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

Abstract

Blood group A/B glycosyltransferases (AT/BTs) and Forssman glycolipid synthase (FS) are encoded by the evolutionarily related (A/B alleles) and 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.

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

“…Similar to the results of DNA transfection with the human AT constructs without the LeuGlyGly266-268GlyGlyAla substitution described above, the positive and negative controls presented anticipated positive and negative results. In addition, the amino acid substitution constructs at codon 69 exhibited substantially higher FORS1 antigen expression when combined with the LeuGlyGly266-268GlyGlyAla substitution, possibly due to the synergistic effects previously reported of the AT constructs containing the deletion of exon 3 or 4, or Met69Ser/Thr substitution in combination with the LeuGlyGly266-268GlyGlyAla substitution 16,24 . However, the Ter construct, H_ABO-A(Met69Ter & GlyGlyAla), did not present any FS activity even with the LeuGlyGly266-268GlyGlyAla substitution.

Figure 6FORS1 antigen expression after DNA transfection of a variety of human AT amino acid substitution constructs at codon 69 with the LeuGlyGly266-268GlyGlyAla substitution into COS1(B3GALNT1 + A4GALT) cells analyzed by immunocytochemistry.

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“…Because altered RNA splicing is frequent in cancer, this mechanism may explain, at least partially, the FORS1 appearance in some cancer cells/tissues of regular individuals in the Forssman antigen-negative human species 17–23 . Additionally, we also serendipitously observed that the methionine to serine/threonine substitutions at codon 69 (Met69Ser/Thr) conferred weak FS activity 24 . Furthermore, co-introduction of one of those deletions or Met69Ser/Thr substitutions with the LeuGlyGly266-268GlyGlyAla substitution strengthened FS activity, possibly by the synergistic effects of altered intra-Golgi localization and/or protein conformation by the former and modified enzymatic specificity by the latter.…”

“…In our previous studies utilizing COS1(B3GALNT1) cells as DNA transfection recipients, we showed that the Met69Ser/Thr or LeuGlyGly266-268GlyGlyAla substitution of the human AT, or the deletion of exon 3 or 4 of the human AT mRNA resulted in the generation of proteins with the FS activity 16,24 . We have also shown that the Met69Ser/Thr substitution or exon 3 or 4 deletion may have a synergistic effect to enhance the FS activity when combined with the LeuGlyGly266-268GlyGlyAla substitution, possibly due to the changes in protein conformation/localization by the former and the changes in sugar specificity by the latter.…”

“…We performed the 2-round PCR using specific primers to introduce amino acid substitutions as previously described 13,15,24 . We used, as templates, H_ABO-A, human AT cDNA expression construct prepared in the pSG5 eukaryotic expression plasmid vector, and its derivative, H_ABO-A(GlyGlyAla), possessing the LeuGlyGly266-268GlyGlyAla tripeptide substitution.…”

“…COS1(B3GALNT1 + A4GALT) cells were created from COS1(B3GALNT1) cells expressing human B3GALNT1 gene-encoded β1,3- N -acetyl- d -galactosaminyltransferase 1 15,24 . We employed the glycan customization technology by modular expression of glycosyltransferases, utilizing the retroviral pMigR1g-A4GALT construct 31 .…”