Salmonid polysialyltransferases to generate a variety of sialic acid polymers.

The human polysialyltransferases ST8Sia II and ST8Sia IV catalyze the transfer of several Neu5Ac residues onto glycoproteins forming homopolymers with essential roles during different physiological processes. In salmonids, heterogeneous set of sialic acids polymers have been described in ovary and on eggs cell surface and three genes st8sia4, st8sia2-r1 and st8sia2-r2 were identified that could be implicated in these heteropolymers. The three polysialyltransferases from the salmonid Coregonus maraena were cloned, recombinantly expressed in HEK293 cells and the ST8Sia IV was biochemically characterized. The MicroPlate Sialyltransferase Assay and the non-natural donor substrate CMP-SiaNAl were used to demonstrate enzyme activity and optimize polysialylation reactions. Polysialylation was also carried out with natural donor substrates CMP-Neu5Ac, CMP-Neu5Gc and CMP-Kdn in cell-free and cell-based assays and structural analyses of polysialylated products using the anti-polySia monoclonal antibody 735 and endoneuraminidase N and HPLC approaches. Our data highlighted distinct specificities of human and salmonid polysialyltransferases with notable differences in donor substrates use and the capacity of fish enzymes to generate heteropolymers. This study further suggested an evolution of the biological functions of polySia. C. maraena ST8Sia IV of particular interest to modify glycoproteins with a variety of polySia chains.

N-Glycan on the Non-Consensus N-X-C Glycosylation Site Impacts Activity, Stability, and Localization of the Sda Synthase B4GALNT2.

The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1.

BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

B4GALNT2 Controls Sda and SLex Antigen Biosynthesis in Healthy and Cancer Human Colon.

The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

Vertebrate Alpha2,8-Sialyltransferases (ST8Sia): A Teleost Perspective.

We identified and analyzed α2,8-sialyltransferases sequences among 71 ray-finned fish species to provide the first comprehensive view of the Teleost ST8Sia repertoire. This repertoire expanded over the course of Vertebrate evolution and was primarily shaped by the whole genome events R1 and R2, but not by the Teleost-specific R3. We showed that duplicated st8sia genes like st8sia7, st8sia8, and st8sia9 have disappeared from Tetrapods, whereas their orthologues were maintained in Teleosts. Furthermore, several fish species specific genome duplications account for the presence of multiple poly-α2,8-sialyltransferases in the Salmonidae (ST8Sia II-r1 and ST8Sia II-r2) and in Cyprinus carpio (ST8Sia IV-r1 and ST8Sia IV-r2). Paralogy and synteny analyses provided more relevant and solid information that enabled us to reconstruct the evolutionary history of st8sia genes in fish genomes. Our data also indicated that, while the mammalian ST8Sia family is comprised of six subfamilies forming di-, oligo-, or polymers of α2,8-linked sialic acids, the fish ST8Sia family, amounting to a total of 10 genes in fish, appears to be much more diverse and shows a patchy distribution among fish species. A focus on Salmonidae showed that (i) the two copies of st8sia2 genes have overall contrasted tissue-specific expressions, with noticeable changes when compared with human co-orthologue, and that (ii) st8sia4 is weakly expressed. Multiple sequence alignments enabled us to detect changes in the conserved polysialyltransferase domain (PSTD) of the fish sequences that could account for variable enzymatic activities. These data provide the bases for further functional studies using recombinant enzymes.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

MicroPlate Sialyltransferase Assay: A Rapid and Sensitive Assay Based on an Unnatural Sialic Acid Donor and Bioorthogonal Chemistry.

Mammalian sialyltransferases transfer sialic acids onto glycoproteins and glycolipids within the Golgi apparatus. Despite their key role in glycosylation, the study of their enzymatic activities is limited by the lack of appropriate tools. Herein, we developed a quick and sensitive sialyltransferase microplate assay based on the use of the unnatural CMP-SiaNAl donor substrate. In this assay, an appropriate acceptor glycoprotein is coated on the bottom of 96-well plate and the sialyltransferase activity is assessed using CMP-SiaNAl. The alkyne tag of SiaNAl enables subsequent covalent ligation of an azido-biotin probe via CuAAC and an antibiotin-HRP conjugated antibody is then used to quantify the amount of transferred SiaNAl by a colorimetric titration. With this test, we evaluated the kinetic characteristics and substrate preferences of two human sialyltransferases, ST6Gal I and ST3Gal I toward a panel of asialoglycoprotein acceptors, and identified cations that display a sialyltransferase inhibitory effect.

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting.

The Sda /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the ß1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase. DATABASE: The proteins ß1,4GalNAcT II, ß1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.

Probing the CMP-Sialic Acid Donor Specificity of Two Human ß-d-Galactoside Sialyltransferases (ST3Gal†I and ST6Gal†I) Selectively Acting on O- and N-Glycosylproteins.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human ß-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate ß-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal).

Cell surface of eukaryotic cells is covered with a wide variety of sialylated molecules involved in diverse biological processes and taking part in cell-cell interactions. Although the physiological relevance of these sialylated glycoconjugates in vertebrates begins to be deciphered, the origin and evolution of the genetic machinery implicated in their biosynthetic pathway are poorly understood. Among the variety of actors involved in the sialylation machinery, sialyltransferases are key enzymes for the biosynthesis of sialylated molecules. This review focus on ß-galactoside α2,3/6-sialyltransferases belonging to the ST3Gal and ST6Gal families. We propose here an outline of the evolutionary history of these two major ST families. Comparative genomics, molecular phylogeny and structural bioinformatics provided insights into the functional innovations in sialic acid metabolism and enabled to explore how ST-gene function evolved in vertebrates.

Differentiation of Crohn's Disease-Associated Isolates from Other Pathogenic Escherichia coli by Fimbrial Adhesion under Shear Force.

Shear force exerted on uropathogenic Escherichia coli adhering to surfaces makes type-1 fimbriae stretch out like springs to catch on to mannosidic receptors. This mechanism is initiated by a disruption of the quaternary interactions between the lectin and the pilin of the two-domain FimH adhesin and transduces allosterically to the mannose-binding pocket of FimH to increase its affinity. Mannose-specific adhesion of 14 E. coli pathovars was measured under flow, using surface plasmon resonance detection on functionalized graphene-coated gold interfaces. Increasing the shear had important differential consequences on bacterial adhesion. Adherent-invasive E. coli, isolated from the feces and biopsies of Crohn's disease patients, consistently changed their adhesion behavior less under shear and displayed lower SPR signals, compared to E. coli opportunistically infecting the urinary tract, intestines or loci of knee and hip prostheses. We exemplified this further with the extreme behaviors of the reference strains UTI89 and LF82. Whereas their FimA major pilins have identical sequences, FimH of LF82 E. coli is marked by the Thr158Pro mutation. Positioned in the inter-domain region known to carry hot spots of mutations in E. coli pathotypes, residue 158 is indicated to play a structural role in the allosteric regulation of type-1 fimbriae-mediated bacterial adhesion.

Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

A forward genetic approach in Chlamydomonas reinhardtii as a strategy for exploring starch catabolism.

A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding ß-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae.

Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria.

*Type II secretion (T2S) systems of many plant-pathogenic bacteria often secrete cell wall-degrading enzymes into the plant apoplast. *Here, we show that the Xps-T2S system from the plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) promotes disease and contributes to the translocation of effector proteins that are delivered into the plant cell by the type III secretion (T3S) system. *The Xcs-T2S system instead lacks an obvious virulence function. However, individual xcs genes can partially complement mutants in homologous xps genes, indicating that they encode functional components of T2S systems. Enzyme activity assays showed that the Xps system contributes to secretion of proteases and xylanases. We identified the virulence-associated xylanase XynC as a substrate of the Xps system. However, homologs of known T2S substrates from other Xanthomonas spp. are not secreted by the T2S systems from Xcv. Thus, T2S systems from Xanthomonas spp. appear to differ significantly in their substrate specificities. *Transcript analyses revealed that expression of xps genes in Xcv is activated by HrpG and HrpX, key regulators of the T3S system. By contrast, expression of xynC and extracellular protease and xylanase activities are repressed by HrpG and HrpX, suggesting that components and substrates of the Xps system are differentially regulated.

The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay.

Dickeya dadantii is a pectinolytic phytopathogen enterobacterium that causes soft rot disease on a wide range of plant species. The virulence of D. dadantii involves several factors, including the osmoregulated periplasmic glucans (OPGs) that are general constituents of the envelope of proteobacteria. In addition to the loss of virulence, opg-negative mutants display a pleiotropic phenotype, including decreased motility and increased exopolysaccharide synthesis. A nitrosoguanidine-induced mutagenesis was performed on the opgG strain, and restoration of motility was used as a screen. The phenotype of the opg mutant echoes that of the Rcs system: high level activation of the RcsCD-RcsB phosphorelay is needed to activate exopolysaccharide synthesis and to repress motility, while low level activation is required for virulence in enterobacteria. Here, we show that mutations in the RcsCDB phosphorelay system restored virulence and motility in a D. dadantii opg-negative strain, indicating a relationship between the Rcs phosphorelay and OPGs.

Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the membrane-bound and the soluble periplasmic phosphoglycerol transferases are encoded by the same gene.

In Escherichia coli, osmoregulated periplasmic glucans (OPGs) are highly substituted by phosphoglycerol, phosphoethanolamine and succinyl residues. A two-step model was proposed to account for phosphoglycerol substitution: first, the membrane-bound phosphoglycerol transferase I transfers residues from membrane phosphatidylglycerol to nascent OPG molecules; second, the periplasmic phosphoglycerol transferase II swaps residues from one OPG molecule to another. Gene opgB was reported to encode phosphoglycerol transferase I. In this study, we demonstrate that the periplasmic enzyme II is a soluble form of the membrane-bound enzyme I. In addition, timing of OPG substitution was investigated. OPG substitution by succinyl residues occurs rapidly, probably during the backbone polymerization, whereas phosphoglycerol addition is a very progressive process. Thus, both phosphoglycerol transferase activities appear biologically necessary for complete OPG substitution.

Characterization of the Erwinia chrysanthemi Gan locus, involved in galactan catabolism.

beta-1,4-Galactan is a major component of the ramified regions of pectin. Analysis of the genome of the plant pathogenic bacteria Erwinia chrysanthemi revealed the presence of a cluster of eight genes encoding proteins potentially involved in galactan utilization. The predicted transport system would comprise a specific porin GanL and an ABC transporter made of four proteins, GanFGK(2). Degradation of galactans would be catalyzed by the periplasmic 1,4-beta-endogalactanase GanA, which released oligogalactans from trimer to hexamer. After their transport through the inner membrane, oligogalactans would be degraded into galactose by the cytoplasmic 1,4-beta-exogalactanase GanB. Mutants affected for the porin or endogalactanase were unable to grow on galactans, but they grew on galactose and on a mixture of galactotriose, galactotetraose, galactopentaose, and galactohexaose. Mutants affected for the periplasmic galactan binding protein, the transporter ATPase, or the exogalactanase were only able to grow on galactose. Thus, the phenotypes of these mutants confirmed the functionality of the gan locus in transport and catabolism of galactans. These mutations did not affect the virulence of E. chrysanthemi on chicory leaves, potato tubers, or Saintpaulia ionantha, suggesting an accessory role of galactan utilization in the bacterial pathogeny.

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active.

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.

The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans.

Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography. One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC. opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis. Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis. In contrast, inactivation of a gene similar to ndvB, encoding the OPG-glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides. Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit. Expression of opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.