Genetic Determinants of Fiber-Associated Traits in Flax Identified by Omics Data Integration.

In this paper, we explore potential genetic factors in control of flax phenotypes associated with fiber by mining a collection of 306 flax accessions from the Federal Research Centre of the Bast Fiber Crops, Torzhok, Russia. In total, 11 traits were assessed in the course of 3 successive years. A genome-wide association study was performed for each phenotype independently using six different single-locus models implemented in the GAPIT3 R package. Moreover, we applied a multivariate linear mixed model implemented in the GEMMA package to account for trait correlations and potential pleiotropic effects of polymorphisms. The analyses revealed a number of genomic variants associated with different fiber traits, implying the complex and polygenic control. All stable variants demonstrate a statistically significant allelic effect across all 3 years of the experiment. We tested the validity of the predicted variants using gene expression data available for the flax fiber studies. The results shed new light on the processes and pathways associated with the complex fiber traits, while the pinpointed candidate genes may be further used for marker-assisted selection.

Human Gb3/CD77 synthase reveals specificity toward two or four different acceptors depending on amino acid at position 211, creating P(k), P1 and NOR blood group antigens.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P(k) synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, P(k) blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with P(k) and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the P(k) and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: P(k) and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized.

Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase.

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1-4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1-4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.

The structures of glycophorin C N-glycans, a putative component of the GPC receptor site for Plasmodium falciparum EBA-140 ligand.

Glycophorins C and D are highly glycosylated integral sialoglycoproteins of human red blood cell membranes carrying the Gerbich blood group antigens. The O- and N-glycosidic chains of the major erythrocyte glycoprotein (Lisowska E. 2001, Antigenic properties of human glycophorins - an update. Adv Exp Med Biol, 491:155-169; Tomita M and Marchesi VT. 1975, Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci USA, 72:2964-2968.) are well characterized but the structure of GPC N-glycans has remained unknown. This problem became important since it was reported that GPC N-glycans play an essential role in the interaction with Plasmodium falciparum EBA-140 merozoite ligand. The elucidation of these structures seems essential for full characterization of the GPC binding site for the EBA-140 ligand. We have employed detailed structural analysis using sequential mass spectrometry to show that many GPC N-glycans contain H2 antigen structures and several contain polylactosamine structures capped with fucose. The results obtained indicate structural heterogeneity of the GPC N-glycans and show the existence of structural elements not found in glycophorin A N-glycans. Our results also open a possibility of new interpretation of the data concerning the binding of P. falciparum EBA-140 ligand to GPC. We hypothesize that preferable terminal fucosylation of N-glycosidic chains containing repeating lactosamine units of the GPC Gerbich variant could be an explanation for why the EBA-140 ligand does not react with GPC Gerbich and an indication that the EBA-140 interaction with GPC is distinctly dependent on the GPC N-glycan structure.

Dynamics of miRNA driven feed-forward loop depends upon miRNA action mechanisms.

BACKGROUND: We perform the theoretical analysis of a gene network sub-system, composed of a feed-forward loop, in which the upstream transcription factor regulates the target gene via two parallel pathways: directly, and via interaction with miRNA. RESULTS: As the molecular mechanisms of miRNA action are not clear so far, we elaborate three mathematical models, in which miRNA either represses translation of its target or promotes target mRNA degradation, or is not re-used, but degrades along with target mRNA. We examine the feed-forward loop dynamics quantitatively at the whole time interval of cell cycle. We rigorously proof the uniqueness of solutions to the models and obtain the exact solutions in one of them analytically. CONCLUSIONS: We have shown that different mechanisms of miRNA action lead to a variety of types of dynamical behavior of feed-forward loops. In particular, we found that the ability of feed-forward loop to dampen fluctuations introduced by transcription factor is the model and parameter dependent feature. We also discuss how our results could help a biologist to infer the mechanism of miRNA action.

The effect of carbohydrate moiety structure on the immunoregulatory activity of lactoferrin in vitro.

The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and that the phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.

A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(ß1-3)Gal(α1-4), and Gal(α1-4)GalNAc(ß1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

Modeling the Flowering Activation Motif during Vernalization in Legumes: A Case Study of M. trancatula.

In many plant species, flowering is promoted by the cold treatment or vernalization. The mechanism of vernalization-induced flowering has been extensively studied in Arabidopsis but remains largely unknown in legumes. The orthologs of the FLC gene, a major regulator of vernalization response in Arabidopsis, are absent or non-functional in the vernalization-sensitive legume species. Nevertheless, the legume integrator genes FT and SOC1 are involved in the transition of the vernalization signal to meristem identity genes, including PIM (AP1 ortholog). However, the regulatory contribution of these genes to PIM activation in legumes remains elusive. Here, we presented the theoretical and data-driven analyses of a feed-forward regulatory motif that includes a vernalization-responsive FT gene and several SOC1 genes, which independently activate PIM and thereby mediate floral transition. Our theoretical model showed that the multiple regulatory branches in this regulatory motif facilitated the elimination of no-sense signals and amplified useful signals from the upstream regulator. We further developed and analyzed four data-driven models of PIM activation in Medicago trancatula in vernalized and non-vernalized conditions in wild-type and fta1-1 mutants. The model with FTa1 providing both direct activation and indirect activation via three intermediate activators, SOC1a, SOC1b, and SOC1c, resulted in the most relevant PIM dynamics. In this model, the difference between regulatory inputs of SOC1 genes was nonessential. As a result, in the M. trancatula model, the cumulative action of SOC1a, SOC1b, and SOC1c was favored. Overall, in this study, we first presented the in silico analysis of vernalization-induced flowering in legumes. The considered vernalization network motif can be supplemented with additional regulatory branches as new experimental data become available.

Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila.

Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. Our recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. Here, we use this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, our results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. Our models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, our modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.

The Genetic Landscape of Fiber Flax.

Genetic diversity in a breeding program is essential to overcome modern-day environmental challenges faced by humanity and produce robust, resilient crop cultivars with improved agronomic characteristics, as well as to trace crop domestication history. Flax (Linum usitatissimum), one of the first crops domesticated by mankind, has been traditionally cultivated for fiber as well as for medicinal purposes and as a nutritional product. The origins of fiber flax are hidden in the mists of time and can be hypothetically traced back to either the Indo-Afghan region or Fertile Crescent. To shed new light on fiber flax genetic diversity and breeding history, in this study, we presented a comprehensive analysis of the core collection of flax (306 accessions) of different morphotypes and geographic origins maintained by the Russian Federal Research Center for Bast Fiber Crops. We observed significant population differentiation between oilseed and fiber morphotypes, as well as mapped genomic regions affected by recent breeding efforts. We also sought to unravel the origins of kryazhs, Russian heritage landraces, and their genetic relatedness to modern fiber flax cultivars. For the first time, our results provide strong genetic evidence in favor of the hypothesis on kryazh's mixed origin from both the Indo-Afghan diversity center and Fertile Crescent. Finally, we showed predominant contribution from Russian landraces and kryazhs into the ancestry of modern fiber flax varieties. Taken together, these findings may have practical implications on the development of new improved flax varieties with desirable traits that give farmers greater choice in crop management and meet the aspirations of breeders.

Lectins as tools in glycoconjugate research.

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate-protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin-carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin-glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, beta-elimination), which extend the applicability of these methods, are also described.

Murine monoclonal anti-s and other anti-glycophorin B antibodies resulting from immunizations with a GPB.s peptide.

BACKGROUND: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti-s reagents are expensive to produce because of the scarcity of human anti-s serum. Our aim was to develop hybridoma cell lines that secrete reagent-grade anti-s monoclonal antibodies (MoAbs) to supplement the supply of human anti-s reagents. STUDY DESIGN AND METHODS: Mice were immunized with the GPB(s) peptide sequence TKSTISSQTNGETGQLVHRF. Hybridomas were produced by fusing mouse splenocytes with mouse myeloma cells (X63.Ag8.653). Screening for antibody production was done on microtiter plates by hemagglutination. Characterization of the MoAbs was done by hemagglutination, immunoblotting, and epitope mapping. RESULTS: Eight immunoglobulin G MoAbs were identified. Five antibodies are specific by hemagglutination for s and two MoAbs, when diluted, are anti-S-like, but additional analyses shows a broad range of reactivity for GPB. Typing red blood cells (RBCs) for s from 35 donors was concordant with molecular analyses as were tests on RBCs with a positive direct antiglobulin test (DAT) from 15 patients. The anti-s MoAbs are most reactive with peptides containing the (31)QLVHRF(36) motif, with (29)Thr. By Pepscan analyses, the anti-S-like MoAbs reacted within the same regions as did anti-s, but independently of (29)Met. One antibody was defined serologically as anti-U; however, its epitope was identified as (21)ISSQT(25), a sequence common for both GPA and GPB. CONCLUSION: In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.

Antibodies against Citrobacter braakii O37 cells recognize the N-glycan of the band 3 glycoprotein of human erythrocyte membrane.

The phenomenon of molecular mimicry was found previously for Citrobacter braakii O37, which shared epitopes with human and horse erythrocytes. The aim of this study was to elucidate the basis of the serological cross-reactivity between anti-C. braakii O37 serum and human erythrocytes. The experiments involved analyzing the epitope on the human erythrocyte membrane, that could be recognized by affinity-purified antibodies. The results indicated a specific glycoprotein fraction in immunoblotting, namely band 3, which interacted with the antibodies purified on lipopolysaccharide from C. braakii O37 LPS (LPS O37) and its core affinity columns. Treating the erythrocytes with trypsin, which cleaves glycophorin A, improved the agglutination because band 3 became more available for antibody binding. Isolated band 3 immobilized on an affinity plate could be used to purify antibodies from the anti-C. braakii O37 serum. These antibodies showed a specific reactivity to LPS O37, but not to the related lipopolysaccharide from Salmonella Toucra O48. Furthermore, the inhibition of agglutination with lactose, the diminished interaction of the specific antibodies purified on LPS O37 with endo-beta-galactosidase-treated band 3, and the reactivity of these antibodies to the 40-kDa fragment of band 3 but not to its trypsin-elaborated 60-kDa fragment, all indicated that the epitope is located on the N-glycan of band 3.

Single nucleotide polymorphisms in A4GALT spur extra products of the human Gb3/CD77 synthase and underlie the P1PK blood group system.

Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.

Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis.

Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.

Synthesis and inhibitory activity of a di- and a trisaccharide corresponding to an erythrocyte glycolipid responsible for the nor polyagglutination.

The polyagglutinable erythrocytes NOR contain unusual neutral glycolipids reactive with anti-NOR antibodies. The disaccharide alpha-D-Galp-(1-->4)-D-GalpNAc and the trisaccharide alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1-->3)-D-Gal corresponding to the non-reducing end of a NOR glycolipid (NOR1) were chemically synthesized. The syntheses were based on a common (1-->4)-beta-D-GalNAc precursor, and utilized benzyl glycoside and benzyl ether functions for persistent blocking of hydroxyls. The alpha-D-Galp-(1-->4)-beta-D-GalpNAc structural element has been found only recently in Nature, and derivatives thereof have not been synthesized before. Both the synthesized oligosaccharides inhibited specifically human anti-NOR antibodies, the trisaccharide being 300 times more active than the disaccharide.

Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.

Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype.

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked alpha1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer. Treatment of NOR2 with alpha-galactosidase gave a glycolipid migrating between NOR1 and NOR2, which did not react with either GSL-IB4 or anti-NOR antibodies but did react with GalNAc-specific soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.

Presence of natural anti-Galalpha1-4GalNAcbeta1-3Gal (anti-NOR) antibodies in animal sera.

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galalpha1-4GalNAcbeta1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and weakly by Galalpha1-4Gal. However, the type 1 antibodies are strongly inhibited by Galalpha1-4Galbeta1-3Gal-R and weakly by Galalpha1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galalpha1-4GalNAcbeta1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several alpha-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-alphaGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.