The A4GALT locus encodes a glycosyltransferase that synthesizes the terminal Gal␣1-4Gal of the P k (Gb3/CD77) glycosphingolipid, important in transfusion medicine, obstetrics, and pathogen susceptibility. Critical nucleotide changes in A4GALT not only abolish P k formation but also another Gal␣1-4Gal-defined antigen, P1, which belongs to the only blood group system for which the responsible locus remains undefined. Since known A4GALT polymorphisms do not explain the P1؊P k ؉ phenotype, P 2 , we set out to elucidate the genetic basis of P 1 /P 2 . Despite marked differences (P 1 > P 2 ) in A4GALT transcript levels in blood, luciferase experiments showed no difference between P 1 /P 2 -related promoter sequences. Investigation of A4GALT mRNA in cultured human bone marrow cells revealed novel transcripts containing only the noncoding exon 1 and a sequence (here termed exon 2a) from intron 1. These 5-capped transcripts include poly-A tails and 3 polymorphic sites, one of which was P 1 /P 2 -specific among > 200 donors and opens a short reading frame in P 2 alleles. We exploited these data to devise the first genotyping assays to predict P1 status. P 1 /P 2 genotypes correlated with both transcript levels and P1/P k expression on red cells. Thus, P 1 zygosity partially explains the well-known interindividual variation in P1 strength. Future investigations need to focus on regulatory mechanisms underlying P1 synthesis. (Blood. 2011; 117(2):678-687)

SUMMARY Sepsis is a life-threatening inflammatory syndrome accompanying a bloodstream infection. Frequently secondary to pathogenic bacterial infections, sepsis remains difficult to treat as a singular disease mechanism. We compared the pathogenesis of murine sepsis experimentally elicited by five bacterial pathogens and report similarities among host responses to Gram-negative Salmonella and E. coli. We observed that a host protective mechanism involving de-toxification of lipopolysaccharide by circulating alkaline phosphatase (AP) isozymes was incapacitated during sepsis caused by Salmonella or E. coli through activation of host Toll-like receptor 4, which triggered Neu1 and Neu3 neuraminidase induction. Elevated neuraminidase activity accelerated the molecular aging and clearance of AP isozymes, thereby intensifying disease. Mice deficient in the sialyltransferase ST3Gal6 displayed increased disease severity, while deficiency of the endocytic lectin hepatic Ashwell-Morell receptor was protective. AP augmentation or neuraminidase inhibition diminished inflammation and promoted host survival. This study illuminates distinct routes of sepsis pathogenesis, which may inform therapeutic development.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1k and P2k phenotype RBCs, expressing high levels of the Pk Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

P1 and P are glycosphingolipid antigens synthesized by the -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 P (P1P) and P (P1P) phenotypes. transcript levels are higher in P individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of , particularly rs5751348, correlate with P/P status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 () haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates expression. Intriguingly, -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of did not affect transcript levels. Instead, protein pull-down experiments using but not oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds alleles selectively, and knockdown of significantly decreased transcription. These data indicate that RUNX1 regulates and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

Intestinal inflammation is the underlying basis of colitis and the inflammatory bowel diseases. These syndromes originate from genetic and environmental factors that remain to be fully identified. Infections are possible disease triggers, including recurrent human food-poisoning by the common foodborne pathogen Salmonella enterica Typhimurium (ST), which in laboratory mice causes progressive intestinal inflammation leading to an enduring colitis. In this colitis model, disease onset has been linked to Toll-like receptor-4–dependent induction of intestinal neuraminidase activity, leading to the desialylation, reduced half-life, and acquired deficiency of anti-inflammatory intestinal alkaline phosphatase (IAP). Neuraminidase (Neu) inhibition protected against disease onset; however, the source and identity of the Neu enzyme(s) responsible remained unknown. Herein, we report that the mammalian Neu3 neuraminidase is responsible for intestinal IAP desialylation and deficiency. Absence of Neu3 thereby prevented the accumulation of lipopolysaccharide-phosphate and inflammatory cytokine expression in providing protection against the development of severe colitis.

Background: Expression of x 2 glycosphingolipid (PX2) is elevated on erythrocytes from individuals with the rare P/P1/P knegative p phenotype. Results: Globoside-deficient individuals with mutated P synthase (␤1,3GalNAc-T1) lack PX2 and have anti-PX2 in plasma. Transfection of B3GALNT1 induces P and PX2 expression. Conclusion: PX2 synthesized by ␤1,3GalNAc-T1 fulfills blood group criteria. Significance: ␤1,3GalNAc-T1 uses different acceptors to form immunologically distinct glycosphingolipids.

The antigens in the P1PK blood group system are carried on glycosphingolipids. The system currently includes three different antigens, P1, Pk, and NOR. The P1 antigen was disovered in 1927 by Landsteiner and Levine, and Pkand NOR were described in 1951 and 1982, respectively. As in the ABO system, naturally occurring antibodies of the immunoglobulin (Ig) M or IgG class, against the missing carbohydrate structures, can be present in the sera of people lacking the corresponding antigen. Anti-P1 is generally a weak and cold-reactive antibody not implicated in hemolytic transfusion reaction (HTR) or hemolytic disease of the fetus and newborn while Pkantibodies can cause HTR, and anti-NOR is regarded as a polyagglutinin. A higher frequency of miscarriage is seen in women with the rare phenotypes p, P1 k, and P2 k. Furthermore, the Pkand P1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. Why p individuals lack not only Pkand P expression but also P1 has been a longstanding enigma. Recently, it was shown that the same A4GALT-encoded galactosyltransferase synthesizes both the P1 and Pkantigens and that a polymorphism in a new exon in this gene predicts the P1and P2phenotypes. Immunohematology 2013;29:25–33.

For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.