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

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

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

Requirement of RIZ1 for cancer prevention by methyl-balanced diet

The typical Western diet is not balanced in methyl nutrients that regulate the level of the methyl donor S-adenosylmethionine (SAM) and its derivative metabolite S-adenosylhomocysteine (SAH), which in turn may control the activity of certain methyltransferases. Feeding rodents with amino acid defined and methyl-imbalanced diet decreases hepatic SAM and causes liver cancers. RIZ1 (PRDM2 or KMT8) is a tumor suppressor and functions in transcriptional repression by methylating histone H3 lysine 9. Here we show that a methyl-balanced diet conferred additional survival benefits compared to a tumor-inducing methyl-imbalanced diet only in mice with wild type RIZ1 but not in mice deficient in RIZ1. While absence of RIZ1 was tumorigenic in mice fed the balanced diet, its presence did not prevent tumor formation in mice fed the imbalanced diet. Unlike most of its related enzymes, RIZ1 was upregulated by methyl-balanced diet. Methyl-balanced diet did not fully repress oncogenes such as c-Jun in the absence of RIZ1. The data identify RIZ1 as a critical target of methyl-balanced diet in cancer prevention. The molecular understanding of dietary carcinogenesis may help people make informed choices on diet, which may greatly reduce the incidence of cancer

Rare and Frequent Promoter Methylation, Respectively, of TSHZ2 and 3 Genes That Are Both Downregulated in Expression in Breast and Prostate Cancers

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.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.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

Emerging glyco-based strategies to steer immune responses

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell-cell communications and in the regulation of immune responses. Through the interaction with glycan-binding receptors, glycans are able to affect the activation status of antigen-presenting cells, leading either to induction of pro-inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco-chemists and glyco-immunologists to develop glycan-based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan-modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen-presenting cells, like dendritic cells. In addition, we address how glycan-based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan-based therapies, including chimeric antigen receptor (CAR)-T cells to achieve targeting of tumor-associated glycan-specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity

Recent advances on smart glycoconjugate vaccines in infections and cancer

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as “tumor-associated carbohydrate antigens”. Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy