评估对人Gb3 / CD77合酶的特异性和活性至关重要的氨基酸残基,Glycoconjugate Journal

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评估对人Gb3 / CD77合酶的特异性和活性至关重要的氨基酸残基
Glycoconjugate Journal ( IF 2.7 ) Pub Date : 2016-08-18 , DOI: 10.1007/s10719-016-9716-9
Radoslaw Kaczmarek , Katarzyna Mikolajewicz , Katarzyna Szymczak , Maria Duk , Edyta Majorczyk , Anna Krop-Watorek , Anna Buczkowska , Marcin Czerwinski

人Gb3 / CD77合酶（α1,4-半乳糖基转移酶）是唯一已知的由于点突变而改变受体特异性的糖基转移酶。该酶由A4GALT基因座编码，负责Gb3（CD77，P ^k抗原）和P1糖鞘脂中Gal（α1-4）Gal部分的生物合成。我们发现之前，单个核苷酸取代c.631C>的G的开放阅读框A4GALT，导致在位置211（取代第Q211E）替换为谷氨酸谷氨酰胺，拓宽了酶受体的特异性，所以它不仅能将半乳糖附着到另一个半乳糖上，也附着在N上-乙酰半乳糖胺。后者的反应导致了NOR抗原的合成，这是具有以前未在哺乳动物中描述的末端Gal（α1-4）GalNAc序列的糖鞘脂。由于第211位对酶活性的重要性，我们用编码Gb3 / CD77合酶的载体用天冬氨酸或天冬酰胺取代的谷氨酰胺稳定转染了2102Ep细胞，并通过定量流式细胞仪，高效薄层色谱法评估了这些细胞和实时PCR。我们发现转染了编码带有替换p的Gb3 / CD77合酶的载体的细胞。Q211D或p。Q211N不表达P ^k，P1和NOR抗原，提示酶活性完全丧失。因此，Gb3 / CD77合酶第211位的氨基酸残基对于参与P ^k，P1和NOR抗原形成的酶的特异性和活性至关重要。总之，这种方法为人类Gb3 / CD77合酶的作用机理提供了新的见解。

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Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1–4)Gal moiety in Gb3 (CD77, P^k antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1–4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express P^k, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of P^k, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.

更新日期：2016-08-18

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