Glycoproteomic and Phenotypic Elucidation of B4GALNT2 Expression Variants in the SID Histo-Blood Group System

Abstract

The Sda histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sda, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SIDnull candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sda synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.

The Sda Carbohydrate Antigen and Its Biosynthetic Enzyme B4GALNT2 in Health and Disease.

“…Reasonably, it is possible that differences in the regulatory regions of the B4GALNT2 gene allow a stronger expression in these individuals. However, neither the coding sequence nor the 2000 bp upstream genomic region of the B4GALNT2 gene in 5 Cad + individuals revealed common alterations potentially accounting for their Cad status [23]. In conclusion, some of the Sd a-phenotypes are due to mutations in the coding sequence of B4GALNT2, but the origin of other cases remains obscure [6].…”

“…Transfection of the p.Cys466Arg mutant form failed to turn a Sd a-cell line into a Sd a+ status. Unexpectedly, both p.Gln436Arg and p.Arg523Trp mutant forms induced a Sd a expression level comparable with wild type [23]. Another important question regards the origin of the Cad status.…”

Truncated glycosyltransferase coding regions in novelABOalleles give rise to weak A or B blood group expression and discrepant typing results

“…The constructs used in this study were obtained from GeneCopoeia (Rockville, MD, USA; vector made by Labomics; Nivelles, Belgium) and based on ABO*A1.01 (NG_006669.1) and B.01 consensus sequences with the addition of the investigated variants (Table 1). Plasmid constructs were amplified and purified according to Stenfelt et al 16 Sequences of vector inserts were confirmed by Eurofins Genomics (Ebersberg, Germany), using Sanger sequencing, with primers M61‐F (5'‐GCGGTAGGCGTGTACGGT) and M61‐R (5'‐AGCAGTCCCCAAGTCAGT).…”

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems

“…Expression and glycoproteomic studies with B4GALNT2 constructs with wild‐type sequence or the predominant null candidate variant c.1396C were performed in HEK293 cells [7]. While the consensus ‘wild‐type’ allele induced expression of the antigen, the c.1396C construct did not.…”