Default biosynthesis pathway for blood group-related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group O Le(a-b-) nonsecretor

Glycoconjugates of the GI tract are important for microbial interactions. The expression of histo-blood group glycosyltransferases governs both the expression of blood group determinants and in part the structure and size of the glycoconjugates. Using neutral glycolipids isolated from the small intestine of a rare blood group O Le(a-b-) ABH secretor-negative (nonsecretor) individual we were able to map the "default" pathway of the individual lacking ABO, Lewis, and secretor glycosyltransferases. Structures were deduced with combined analysis of mass spectrometry (MALDI-TOF and ESI-MS/MS), and 1H NMR (500 and 600 MHz). All structures present at a level >5% were structurally resolved and included two extended structures: Galbeta4(Fucalpha3)GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer and Galbeta3GlcNAcbeta3(Galbeta4[Fucalpha3]GlcNAcbeta6)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer. The first, a novel component, is based on a type 2 chain and bears the Lex glycotopes on both its branches. The second, a major component, is based on a type 1 chain, which bears a 3-linked type 1 precursor (Lec) glycotope and a 6-linked Lex glycotope on its branches. This latter structure is identical to that previously isolated from plasma and characterized by MS and GC-MS but not by NMR. Structural resolution of these structures was supported by reanalysis of the blood group H-active decaosylceramides previously isolated from rat small intestine. Other minor linear monofucosylated penta-, hepta-, and difucosylated octaosylceramides, some bearing blood group determinants, were also identified. The cumulative data were used to define a default biosynthesis pathway where it can be seen that carbohydrate chain extension, in the absence of blood group glycosyltransferases, is controlled and regulated by non-blood group fucosylation and branching with type 2 Galbeta4GlcNAc branches.     

Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis of type III and IV structures and NMR characterization of type I-VI antigens

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to VI antigens, which were carried out to probe differences in overall conformation of the molecules. These NMR investigations showed very little difference in the ¹H chemical shifts, as well as ¹H–¹H coupling constants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in solution.     

Long-term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals--a birth-and-death evolution model

Functional glycosyltransferase 6 (GT6) family members catalyze the transfer of galactose or N-acetylgalactosamine in alpha1,3 linkage to various substrates and synthesize structures related to the A and B histo-blood group antigens, the Forssman antigen, alphaGal epitope, and iGb3 glycolipid. In rat, mouse, dog, and cow genomes, we have identified three new mammalian genes (GT6m5, GT6m6, and GT6m7) encoding putative proteins belonging to the GT6 family. Among these, GT6m6 protein does not display major alterations of the GT6 motifs involved in binding of the divalent cation and the substrate. Based on protein sequence comparison, gene structure, and synteny, GT6 homologous sequences were also identified in bird, fish, and amphibian genomes. Strikingly, the number and type of GT6 genes varied widely from species to species, even within phylogenetically related groups. In human, except ABO functional alleles, all other GT6 genes are either absent or nonfunctional. Human, mouse, and cow have only one ABO gene, whereas rat and dog have several. In the chicken, the Forssman synthase-like is the single GT6 family member. Five Forssman synthase-like genes were found in zebrafish, but are absent from three other fishes (fugu, puffer fish, and medaka). Two iGb3 synthase-like genes were found in medaka, which are absent from zebrafish. Fugu, puffer fish, and medaka have an additional GT6 gene that we termed GT6m8, which is absent from all other species analyzed here. These observations indicate that individual GT6 genes have expanded and contracted by recurrent duplications and deletions during vertebrate evolution, following a birth-and-death evolution type.     

Cloning of a rat gene encoding the histo-blood group A enzyme. Tissue expression of the gene and of the A and B antigens.

The complete coding sequence of a BDIX rat gene homologous to the human ABO gene was determined. Identification of the exon-intron boundaries, obtained by comparison of the coding sequence with rat genomic sequences from data banks, revealed that the rat gene structure is identical to that of the human ABO gene. It localizes to rat chromosome 3 (q11-q12), a region homologous to human 9q34. Phylogenetic analysis of a set of sequences available for the various members of the same gene family confirmed that the rat sequence belongs to the ABO gene cluster. The cDNA was transfected in CHO cells already stably transfected with an alpha1,2fucosyltransferase in order to express H oligosaccharide acceptors. Analysis of the transfectants by flow cytometry indicated that A but not B epitopes were synthesized. Direct assay of the enzyme activity using 2' fucosyllactose as acceptor confirmed the strong UDP-GalNAc:Fucalpha1,2GalalphaGalNAc transferase (Atransferase) activity of the enzyme product and allowed detection of a small UDP-Gal:Fucalpha1,2GalalphaGal transferase (B transferase) activity. The presence of the mRNA and of the A and B antigens was searched in various BDIX rat tissues. There was a general good concordance between the presence of the mRNA and that of the A antigen. Tissue distributions of the A and B antigens in the homozygous BDIX rat strain were largely different, indicating that these antigens cannot be synthesized by alleles of the same gene in this rat inbred strain.     

Association between expression of the H histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus

RHDV (rabbit hemorrhagic disease virus) is a highly virulentcalicivirus that has become a major cause of mortality in wildrabbit populations (Oryctolagus cuniculus). It binds to thehisto-blood group antigen (HBGA) H type 2 which requires an1,2fucosyltransferase for its synthesis. In rabbit, three 1,2fucosyltransferasesgenes are known, Fut1, Fut2, and Sec1. Nonfunctional allelesat any of these loci could potentially confer resistance toRHDV, similar to human FUT2 alleles that determine the nonsecretorphenotype and resistance to infection by various NoV strains.In this study, we looked for the presence of H type 2 on buccalepithelial cells of wild rabbits from two geographic areas underRHDV pressure and from one RHDV-free area. Some animals withdiminished H type 2 expression were found in the three populations(nonsecretor-like phenotype). Their frequency markedly increasedaccording to the RHDV impact, suggesting that outbreaks selectedsurvivors with low expression of the virus ligand. Polymorphismsof the Fut1, Fut2, and Sec1 coding regions were determined amonganimals that either died or survived outbreaks. The Fut2 andSec1 genes presented a high polymorphism and the frequency ofone Sec1 allele was significantly elevated, over 6-fold, amongsurvivors. Sec1 enzyme variants showed either moderate, low,or undetectable catalytic activity, whereas all variant Fut2enzymes showed strong catalytic activity. This functional analysisof the enzymes encoded by each Fut2 and Sec1 allele suggeststhat the association between one Sec1 allele and survival mightbe explained by a deficit of 1,2fucosyltransferase expressionrather than by impaired catalytic activity.     

产甘油假丝酵母甘油合成关键酶编码基因的克隆

产甘油假丝酵母(Candida glycerinogenes)作为优良的甘油生产菌株已经成功应用于工业化生产。但相对于酿酒酵母, 该菌株的耐高渗机理和甘油代谢的分子机制还不甚清楚。本文根据已公布的3-磷酸甘油脱氢酶基因的序列信息, 设计出一组寡核苷酸, 再运用简并PCR结合反向PCR技术从C. glycerinogenes的基因组DNA中获得了4 900 bp的核苷酸序列, 递交GenBank (No. EU186536)。该序列包含完整的编码胞浆3-磷酸甘油脱氢酶编码基因(CgGPD)开放阅读框及其上、下游调控序列。1 167 bp的开放阅读框编码388个氨基酸残基的蛋白。所演绎出氨基酸序列分析比对结果表明该基因产物的序列具有典型的胞浆3-磷酸甘油脱氢酶结构特征, 但与已鉴定的相关基因存在中等程度的同源性并在相应的辅酶催化位点和底物结合位点区域具有高度的保守性, 在氨基酸水平上与安格斯毕赤酵母的相似性最高, 达到70.9%。该基因在Saccharomyces cerevisiae W303A中异源表达能够显著提高细胞的甘油合成能力。     

Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a glycosyltransferase with both A and B transferase activity

We have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and this cis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future.     

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.     

Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and human colon cancer cell lines

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAcα1–3[Fucα1–2]Galβ1–4GlcNAc-R) and B (Galα1–3[Fucα1–2]Galβ1–4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4–21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A-binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer.     

Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme.

Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

Cloning of the gene encoding quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus: evidence for the presence of a second enzyme.

We cloned the gene coding for the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. This clone complements gdh mutations in A. calcoaceticus, Pseudomonas aeruginosa, and Escherichia coli. The gene codes for a protein with an Mr of 83,000. Evidence is presented for the presence of two different glucose dehydrogenase enzymes in A. calcoaceticus: a protein with an Mr of 83,000 and a dimer of two identical subunits with an Mr of 50,000.     

Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain

Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion proteins from the lysates of lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain. The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.     

Inventory of the cichlid olfactory receptor gene repertoires: identification of olfactory genes with more than one coding exon

Background

To help understand the molecular mechanisms underlying the remarkable phenotypic diversity displayed by cichlids, the genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were recently determined. Here, we present the contents of the olfactory receptor (OR) repertoires in the genomes of these five fishes.

Results

We performed an exhaustive TBLASTN search of the five cichlid genomes to identify their OR repertoires as completely as possible. We used as bait a set of ORs described in the literature. The cichlid repertoires thereby extracted contained large numbers of complete genes (O. niloticus 158; H. burtoni 90; M. zebra 102; N. brichardi 69; P. nyererei 88), a small numbers of pseudogenes and many “edge genes” corresponding to incomplete genes located at the ends of contigs. A phylogenetic tree was constructed and showed these repertoires include a large number of families and subfamilies. It also allowed the identification of a large number of OR analogues between cichlids with very high amino-acid identity (≥99%). Nearly 9% of the full-length cichlid OR genes are composed of several coding exons. This is very unusual for vertebrate OR genes. Nevertheless, the evidence is strong, and includes the donor and acceptor splice junction sequences; also, the positions of these genes in the phylogenetic tree indicate that they constitute subfamilies well apart from non-OR G protein-coupled receptor families.

Conclusions

Cichlid OR repertoires are made up of a larger number of genes and fewer pseudogenes than those in other teleosts except zebrafish. These ORs share all identified properties common to all fish ORs; however, the large number of families and subfamilies, each containing few ORs implies that they have evolved more rapidly. This high level of OR diversity is consistent with the substantial phenotypic diversity that characterizes cichlids.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-586) contains supplementary material, which is available to authorized users.     

Cloning,expression, and protective immunity in mice of a gene encoding the diagnostic antigen P-29 of Echinococcus granulosus

Taeniid tapeworm Echinococcus granulosus is the causative agent of Echinococcosis, an important zoonosis with worldwide distribution. In this study, a diagnostic antigen P-29 was cloned from E. granulosus and expressed in Escherichia coil Sequence analysis showed that EgP-29 contains 717-bp open reading frame and encodes a protein of 238 amino acid residues with a predicted molecular weight of 27.1 kDa. The recombinant EgP-29 （rEgP-29） could be recognized with antimice sera in Western blotting. The specific antibody was detected by enzyme-linked immunosorbent assay. Mice vaccinated with rEgP-29 and challenged intrapero itoneally with E. granulosus protoscoleces revealed sig- nificant protective immunity of 96.6% （P〈0.05）, compared with the control group. Thus, rEgP-29 protein is a promising candidate for an effective vaccine to prevent secondary echinococcosis.     

Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris

N-glycans are synthesized in both yeast and mammals through the ordered assembly of a lipid-linked core Glc(3)Man(9)GlcNAc(2) structure that is subsequently transferred to a nascent protein in the endoplasmic reticulum. Once folded, glycoproteins are then shuttled to the Golgi, where additional but divergent processing occurs in mammals and fungi. We cloned the Pichia pastoris homolog of the ALG3 gene, which encodes the enzyme that converts Man(5)GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP. Deletion of this gene in an och1 mutant background resulted in the secretion of glycoproteins with a predicted Man(5)GlcNAc(2) structure that could be trimmed to Man(3)GlcNAc(2) by in vitro alpha-1,2-mannosidase treatment. However, several larger glycans ranging from Hex(6)GlcNAc(2) to Hex(12)GlcNAc(2) were also observed that were recalcitrant to an array of mannosidase digests. These results contrast the far simpler glycan profile found in Saccharomyces cerevisiae alg3-1 och1, indicating diverging Golgi processing in these two closely related yeasts. Finally, analysis of the P. pastoris alg3 deletion mutant in the presence and absence of the outer chain initiating Och1p alpha-1,6-mannosyltransferase activity suggests that the PpOch1p has a broader substrate specificity compared to its S. cerevisiae counterpart.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.