Reduced α-galactosidase A activity in zebrafish (Danio rerio) mirrors distinct features of Fabry nephropathy phenotype.

Fabry disease (FD) is a rare genetic lysosomal storage disorder, resulting from partial or complete lack of alpha-galactosidase A (α-GAL) enzyme, leading to systemic accumulation of substrate glycosphingolipids with a broad range of tissue damage. Current in vivo models are laborious, expensive, and fail to adequately mirror the complex FD physiopathology. To address these issues, we developed an innovative FD model in zebrafish. Zebrafish GLA gene encoding α-GAL enzyme presents a high (>70%) homology with its human counterpart, and the corresponding protein has a similar tissue distribution, as evaluated by immunohistochemistry. Moreover, a similar enzymatic activity in different life stages could be demonstrated. By using CRISPR/Cas9 technology, we generated a mutant zebrafish with decreased GLA gene expression, and decreased expression of the specific gene product in the kidney. Mutant animals showed higher plasma creatinine levels and proteinuria. Transmission electron microscopy (TEM) studies documented an increased podocyte foot process width (FPW) in mutant, as compared to wild type zebrafish. This zebrafish model reliably mirrors distinct features of human FD and could be advantageously used for the identification of novel biomarkers and for an effective screening of innovative therapeutic approaches.

The alpha1,3GalT knockout/alpha1,2FucT transgenic pig does not appear to have an advantage over the alpha1,3GalT knockout pig with respect to glycolipid reactivity with human serum antibodies.

BACKGROUND: The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective. METHODS: Neutral and acidic glycolipids from GalT-KO/FucT-TG small intestine were compared with glycolipids from two wildtype and two GalT-KO pig intestines. Glycolipid reactivity was tested on thin layer chromatography plates using chemical reagents, antibodies, lectins, and human serum. Structural characterization of neutral glycolipids was performed by LC-ESI/MS and proton NMR spectroscopy. RESULTS: Characterization of the glycolipid expression in GalT-KO/FucT-TG intestine showed absence of Gal antigens and decreased/unchanged levels of the N-acetyllactosamine precursor and the blood group H type 2 expression, when compared with the wildtype. The reactivity of human serum antibodies to GalT-KO/FucT-TG derived glycolipids was similar or slightly elevated when compared with GalT-KO glycolipids. Results from LC-ESI/MS and proton NMR spectroscopy revealed no established neutral xenogenic antigens in the GalT-KO/FucT-TG pig, and could thus not explain the immunologic reactivity to human serum antibodies. The antibody binding to acidic glycolipids is most likely to be explained by the abundance of N-glycolylneuraminic acid epitopes in pig tissues. Six neutral complex biantennary glycolipids with blood group H type 1, 2, Lewis(x) and Lewis(y) determinants were found, of which three were identified in this work for the first time. One of these was a nonaglycosylceramide with blood group H type 2 and lactosyl determinants linked to a lactotetraosyl core, and the other two were decaglycosylceramides with blood group H type 1 and H type 2 determinants linked to a neolactotetraosyl core, and Lewis(x) and blood group H type 1 determinants on a lactotetraosyl core, respectively. CONCLUSIONS: Lipid-linked carbohydrate antigens in the GalT-KO/FucT-TG pig intestine showed no or minor qualitative difference when compared with GalT-KO pigs. The GalT-KO/FucT-TG pig did not appear to have an advantage over the GalT-KO pig with respect to reactivity with human antibodies from a xenotransplantation perspective.

3-fluoro- and 3,3-difluoro-3,4-dideoxy-KRN7000 analogues as new potent immunostimulator agents: total synthesis and biological evaluation in human invariant natural killer T cells and mice.

We propose here the synthesis and biological evaluation of 3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on the sphingoid base is combined with the introduction of mono or difluoro substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect on the stability of the ternary CD1d/GalCer/TCR complex which strongly modulate the immune responses. Biological evaluations were performed in vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing T(H)1 or T(H)2 biases that strongly depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore human iNKT activation.

Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine.

BACKGROUND: αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential. METHODS: The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays. RESULTS: All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6-12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1). CONCLUSIONS: Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine.

Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies.

BACKGROUND: alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids. METHODS: Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. RESULTS: GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues. CONCLUSIONS: GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.

Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli.

F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucalpha2Galbeta3GlcNAc), while an optimal binding epitope was created by addition of the terminal alpha3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galalpha3(Fucalpha2)Galbeta3GlcNAc) and the blood group A type 1 determinant (GalNAcalpha3(Fucalpha2)-Galbeta3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucalpha2Galbeta3GlcNAc-beta3Galbeta4Glcbeta1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), A type 4 heptaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), A type 1 octaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcalpha3-(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants.

Blood group ABO antigen expression in human embryonic stem cells and in differentiated hepatocyte- and cardiomyocyte-like cells.

BACKGROUND: The use of stem cells in regenerative medicine and transplantation may require grafting of cells that will challenge the recipient's immune system. Our knowledge of tissue antigen expression in human embryonic stem cells (hESC) and during their differentiation is limited, especially regarding histo-blood group AB(O)H antigens. METHODS: Nine different hESC lines, and hESC-derived hepatocyte- and cardiomyocyte-like cells, were blood group ABO genotyped and A/B antigen expression was studied by immunohistochemistry. RESULTS: This study reveals, for the first time, that A and B antigens in hESC were expressed according to the ABO genotype and that the antigens had a different cellular/sub-cellular distribution. In addition, several genotype A hESC lines stained positive with one anti-B antibody. Furthermore, studies of hepatocyte- and cardiomyocyte-like cells of different maturation state, originating from a blood group B hESC line, showed that hepatocyte-like cells expressed B antigens whereas cardiomyocyte-like cells were negative. CONCLUSION: Since clinical stem-cell therapy is likely to be performed with immature progenitor cells, blood group ABO compatibility of donor cells/recipients should be favorable to avoid unnecessary rejection problems caused by ABO incompatibility. The in vitro loss of B antigens in a genotype B hESC line indicates that loss of ABH antigens occurs early during human embryogenesis since these antigens are lacking in adult cardiomyocytes.

Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants.

Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.

Studies on glycolipid antigens in small intestine and pancreas from alpha1,3-galactosyltransferase knockout miniature swine.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Galalpha1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. METHODS: Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. RESULTS: Using Galalpha1,3nLc4 glycolipid reference, total Galalpha1,3Gal glycolipid antigens in the WT animal was estimated at about 30 microg (small intestine) and 3 microg (pancreas) per gram of dry tissue. Galalpha1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The alpha1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Galalpha1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. CONCLUSIONS: Knockout of porcine alpha1,3-galactosyltransferase gene results in elimination of Galalpha1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.