Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.

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

The Sda histo-blood group antigen (GalNAcß1-4(NeuAcα2-3)Galß-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 ß4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.

The P1PK blood group system: revisited and resolved.

CONCLUSIONS: This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25-33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

Missense mutations in the C-terminal portion of the B4GALNT2-encoded glycosyltransferase underlying the Sd(a-) phenotype.

Sda is a high-frequency carbohydrate histo-blood group antigen, GalNAcß1-4(NeuAcα2-3)Galß, implicated in pathogen invasion, cancer, xenotransplantation and transfusion medicine. Complete lack of this glycan epitope results in the Sd(a-) phenotype observed in 4% of individuals who may produce anti-Sda. A candidate gene (B4GALNT2), encoding a Sda-synthesizing ß-1,4-N-acetylgalactosaminyltransferase (ß4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sda deficiency was never elucidated. Experimental and bioinformatic approaches were used to identify and characterize B4GALNT2 variants in nine Sd(a-) individuals. Homozygosity for rs7224888:T > C dominated the cohort (n = 6) and causes p.Cys466Arg, which targets a highly conserved residue located in the enzymatically active domain and is judged deleterious to ß4GalNAc-T2. Its allele frequency was 0.10-0.12 in different cohorts. A Sd(a-) compound heterozygote combined rs7224888:T > C with a splice-site mutation, rs72835417:G > A, predicted to alter splicing and occurred at a frequency of 0.11-0.12. Another compound heterozygote had two rare nonsynonymous variants, rs148441237:A > G (p.Gln436Arg) and rs61743617:C > T (p.Arg523Trp), in trans. One sample displayed no differences compared to Sd(a+). When investigating linkage disequilibrium between B4GALNT2 variants, we noted a 32-kb block spanning intron 9 to the intergenic region downstream of B4GALNT2. This block includes RP11-708H21.4, a long non-coding RNA recently reported to promote tumorigenesis and poor prognosis in colon cancer. The expression patterns of B4GALNT2 and RP11-708H21.4 correlated extremely well in >1000 cancer cell lines. In summary, we identified a connection between variants of the cancer-associated B4GALNT2 gene and Sda, thereby establishing a new blood group system and opening up for the possibility to predict Sd(a+) and Sd(a‒) phenotypes by genotyping.

The P1 histo-blood group antigen is present on human red blood cell glycoproteins.

BACKGROUND: The P1 antigen was first described in 1927 and belongs to the P1PK histo-blood group system, together with Pk and NOR. The A4GALT-encoded 4-α-galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS: RBCs of known P1 /P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α-galactosidases, papain, and/or peptide-N-glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblot. A new P 1 /P 2 -allelic discrimination assay based on rs5751348 was validated. RESULTS: P1 occurs on various glycoproteins, seen as smearlike patterns in anti-P1-stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P 1 -homozygous and P 1 -heterozygous RBCs (P 1 P 1 > P 1 P 2 ), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α-galactosidase of broad Galα-1,3/4/6-specificity. Peptide-N-glycosidase F treatment reduced the P1 signal, while papain or α-1,3-specific galactosidase did not. P 1 /P 2 status was confirmed by a new rs5751348 assay. CONCLUSION: Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N-glycans.

Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT.

P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 (KLF1) haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1 -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively, and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.