Laboratory of Immunohematology and Glycobiology, Josep Carreras Leukaemia Research Institute (IJC), Campus Can Ruti, Camí de les Escoles, Badalona, Barcelona, 08916, Spain.
Program of Predictive and Personalized Medicine of Cancer (PMPPC), Institut d'Investigació Germans Trias i Pujol (IGTP), Campus Can Ruti, Camí de les Escoles, Badalona, Barcelona, 08916, Spain.
Sci Rep. 2019 Jan 29;9(1):846. doi: 10.1038/s41598-018-37515-5.
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.
功能平行 ABO、GBGT1、A3GALT2 和 GGTA1 基因分别编码血型 A 和 B 转移酶（AT 和 BT）、福斯曼糖脂合成酶（FS）、同三聚半乳糖基神经酰胺合成酶（iGb3S）和 α1,3-半乳糖基转移酶（GT）。这些糖基转移酶将 N-乙酰-d-半乳糖胺（GalNAc）或 d-半乳糖转移形成 α1,3-糖苷键。然而，它们的受体底物是多样的。之前，我们证明了人类 AT/BT 密码子 266 和 268 处的氨基酸对其独特的糖特异性至关重要，阐明了 ABO 糖基化多态性在输血和移植医学中的重要分子遗传学基础。我们还制备了具有 20 种可能氨基酸的体外突变 AT/BT，表明这些密码子决定了转移酶活性和糖特异性。我们已经将结构分析扩展到包括进化上相关的 α1,3-Gal(NAc)转移酶。制备了具有 AT 特异性 AlaGlyGly 或 LeuGlyGly、BT 特异性 MetGlyAla、FS 特异性 GlyGlyAla 或 iGb3S 和 GT 特异性 HisAlaAla 的选定三肽的 AT、FS、iGb3S 和 GT 的真核表达构建体，这些三肽对应于人类 AT/BT 的 266-268 密码子。使用现有的和新创建的适当受体细胞进行 DNA 转染，并通过免疫检查细胞表面寡糖抗原的出现来检查。结果表明，除了原始三肽之外，还有几种三肽也赋予了转移酶活性。然而，这些转移酶的功能氨基酸谱不同，这表明这些密码子周围的结构差异影响了供体核苷酸-糖和受体底物之间的相互作用。结论是，底物结合口袋中的不同三肽序列有助于产生具有多样化特异性的 α1,3-Gal(NAc)转移酶。
