The stem region of group A transferase is crucial for its specificity, and its alteration promotes heterologous Forssman synthase activity.

Some stem region mutants of human blood group A transferase (hAT) possess Forssman synthase (FS) activity, but very little is known about the mechanisms responsible for this enzymatic crosstalk. We performed confocal microscopy and image analysis to determine whether different intra-Golgi localization was accountable for this acquired activity. We also performed structural modeling and mutational and normal mode analyses. We introduced new mutations in the stem region and tested its FS and AT activities. No differences in subcellular localization were found between hAT and FS-positive mutants. AlphaFold models of hAT and mFS (mouse Forssman synthase) showed that the hAT stem region has a tether-like stem region, while in mFS, it encircles its catalytic domain. In silico analysis of FS-positive mutants indicated that stem region mutations induced structural changes, decreasing interatomic interactions and mobility of hAT that correlated with FS activity. Several additional mutations introduced in that region also bestowed FS activity without altering the AT activity: hAT 37-55 aa substitution by mFS 34-52, 37-55 aa deletion, and missense mutations: S46P, Q278Y, and Q286M. Stem region structure, mobility, and interactions are crucial for hAT specificity. Moreover, stem region mutations can lead to heterologous Forssman activity without changes in the catalytic machinery.

Mixed-Up Sugars: Glycosyltransferase Cross-Reactivity in Cancerous Tissues and Their Therapeutic Targeting.

The main categories of glycan changes in cancer are: (1) decreased expression of histo-blood group A and/or B antigens and increased Lewis-related antigens, (2) appearance of cryptic antigens, such as Tn and T, (3) emergence of genetically incompatible glycans, such as A antigen expressed in tumors of individuals of group B or O and heterophilic expression of Forssman antigen (FORS1), and (4) appearance of neoglycans. This review focuses on the expression of genetically incompatible A/B/FORS1 antigens in cancer. Several possible molecular mechanisms are exemplified, including missense mutations that alter the sugar specificity of A and B glycosyltransferases (AT and BT, respectively), restoration of the correct codon reading frame of O alleles, and modification of acceptor specificity of AT to synthesize the FORS1 antigen by missense mutations and/or altered splicing. Taking advantage of pre-existing natural immunity, the potential uses of these glycans for immunotherapeutic targeting will also be discussed.

ABO blood group A transferase and its codon 69 substitution enzymes synthesize FORS1 antigen of FORS blood group system.

Human histo-blood group A transferase (AT) catalyzes the biosynthesis of oligosaccharide A antigen important in blood transfusion and cell/tissue/organ transplantation. This enzyme may synthesize Forssman antigen (FORS1) of the FORS blood group system when exon 3 or 4 of the AT mRNA is deleted and/or the LeuGlyGly tripeptide at codons 266-268 of AT is replaced by GlyGlyAla. The Met69Ser/Thr substitutions also confer weak Forssman glycolipid synthase (FS) activity. In this study, we prepared the human AT derivative constructs containing any of the 20 amino acids at codon 69 with and without the GlyGlyAla substitution, transfected DNA to newly generated COS1(B3GALNT1 + A4GALT) cells expressing an enhanced level of globoside (Gb4), the FS acceptor substrate, and immunologically examined the FORS1 expression. Our results showed that all those substitution constructs at codon 69 exhibited FS activity. The combination with GlyGlyAla significantly increased the activity. The conserved methionine residue in the ABO, but not GBGT1, gene-encoded proteins may implicate its contribution to the separation of these genes in genetic evolution. Surprisingly, with increased Gb4 availability, the original human AT with the methionine residue at codon 69 was also demonstrated to synthesize FORS1, providing another molecular mechanism of FORS1 appearance in cancer of ordinary FORS1-negative individuals.

Amino acid substitutions at sugar-recognizing codons confer ABO blood group system-related α1,3 Gal(NAc) transferases with differential enzymatic activity.

Functional paralogous ABO, GBGT1, A3GALT2, and GGTA1 genes encode blood group A and B transferases (AT and BT), Forssman glycolipid synthase (FS), isoglobotriaosylceramide synthase (iGb3S), and α1,3-galactosyltransferase (GT), respectively. These glycosyltransferases transfer N-acetyl-d-galactosamine (GalNAc) or d-galactose forming an α1,3-glycosidic linkage. However, their acceptor substrates are diverse. Previously, we demonstrated that the amino acids at codons 266 and 268 of human AT/BT are crucial to their distinct sugar specificities, elucidating the molecular genetic basis of the ABO glycosylation polymorphism of clinical importance in transfusion and transplantation medicine. We also prepared in vitro mutagenized ATs/BTs having any of 20 possible amino acids at those codons, and showed that those codons determine the transferase activity and sugar specificity. We have expanded structural analysis to include evolutionarily related α1,3-Gal(NAc) transferases. Eukaryotic expression constructs were prepared of AT, FS, iGb3S, and GT, possessing selected tripeptides of AT-specific AlaGlyGly or LeuGlyGly, BT-specific MetGlyAla, FS-specific GlyGlyAla, or iGb3S and GT-specific HisAlaAla, at the codons corresponding to 266-268 of human AT/BT. DNA transfection was performed using appropriate recipient cells existing and newly created, and the appearance of cell surface oligosaccharide antigens was immunologically examined. The results have shown that several tripeptides other than the originals also bestowed transferase activity. However, the repertoire of functional amino acids varied among those transferases, suggesting that structures around those codons differentially affected the interactions between donor nucleotide-sugar and acceptor substrates. It was concluded that different tripeptide sequences at the substrate-binding pocket have contributed to the generation of α1,3-Gal(NAc) transferases with diversified specificities.

Blood group ABO gene-encoded A transferase catalyzes the biosynthesis of FORS1 antigen of FORS system upon Met69Thr/Ser substitution.

Blood group A/B glycosyltransferases (AT/BTs) and Forssman glycolipid synthase (FS) are encoded by the evolutionarily related ABO (A/B alleles) and GBGT1 genes, respectively. AT/BT and FS catalyze the biosynthesis of A/B and Forssman (FORS1) oligosaccharide antigens that are responsible for the distinct blood group systems of ABO and FORS. Using genetic engineering, DNA transfection, and immunocytochemistry and immunocytometry, we have previously shown that the eukaryotic expression construct encoding human AT, whose LeuGlyGly tripeptide at codons 266 to 268 was replaced with FS-specific GlyGlyAla tripeptide, induced weak appearance of FORS1 antigen. Recently, we have shown that the human AT complementary DNA constructs deleting exons 3 or 4, but not exons 2 or 5, induced moderate expression of FORS1 antigen. The constructs containing both the GlyGlyAla substitution and the exon 3 or 4 deletion exhibited an increased FS activity. Here, we report another molecular mechanism in which an amino acid substitution at codon 69 from methionine to threonine or serine (Met69Thr/Ser) also modified enzymatic specificity and permitted FORS1 biosynthesis. Considering that codon 69 is the first amino acid of exon 5 and that the cointroduction of Met69Thr and GlyGlyAla substitutions also enhanced FS activity, the methionine substitutions may affect enzyme structure in a mode similar to the exon 3 or 4 deletion but distinct from the GlyGlyAla substitution.

Non-AUG start codons responsible for ABO weak blood group alleles on initiation mutant backgrounds.

Histo-blood group ABO gene polymorphism is crucial in transfusion medicine. We studied the activity and subcellular distribution of ABO gene-encoded A glycosyltransferases with N-terminal truncation. We hypothesized that truncated enzymes starting at internal methionines drove the synthesis of oligosaccharide A antigen in those already described alleles that lack a proper translation initiation codon. Not only we tested the functionality of the mutant transferases by expressing them and assessing their capacity to drive the appearance of A antigen on the cell surface, but we also analyzed their subcellullar localization, which has not been described before. The results highlight the importance of the transmembrane domain because proteins deprived of it are not able to localize properly and deliver substantial amounts of antigen on the cell surface. Truncated proteins with their first amino acid well within the luminal domain are not properly localized and lose their enzymatic activity. Most importantly, we demonstrated that other codons than AUG might be used to start the protein synthesis rather than internal methionines in translation-initiation mutants, explaining the molecular mechanism by which transferases lacking a classical start codon are able to synthesize A/B antigens.

Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases.

A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars.

ABO blood group A transferases catalyze the biosynthesis of FORS blood group FORS1 antigen upon deletion of exon 3 or 4.

Evolutionarily related ABO and GBGT1 genes encode, respectively, A and B glycosyltransferases (AT and BT) and Forssman glycolipid synthase (FS), which catalyze the biosynthesis of A and B, and Forssman (FORS1) oligosaccharide antigens responsible for the ABO and FORS blood group systems. Humans are a Forssman antigen-negative species; however, rare individuals with Apae phenotype express FORS1 on their red blood cells. We previously demonstrated that the replacement of the LeuGlyGly tripeptide sequence at codons 266 to 268 of human AT with GBGT1-encoded FS-specific GlyGlyAla enabled the enzyme to produce FORS1 antigen, although the FS activity was weak. We searched for additional molecular mechanisms that might allow human AT to express FORS1. A variety of derivative expression constructs of human AT were prepared. DNA was transfected into COS1 (B3GALNT1) cells, and cell-surface expression of FORS1 was immunologically monitored. To our surprise, the deletion of exon 3 or 4, but not of exon 2 or 5, of human AT transcripts bestowed moderate FS activity, indicating that the A allele is inherently capable of producing a protein with FS activity. Because RNA splicing is frequently altered in cancer, this mechanism may explain, at least partially, the appearance of FORS1 in human cancer. Furthermore, strong FS activity was attained, in addition to AT and BT activities, by cointroducing 1 of those deletions and the GlyGlyAla substitution, possibly by the synergistic effects of altered intra-Golgi localization/conformation by the former and modified enzyme specificity by the latter.

An integrative evolution theory of histo-blood group ABO and related genes.

The ABO system is one of the most important blood group systems in transfusion/transplantation medicine. However, the evolutionary significance of the ABO gene and its polymorphism remained unknown. We took an integrative approach to gain insights into the significance of the evolutionary process of ABO genes, including those related not only phylogenetically but also functionally. We experimentally created a code table correlating amino acid sequence motifs of the ABO gene-encoded glycosyltransferases with GalNAc (A)/galactose (B) specificity, and assigned A/B specificity to individual ABO genes from various species thus going beyond the simple sequence comparison. Together with genome information and phylogenetic analyses, this assignment revealed early appearance of A and B gene sequences in evolution and potentially non-allelic presence of both gene sequences in some animal species. We argue: Evolution may have suppressed the establishment of two independent, functional A and B genes in most vertebrates and promoted A/B conversion through amino acid substitutions and/or recombination; A/B allelism should have existed in common ancestors of primates; and bacterial ABO genes evolved through horizontal and vertical gene transmission into 2 separate groups encoding glycosyltransferases with distinct sugar specificities.

Murine cell glycolipids customization by modular expression of glycosyltransferases.

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Molecular genetic basis of the human Forssman glycolipid antigen negativity.

Forssman heterophilic glycolipid antigen has structural similarity to the histo-blood group A antigen, and the GBGT1 gene encoding the Forssman glycolipid synthetase (FS) is evolutionarily related to the ABO gene. The antigen is present in various species, but not in others including humans. We have elucidated the molecular genetic basis of the Forssman antigen negativity in humans. In the human GBGT1 gene, we identified two common inactivating missense mutations (c.688G>A [p.Gly230Ser] and c.887A>G [p.Gln296Arg]). The reversion of the two mutations fully restored the glycosyltransferase activity to synthesize the Forssman antigen in vitro. These glycine and glutamine residues are conserved among functional GBGT1 genes in Forssman-positive species. Furthermore, the glycine and serine residues represent those at the corresponding position of the human blood group A and B transferases with GalNAc and galactose specificity, respectively, implicating the crucial role the glycine residue may play in the FS α1,3-GalNAc transferase activity.

ABO research in the modern era of genomics.

Research on ABO has advanced significantly in recent years. A database was established to manage the sequence information of an increasing number of novel alleles. Genome sequencings have identified ABO orthologues and paralogues in various organisms and enhanced the knowledge on the evolution of the ABO and related genes. The most prominent advancements include clarification of the association between ABO and different disease processes. For instance, ABO status affects the infectivity of certain strains of Helicobacter pylori and Noroviruses as well as the sequestration and rosetting of red blood cells infected with Plasmodium falciparum. Genome-wide association studies have conclusively linked the ABO locus to pancreatic cancer, venous thromboembolism, and myocardial infarction in the presence of coronary atherosclerosis. These findings suggest ABO's important role in determining an individual's susceptibility to such diseases. Furthermore, our understanding of the structures of A and B transferases and their enzymology has been dramatically improved. ABO has also become a research subject in neurobiology and the preparation of artificial/universal blood and became a topic in the pseudoscience of "blood type diets." With such new progress, it has become evident that ABO is a critical player in the modern era of genomic medicine. This article provides the most up-to-date information regarding ABO genomics.

Rare and frequent promoter methylation, respectively, of TSHZ2 and 3 genes that are both downregulated in expression in breast and prostate cancers.

BACKGROUND: Neoplastic cells harbor both hypomethylated and hypermethylated regions of DNA. Whereas hypomethylation is found mainly in repeat sequences, regional hypermethylation has been linked to the transcriptional silencing of certain tumor suppressor genes. We attempted to search for candidate genes involved in breast/prostate carcinogenesis, using the criteria that they should be expressed in primary cultures of normal breast/prostate epithelial cells but are frequently downregulated in breast/prostate cancer cell lines and that their promoters are hypermethylated. METHODOLOGY/PRINCIPAL FINDINGS: We identified several dozens of candidates among 194 homeobox and related genes using Systematic Multiplex RT-PCR and among 23,000 known genes and 23,000 other expressed sequences in the human genome by DNA microarray hybridization. An additional examination, by real-time qRT-PCR of clinical specimens of breast cancer, further narrowed the list of the candidates. Among them, the most frequently downregulated genes in tumors were NP_775756 and ZNF537, from the homeobox gene search and the genome-wide search, respectively. To our surprise, we later discovered that these genes belong to the same gene family, the 3-member Teashirt family, bearing the new names of TSHZ2 and TSHZ3. We subsequently determined the methylation status of their gene promoters. The TSHZ3 gene promoter was found to be methylated in all the breast/prostate cancer cell lines and some of the breast cancer clinical specimens analyzed. The TSHZ2 gene promoter, on the other hand, was unmethylated except for the MDA-MB-231 breast cancer cell line. The TSHZ1 gene was always expressed, and its promoter was unmethylated in all cases. CONCLUSIONS/SIGNIFICANCE: TSHZ2 and TSHZ3 genes turned out to be the most interesting candidates for novel tumor suppressor genes. Expression of both genes is downregulated. However, differential promoter methylation suggests the existence of distinctive mechanisms of transcriptional inactivation for these genes.

Determinants of the nucleocytoplasmic shuttling of muscle glycogen synthase.

Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3-L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose-induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555-633 of human MGS, which encompasses an Arg-rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS co-localizes with the promyelocytic leukaemia oncoprotein and p80-coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes.

Crystallization and preliminary X-ray analysis of the glycogen synthase from Pyrococcus abyssi.

Glycogen synthase catalyzes the transfer of glucosyl residues from ADP- or UDP-glucose to the non-reducing end of a growing alpha-1,4-glucan chain. To date, no crystallographic structure of an animal/fungal glycogen synthase (family 3 of the glycosyl transferases) or a bacterial/plant glycogen/starch synthase (family 5) has been reported. This paper describes the recombinant expression, crystallization and preliminary X-ray analysis of the glycogen synthase from the hyperthermophilic archaeon Pyrococcus abyssi, the smallest enzyme of the members of families 3 and 5 of the glycosyl transferases. Crystals from this protein and from its selenomethionyl variant were grown in 100 mM sodium citrate pH 5.6 containing 20% PEG and 20% dioxane by the hanging-drop vapour-diffusion method at 293 K. The crystals, which grew as thin needles, diffracted to 3.5 A resolution and belong to space group C2, with unit-cell parameters a = 202, b = 73, c = 149 A, beta = 131 degrees. The crystallographic and biochemical data are consistent with either a dimer or a tetramer in the crystal asymmetric unit and a volume solvent content of 70 or 39%, respectively.

Control of glycogen deposition.

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.

Glycogen synthase: towards a minimum catalytic unit?

Classically, alpha-1,4-glucan synthases have been divided into two families, animal/fungal glycogen synthases (GS) and bacterial/plant starch synthases (G(S)S), according to differences in sequence, sugar donor specificity and regulatory mechanisms. Detailed sequence analysis, predicted secondary structure comparison and threading analysis show that these two families are structurally related and that some domains of GSs were acquired to meet regulatory requirements. Archaeal G(S)S present structural and functional features that are conserved in one, the other or both families. Therefore, they are the link between GS and G(S)S and harbor the minimal sequence and structural features that constitute the minimum catalytic unit of the alpha-1,4-glucan synthase superfamily.

Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase.

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.