Compartmentalization of galactan biosynthesis in mycobacteria.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

Further defining the phenotypic spectrum of B4GALT7 mutations.

Proteoglycans are components of the extracellular matrix with diverse biological functions. Defects in proteoglycan synthesis have been linked to several human diseases with common features of short stature, hypermobility, joint dislocations, and skeletal dysplasia. B4GALT7 encodes galactosyltransferase-I that catalyzes the addition of a galactose moiety to a xylosyl group in the tetrasaccharide linker of proteoglycans. Mutations in this gene have been associated with the rare progeroid form of Ehlers Danlos syndrome and in addition more recently found to underlie Larsen of Reunion Island syndrome. Nine individuals have been reported with a diagnosis of the progeroid form of Ehlers Danlos syndrome, four of whom have had molecular characterization showing homozygous or compound heterozygous mutations in B4GALT7. We report two newly described patients with compound heterozygous mutations in B4GALT7, and show that the six individuals with confirmed mutations do not have the progeroid features described in the original five patients with a clinical diagnosis of the progeroid form of Ehlers Danlos syndrome. We suggest that galactosyltransferase-I deficiency does not cause the progeroid form of Ehlers Danlos syndrome, but instead results in a clinically recognizable syndrome comprising short stature, joint hypermobility, radioulnar synostosis, and severe hypermetropia. This group of syndromic patients are on a phenotypic spectrum with individuals who have Larsen of Reunion Island syndrome, although the key features of osteopenia, fractures and hypermetropia have not been reported in patients from Reunion Island. © 2016 Wiley Periodicals, Inc.

Galactosylation of glycoconjugates using Pacific oyster ß-1,3-galactosyltransferases.

The Pacific oyster (Magallana gigas) exhibits an extensive diversity of N- and O-linked glycoconjugates, offering significant potential for biotechnological applications. Through genomic data mining, we have identified and characterized a suite of ß-1,3-galactosyltransferase enzymes, pivotal for the synthesis of glycan structures. Out of ten cloned gene candidates, six enzymes were successfully expressed recombinantly in Escherichia coli. Four of these enzymes exhibited measurable catalytic activity in the transfer of galactose to various acceptor substrates. Notably, MgB3GalT1 demonstrated the highest efficiency, achieving a 91.2 % conversion rate. This enzyme was proficient in glycosylating diverse glycan structures, including Core 2 O-glycans and several di-, tri-, and tetra-antennary complex N-glycan standards. Mass spectrometric analysis confirmed the successful modification of N-glycans. These findings open new approaches for utilizing oyster-derived enzymes in glycan-based therapeutics and molecular glycoengineering, highlighting their utility in synthetic applications and biotechnological advancements.

Eight hydroxyproline-O-galactosyltransferases play essential roles in female reproductive development.

In angiosperms, ovules give rise to seeds upon fertilization. Thus, seed formation is dependent on both successful ovule development and tightly controlled communication between female and male gametophytes. During establishment of these interactions, cell walls play a pivotal role, especially arabinogalactan-proteins (AGPs). AGPs are highly glycosylated proteins decorated by arabinogalactan side chains, representing 90 % of the AGP molecule. AGP glycosylation is initiated by a reaction catalysed by hydroxyproline-O-galactosyltransferases (Hyp-GALTs), specifically eight of them (GALT2-9), which add the first galactose to Hyp residues. Five Hyp-GALTs (GALT2, 5, 7, 8 and 9) were previously described as essential for AGP functions in pollen and ovule development, pollen-pistil interactions, and seed morphology. In the present work, a higher order Hyp-GALT mutant (23456789) was studied, with a high degree of under-glycosylated AGPs, to gain deeper insight into the crucial roles of these eight enzymes in female reproductive tissues. Notably, the 23456789 mutant demonstrated a high quantity of unfertilized ovules, displaying abnormal callose accumulation both at the micropylar region and, sometimes, throughout the entire embryo sac. Additionally, this mutant displayed ovules with abnormal embryo sacs, had a disrupted spatiotemporal distribution of AGPs in female reproductive tissues, and showed abnormal seed and embryo development, concomitant with a reduction in AGP-GlcA levels. This study revealed that at least three more enzymes exhibit Hyp-O-GALT activity in Arabidopsis (GALT3, 4 and 6), and reinforces the crucial importance of AGP carbohydrates in carrying out the biological functions of AGPs during plant reproduction.

Knockout of eight hydroxyproline-O-galactosyltransferases cause multiple vegetative and reproductive growth defects.

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of ß-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation.

Hydroxyproline-O-Galactosyltransferases Synthesizing Type II Arabinogalactans Are Essential for Male Gametophytic Development in Arabidopsis.

In flowering plants, male reproductive function is determined by successful development and performance of stamens, pollen grains, and pollen tubes. Despite the crucial role of highly glycosylated arabinogalactan-proteins (AGPs) in male gamete formation, pollen grain, and pollen tube cell walls, the underlying mechanisms defining these functions of AGPs have remained elusive. Eight partially redundant Hyp-galactosyltransferases (named GALT2-GALT9) genes/enzymes are known to initiate Hyp-O-galactosylation for Hyp-arabinogalactan (AG) production in Arabidopsis thaliana. To assess the contributions of these Hyp-AGs to male reproductive function, we used a galt2galt5galt7galt8galt9 quintuple Hyp-GALT mutant for this study. Both anther size and pollen viability were compromised in the quintuple mutants. Defects in male gametogenesis were observed in later stages of maturing microspores after meiosis, accompanied by membrane blebbing and numerous lytic vacuoles. Cytological and ultramicroscopic observations revealed that pollen exine reticulate architecture and intine layer development were affected such that non-viable collapsed mature pollen grains were produced, which were devoid of cell content and nuclei, with virtually no intine. AGP immunolabeling demonstrated alterations in cell wall architecture of the anther, pollen grains, and pollen tube. Specifically, the LM2 monoclonal antibody (which recognized ß-GlcA epitopes on AGPs) showed a weak signal for the endothecium, microspores, and pollen tube apex. Pollen tube tips also displayed excessive callose deposition. Interestingly, expression patterns of pollen-specific AGPs, namely AGP6, AGP11, AGP23, and AGP40, were determined to be higher in the quintuple mutants. Taken together, our data illustrate the importance of type-II AGs in male reproductive function for successful fertilization.

The contributions of individual galactosyltransferases to protein specific N-glycan processing in Chinese Hamster Ovary cells.

Galactosylation as part of N-glycan processing is conducted by a set of beta-1,4-galactosyltransferases (B4GALTs), with B4GALT1 as the dominant isoenzyme for this reaction. Nevertheless, the exact contributions of this key-player as well as of the other isoenzymes involved in N-glycosylation, B4GALT2, B4GALT3 and B4GALT4, have not been studied in-depth. To increase the understanding of the protein- and site-specific activities of individual galactosyltransferases in Chinese Hamster Ovary cells, a panel of triple deletion cell lines was generated that expressed only one isoform of B4GALT each. Two model proteins were selected for this study to cover a large spectrum of possible N-glycan structures: erythropoietin and deamine-oxidase. They were expressed as Fc-fusion constructs (EPO-Fc and Fc-DAO) and their N-glycan processing status was analyzed by site-specific mass spectrometry. The sole activity of B4GALT1 resulted in a decrease of 15-21 % of fully galactosylated structures for erythropoietin, emphasizing the involvement of other isoenzymes. Interestingly, the contributions of B4GALT2 and B4GALT3 differed for the two model proteins. Unexpectedly, removal of galactosyltransferases influenced the overall process of N-glycan maturation, with the result of a higher occurrence of poorly processed oligosaccharides. In the context of high productivity cell lines, which can push N-glycan maturation towards incomplete galactosylation, galactosyltransferases are potential targets to ensure stable product quality. In view of our results, specifically engineered "designer" cell lines may be required for different proteins.

ß4GalT1 Mediates PPARγ N-Glycosylation to Attenuate Microglia Inflammatory Activation.

The inflammatory activation of microglia has double-edged effects in central nervous system (CNS) diseases. The ligand-activated transcriptional factor peroxisome proliferator-activated receptor γ (PPARγ) inhibits the inflammatory response. ß-1,4-Galactosyltransferase Ι (ß1, 4GalT1) mediates N-glycosylation. In this study, the N-glycosylation of PPARγ, as well as two N-linked glycosylation sites in its DNA binding domain (DBD), was identified. Disruption of both sites by site-directed mutagenesis completely abrogated the N-glycosylation of PPARγ. PPAR wild-type (WT) transfection inhibited the inflammatory activation of microglia, while the anti-inflammatory function of unglycosylated PPARγ was down-regulated. In addition, ß1, 4GalT1 was shown to interact with PPARγ and to mediate PPARγ glycosylation. ß1, 4GalT1 promoted PPARγ's anti-transcription and anti-inflammatory functions. Collectively, our findings define that ß-1, 4GalT1 mediated PPARγ glycosylation to attenuate the inflammatory activation of microglia, which has implications for potential therapies for CNS inflammatory diseases.

Identification of novel plasma glycosylation-associated markers of aging.

The pro- or anti-inflammatory activities of immunoglobulins G (IgGs) are controlled by the structure of the glycan N-linked to Asn297 of their heavy chain. The age-associated low grade inflammation (inflammaging) is associated with increased plasmatic levels of agalactosylated IgGs terminating with N-acetylglucosamine (IgG-G0) whose biogenesis has not been fully explained. Although the biosynthesis of glycans is in general mediated by glycosyltransferases associated with internal cell membranes, the extracellular glycosylation of circulating glycoproteins mediated by plasmatic glycosyltransferases has been recently demonstrated. In this study we have investigated the relationship between plasmatic glycosyltransferases, IgG glycosylation and inflammatory and aging markers. In cohorts of individuals ranging from infancy to centenarians we determined the activity of plasmatic ß4 galactosyltransferase(s) (B4GALTs) and of α2,6-sialyltransferase ST6GAL1, the glycosylation of IgG, the GlycoAge test (a glycosylation-based marker of aging) and the plasma level of inflammatory and liver damage markers. Our results show that: 1) plasmatic B4GALTs activity is a new marker of aging, showing a linear increase throughout the whole age range. 2) plasmatic ST6GAL1 was high only in children and in people above 80, showing a quadratic relationship with age. 3) Neither plasmatic glycosyltransferase correlated with markers of liver damage. 4) plasmatic ST6GAL1 showed a positive association with acute phase proteins in offspring of short lived parents, but not in centenarians or in their offspring. 5) Although the glycosylation of IgGs was not correlated with the level of the two plasmatic glycosyltransferases, it showed progressive age-associated changes consistent with a shift toward a pro-inflammatory glycotype.

Raffinose Family Oligosaccharides Act As Galactose Stores in Seeds and Are Required for Rapid Germination of Arabidopsis in the Dark.

Raffinose synthase 5 (AtRS5, At5g40390) was characterized from Arabidopsis as a recombinant enzyme. It has a far higher affinity for the substrates galactinol and sucrose than any other raffinose synthase previously reported. In addition raffinose synthase 5 is also working as a galactosylhydrolase, degrading galactinol, and raffinose under certain conditions. Together with raffinose synthase 4, which is predominantly a stachyose synthase, both enzymes contribute to the raffinose family oligosaccharide (RFO) accumulation in seeds. A double knockout in raffinose synthase 4 and raffinose synthase 5 (ΔAtRS4,5) was generated, which is devoid of RFOs in seeds. Unstressed leaves of 4 week old ΔAtRS4,5 plants showed drastically 23.8-fold increased concentrations of galactinol. Unexpectedly, raffinose appeared again in drought stressed ΔAtRS4,5 plants, but not under other abiotic stress conditions. Drought stress leads to novel transcripts of raffinose synthase 6 suggesting that this isoform is a further stress inducible raffinose synthase in Arabidopsis. ΔAtRS4,5 seeds showed a 5 days delayed germination phenotype in darkness and an elevated expression of the transcription factor phytochrome interacting factor 1 (AtPIF1) target gene AtPIF6, being a repressor of germination. This prolonged dormancy is not seen during germination in the light. Exogenous galactose partially promotes germination of ΔAtRS4,5 seeds in the dark suggesting that RFOs act as a galactose store and repress AtPIF6 transcripts.

The relationship of Bel subgroup and the G952A mutation of the α1,3 galactosyltransferase gene / 中华检验医学杂志

Objective To study the molecular genetic background of Bel subtype at ABO blood group.Methods Three samples and fifteen samples were diagnosed as Bel subgroup and normal control samples by serological test,respectively.The extracted DNA was genotyped by sequence specific primer- polymerase chain reaction foilowed by sequencing for Exon6 and exon7 at ABO locus and clones were sequenced.Results A novel Bel variant allele(GenBank EF117687) was identified in a Bel individual.The Bel allele was different from the regular B101 allele by single 952G>A missense mutation in exon7.resulting in an amino acid subsfitution of Val for Met at 318 locus.No mutations were detected in the fifteen control samples and the other two Bel allele samples.Conclusions The mutation position was fimt found to lie on coding region of ABO gene behind nucleotide 930.The mutation of G952A in the al,3 galactosyhransferase gene may be one of the molecular genetic basis of Bel ohenotype.

Primary identification of the expression of ?Gal epitope in porcine embryonic fibroblast / 中华器官移植杂志

Objective To study the synthesis of ?1,3galactosyltransferase gene in porcine embryonic fibroblast.Methods The transcription and translation of ?1,3galactosyltransferase gene were identified in porcine embryonic fibroblast by RT-PCR and Western blot.Results It was identified that there was the expression of ?1,3 galactosyltransferase gene in the cultured porcine embryonic fibroblasts.Conclusion ?1,3 galactosyltransferase gene can be synthesized in porcine embryonic fibroblast. RT-PCR and Western blot can be applied to identify the expression of ?1,3 galactosyltransferase gene in the porcine embryonic fibroblast.

?-1,4-galactosyl transferase I induces acrosome reaction via G protein signal transduction system / 中国病理生理杂志

After the first recognition occurs between the activated sperm and zona pellucida of the oocyte from the mammalians, ?-1,4-galactosyltransferase I (? 4GalT I) combines the N-GlcNAc terminals by O-ligands on the ZP3 of the zona pellucida, which plays a difunctional role in the fertilization. The G protein signal system on the sperm membrane then is consequently activated by ZP3 via ?4GalT I, contributing to the induction of acrosome reaction. It was proved that in the activation of the G protein system, both the BBXB and BBXXB motifs on the N terminal of the long ? 4GalT I are necessary.

Targeting efficiency of a-1,3-galactosyl transferase gene in pig fetal fibroblast cells

Animal cloning technology with somatic cells provides an alternative tool to conventional methods for producing transgenic animals. Gene targeting in animals is made feasible using somatic cells with homologous recombination procedure that is a major technique in embryonic stem cells for knocking-out genes. Homologous recombination events in somatic cells are relatively inefficient as compared to those in ES cells, suggesting the need for establishment of efficient gene targeting system in somatic cells. To investigate the efficiency of positive and negative selection for gene targeting in pig fetal fibroblast cells, pig alpha-1,3-galactosyl transferase (13-GT) gene was used for gene targeting. The neomycin phosphotransferase (Neo(r)) and herpes simplex virus-thymidine kinase (HSV-tk) genes were used as positive and negative selection markers in this experiment. Following transfection with targeting DNA construct, the pig fetal fibroblast cells were selected against resistance of G418 and gancyclovir. In DMEM medium containing 5 to 10% serum, Pig fetal fibroblast cells failed to proliferate during drug selection. Increasing serum concentration to 15% of medium yielded less senescent colonies of pig fetal fibroblast cells following drug selection that allowed enough cell colonies to screen genomic DNA. The frequency of gene targeting in pig fetal fibroblast cells with double drug selection was more than 10-fold efficient compared to that with G418 single selection. Double selection method with Neo' and HSV-tk genes could be useful for gene targeting in somatic cells for production of cloned animals carrying targeted endogenous genes.