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

Abstract

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, Pk 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 Pk, 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 Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.

Tick galactosyltransferases are involved in α-Gal synthesis and play a role during Anaplasma phagocytophilum infection and Ixodes scapularis tick vector development

“…S2 ). Interestingly, α1-4 GALT, also known as Gb3 synthase, is the only known GT enzyme that changes acceptor specificity because of point mutations, and therefore is considered to be a highly promiscuous enzyme 31 , 32 . In addition, a β1-4 GALT gene from Neisseria meningitidis , pglA , catalyses both β1-4 and α1-3 linkages between the galactose and deoxyhexose in a trisaccharide structure 14 .…”

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system

“…We also attempted to evaluate the effect of NOR mutation (c.631C>G) on activity of the Gb3/CD77 synthase in vivo and how it relates to our earlier studies ex vivo and in vitro [ 5 – 7 ]. In our study on a transfected cell line[ 7 ], activity of the consensus Gb3/CD77 synthase was higher than activity of the p.Q211E mutein, in contrast to our results with the purified recombinant variants of the enzyme. The results of this study suggest that the p.Q211E enzyme’s superpromiscuity comes at the cost of P1 synthesis.…”

“…In addition, c.631C>G mutation in A4GALT , leading to p.Q211E substitution in Gb3/CD77 synthase (rs397514502), alters the enzyme specificity, rendering it able to synthesise all the three P1PK system antigens: P k , P1 and NOR[ 6 ]. This makes Gb3/CD77 synthase a unique case of a promiscuous glycosyltransferase, radically unmoored from the old ‘one enzyme-one linkage’ tenet[ 7 ].…”

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions

“…P k and P1 are both expressed on the surface of human red blood cells. Recently, it has been shown that the p.Q211E variant of A4GALT is also able to synthesize NOR antigens, which are rare glycosphingolipids with a terminal Gal(α1–4)GalNAc moiety present in erythrocytes of patients with NOR polyagglutination syndrome [ 135 , 136 ] ( Figure 6 A). Importantly, the existence of some individuals with a genetic deficiency in A4GALT without obvious clinical consequences suggests that selective inhibition of this enzyme could be well tolerated by FD patients [ 137 , 138 ].…”