Abstract
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.
Highlights
Histo-blood group ABO gene polymorphism is crucial in transfusion medicine
N-terminal deletion constructs starting at each internal methionine in order to explain the capacity of these methionines to act as start codons when their upstream initiation was impaired, due to missense mutations or early termination
We have evaluated the subcellular distribution of Δ95-W96M, Δ112-N113M and Δ122-I123M mutants that, even though they do not represent transcripts initiated by any internal methionine, they would add more information about the dependence of the protein distribution on the N-terminal domain length
Summary
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. There are three major alleles: A (A1) alleles that encode N-acetyl-d-galactosaminyltransferases (histo-blood group A transferases), B alleles that encode d-galactosyltransferases (B transferases), and non-functional O alleles Both the A and B transferases utilize the same acceptor substrate H (Fucα1-2Gal-), and transfer by the same α1 ,3-glycosidic linkage an n-acetyl-d-galactosamine (GalNAc) and a galactose (Gal) to synthesize oligosaccharides A and B antigens, GalNAcα1-3(Fucα1 -2)Gal- and Galα1-3(Fucα1-2)Gal-, respectively. The O proteins possess neither activities, and H substance remains without further modifications In addition to those 3 alleles, there are additional alleles that specify weak expression of A or B antigens (phenotypically called as A2, A3, Aweak, Ael, B3, Bel, etc.). A priori different group of weak alleles exist In this group, structural changes outside of the catalytic domain were identified. Analyzing the effects of a translation-initiator mutation, a separate study by others showed that the mutation causes the Aweak phenotype[6]
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