases

The phylogeny of highly divergent multigene families is often difficult to validate but can be substantiated by inclusion of data outside of the phylogeny, such as signature motifs, intron splice site conservation, unique substitutions of conserved residues, similar gene functions, and out groups. The Family 1 Glycosyltransferases (UGTs) comprises such a highly divergent, polyphyletic multigene family. Phylogenetic comparisons of UGTs from plants, animals, fungi, bacteria, and viruses reveal that plant UGTs represent three distinct clades. The majority of the plant sequences appears to be monophyletic and have diverged after the bifurcation of the animal/fungi/plant kingdoms. The two minor clades contain the sterol and lipid glycosyltransferases and each show more homology to non-plant sequences. The lipid glycosyltransferase clade is homologous to bacterial lipid glycosyltransferases and reflects the bacterial origin of chloroplasts. The fully sequenced Arabidopsis thaliana genome contains 120 UGTs including 8 apparent pseudogenes. The phylogeny of plant glycosyltransferases is substantiated with complete phylogenetic analysis of the A. thaliana UGT multigene family, including intron-exon organization and chromosomal localization.     

Mammalian glycosyltransferases: genomic organization and protein structure.

In recent years, several glycosyltransferase genes and cDNAs have been cloned and characterized. Although the glycosyltransferases seem to share the same general architecture, there is only little sequence similarity between the various enzymes. Moreover, a comparison of the organization of the genes shows that there is no common pattern of intron-exon structure. In addition, there seems to be little or no correlation between glycosyltransferase exons and protein domains. Taken together, these observations suggest that many of the glycosyltransferase genes evolved independently. So far, only two glycosyltransferase gene families have been described. These families may have evolved by exon-shuffling, or by gene duplication and subsequent divergence. For specific glycosyltransferases, mechanisms such as alternative splicing and alternative promoter usage play a role in the production of multiple protein isoforms from a single gene. These isoenzymes may differ in their enzymatic properties or cellular localization.     

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.     

Molecular cloning, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.     

Diverse Functions for Six Glycosyltransferases in Caulobacter crescentus Cell Wall Assembly

The essential process of peptidoglycan synthesis requires two enzymatic activities, transpeptidation and transglycosylation. While the PBP2 and PBP3 transpeptidases perform highly specialized functions that are widely conserved, the specific roles of different glycosyltransferases are poorly understood. For example, Caulobacter crescentus encodes six glycosyltransferase paralogs of largely unknown function. Using genetic analyses, we found that Caulobacter glycosyltransferases are primarily redundant but that PbpX is responsible for most of the essential glycosyltransferase activity. Cells containing PbpX as their sole glycosyltransferase are viable, and the loss of pbpX leads to a general defect in the integrity of the cell wall structure even in the presence of the other five glycosyltransferases. However, neither PbpX nor any of its paralogs is required for the specific processes of cell elongation or division, while the cell wall synthesis required for stalk biogenesis is only partially disrupted in several of the glycosyltransferase mutants. Despite their genetic redundancy, Caulobacter glycosyltransferases exhibit different subcellular localizations. We suggest that these enzymes have specialized roles and normally function in distinct subcomplexes but retain the ability to substitute for one another so as to ensure the robustness of the peptidoglycan synthesis process.     

MIR: Family of repeats common to vertebrate genomes

We studied the occurrence of mammalian interspersed repeats (MIRs) in DNA and RNA of vertebrates, invertebrates, and bacteria using the data from GenBank. A special algorithm based on a weight position matrix with optimal alignment using dynamic programming was developed to search for the traces of MIR dissemination. This allowed us to search for highly divergent MIRs carrying deletions and insertions. MIRs were detected in genomes of various fishes, includingLatimeria. This suggests that the origin of MIRs dates back more than 400 million years. The method to search for similarity between highly divergent sequences may be used to find the genome fragments from various ancient repeat families and from various gene families.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.     

Identification of catalytic amino acids in the human GTP fucose pyrophosphorylase active site

GTP-l-fucose pyrophosphorylase(GFPP) catalyzes the reversible formation of the nucleotide-sugar GDP-beta-l-fucose from guanosine triphosphate and beta-l-fucose-1-phosphate. The enzyme functions primarily in the mammalian liver and kidney to salvage free fucose during the breakdown of glycoproteins and glycolipids. GFPP shares little primary sequence identity with other nucleotide-sugar metabolizing enzymes, and the three-dimensional structure of the protein is unknown. The enzyme does contain several sequences that could be nucleotide binding sites, but none of them are an exact match to consensus sequences. Using a combination of site-directed mutagenesis and UV photoaffinity cross-linking, we have identified five amino acid residues that are critical for catalysis. Some of these amino acids are found within the poorly conserved nucleotide binding consensus structures, while others represent new motifs. Two active site lysines can be cross-linked to photoaffinity probes. The site of cross-linking depends on the probe used. The identification of these critical residues highlights how distinct GFPP is from other nucleotide-sugar pyrophosphorylases.     

Allelic divergence precedes and promotes gene duplication

One of the striking observations from recent whole-genome comparisons is that changes in the number of specialized genes in existing gene families, as opposed to novel taxon-specific gene families, are responsible for the majority of the difference in genome composition between major taxa. Previous models of duplicate gene evolution focused primarily on the role that neutral processes can play in evolutionary divergence after the duplicates are already fixed in the population. By instead including the entire cycle of duplication and divergence, we show that specialized functions are most likely to evolve through strong selection acting on segregating alleles at a single locus, even before the duplicate arises. We show that the fitness relationships that allow divergent alleles to evolve at a single locus largely overlap with the conditions that allow divergence of previously duplicated genes. Thus, a solution to the paradox of the origin of organismal complexity via the expansion of gene families exists in the form of the deterministic spread of novel duplicates via natural selection.     

Phylogeny and Systematics of Multituberculate Mammals

We present a synopsis of high-rank multituberculate systematicsand a manually generated cladogram illustrating multituberculate interrelationships. We divide the Multituberculata into the paraphyletic suborder 'Plagiaulacida', an apparently monophyletic suborder Cimolodonta, and one family incertae sedis. Within 'Plagiaulacida' we recognise three informal lines: paulchoffatiid (three families), plagiaulacid (three families) and allodontid (two families and the genus Glirodon). The Cimolodonta are divided into an informal Paracimexomys group; three superfamilies: Ptilodontoidea, Djadochtatherioidea (new), and Taeniolabidoidea (restricted to Taeniolabididae); and five families (superfamily incertae sedis): Eucosmodontidae, Microcosmodontidae, Cimolodontidae, Boffiidae, and Kogaionidae; and some genera incertae sedis. New characters used in our analysis are (1) a tendency of molar cusps to coalesce; and (2) ornamentation of grooves, pits, and ridges on the molars. We argue that the Ptilodontoidea, and less certainly also the Cimolodontidae and Boffiidae, might have originated from amongthe plagiaulacid line, a possible intermediate link being the Paracimexomys group. The remaining Cimolodonta might have originated from unknown members of the Paracimexomys group with separated molar cusps and smooth enamel. The origin of two types of prismatic enamel and a relationship between them are stumbling blocks in understanding the origin of the Cimolodonta; we conclude that microprismatic enamel made its appearance only once. Revised diagnoses of high-rank multituberculate taxa, including lists of all known genera, are given.     

Synthesis and biological evaluation of new UDP-GalNAc analogues for the study of polypeptide-alpha-GalNAc-transferases

A series of three O-methylated UDP-GalNAc analogues have been synthesised using a divergent strategy from a 3,6-di-O-pivaloyl GlcNAc derivative. The biological activity of these probes toward polypeptide-alpha-GalNAc-transferase T1 has been investigated. This study shows that this glycosyltransferase exhibits a very high substrate specificity.     

Ancestral Origin of the ATTCT Repeat Expansion in Spinocerebellar Ataxia Type 10 (SCA10)

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurodegenerative disease characterized by cerebellar ataxia and seizures. The disease is caused by a large ATTCT repeat expansion in the ATXN10 gene. The first families reported with SCA10 were of Mexican origin, but the disease was soon after described in Brazilian families of mixed Portuguese and Amerindian ancestry. The origin of the SCA10 expansion and a possible founder effect that would account for its geographical distribution have been the source of speculation over the last years. To unravel the mutational origin and spread of the SCA10 expansion, we performed an extensive haplotype study, using closely linked STR markers and intragenic SNPs, in families from Brazil and Mexico. Our results showed (1) a shared disease haplotype for all Brazilian and one of the Mexican families, and (2) closely-related haplotypes for the additional SCA10 Mexican families; (3) little or null genetic distance in small normal alleles of different repeat sizes, from the same SNP lineage, indicating that they are being originated by a single step mechanism; and (4) a shared haplotype for pure and interrupted expanded alleles, pointing to a gene conversion model for its generation. In conclusion, we show evidence for an ancestral common origin for SCA10 in Latin America, which might have arisen in an ancestral Amerindian population and later have been spread into the mixed populations of Mexico and Brazil.     

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

A substantial body of work has been devoted to the design and synthesis of glycosyltransferase inhibitors. A major obstacle has always been the demanding chemistry. Therefore, only few potent and selective inhibitors are known to date. Glycosyltransferases possess two distinct binding sites, one for the donor substrate, and one for the acceptor substrate. In many cases binding to the donor site is well defined but data for acceptor binding is sparse. In particular, acceptor binding sites are often shallow, and in many cases the dimensions of the binding pocket are not well defined. One approach to glycosyltransferase inhibitors is to chemically link donor site and acceptor site ligands to generate high affinity binders. Here, we describe a novel approach to identify acceptor site ligands from a fragment library. We have chosen human blood group B galactosyltransferase (GTB) as a biologically important model target. The approach utilizes a combination of STD NMR, spin-lock filtered NMR experiments and surface plasmon resonance measurements. Following this route we have identified molecular fragments from a fragment library that bind to the acceptor site of GTB with affinities of the order of a natural acceptor substrate. Unlike natural substrates these fragments allow for straightforward chemical modifications and, therefore will serve as scaffolds for potent GTB inhibitors. In general, the approach described is applicable to any glycosyltransferase and may assist in the development of novel glycosyltransferase inhibitors.     

Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi.

Glucosylceramides are membrane lipids in most eukaryotic organisms and in a few bacteria. The physiological functions of these glycolipids have only been documented in mammalian cells, whereas very little information is available of their roles in plants, fungi, and bacteria. In an attempt to establish appropriate experimental systems to study glucosylceramide functions in these organisms, we performed a systematic functional analysis of a glycosyltransferase gene family with members of animal, plant, fungal, and bacterial origin. Deletion of such putative glycosyltransferase genes in Candida albicans and Pichia pastoris resulted in the complete loss of glucosylceramides. When the corresponding knock-out strains were used as host cells for homologous or heterologous expression of candidate glycosyltransferase genes, five novel glucosylceramide synthase (UDP-glucose:ceramide glucosyltransferase) genes were identified from the plant Gossypium arboreum (cotton), the nematode Caenorhabditis elegans, and the fungi Magnaporthe grisea, Candida albicans, and P. pastoris. The glycosyltransferase gene expressions led to the biosynthesis of different molecular species of glucosylceramides that contained either C18 or very long chain fatty acids. The latter are usually channeled exclusively into inositol-containing sphingolipids known from Saccharomyces cerevisiae and other yeasts. Implications for the biosynthesis, transport, and function of sphingolipids will be discussed.     

Survey of <Emphasis Type="Italic">O</Emphasis>-GlcNAc level variations in <Emphasis Type="Italic">Xenopus laevis</Emphasis> from oogenesis to early development

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I–V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.     

A minimal TrpRS catalytic domain supports sense/antisense ancestry of class I and II aminoacyl-tRNA synthetases

The emergence of polypeptide catalysts for amino acid activation, the slowest step in protein synthesis, poses a significant puzzle associated with the origin of biology. This problem is compounded as the 20 contemporary aminoacyl-tRNA synthetases belong to two quite distinct families. We describe here the use of protein design to show experimentally that a minimal class I aminoacyl-tRNA synthetase active site might have functioned in the distant past. We deleted the anticodon binding domain from tryptophanyl-tRNA synthetase and fused the discontinuous segments comprising its active site. The resulting 130 residue minimal catalytic domain activates tryptophan. This residual catalytic activity constitutes the first experimental evidence that the conserved class I signature sequences, HIGH and KMSKS, might have arisen in-frame, opposite motifs 2 and 1 from class II, as complementary sense and antisense strands of the same ancestral gene.