Targeted Disruption of Gb3/CD77 Synthase Gene Resulted in the Complete Deletion of Globo-series Glycosphingolipids and Loss of Sensitivity to Verotoxins

Abstract

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 μg of VT-2 or 1.0 μg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs. To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 μg of VT-2 or 1.0 μg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs. Globotriaosylceramide (Gb3/CD77) 2The abbreviations used are: Gb3/CD77, globotriaosylceramide; α1,4Gal-T, α1,4-galactosyltransferase, Gb3/CD77 synthase; VT, verotoxin; HUS, hemolytic uremic syndrome; LPS, lipopolysaccharide; mAb, monoclonal antibody; TNF, tumor necrosis factor, IL, interleukin. is synthesized from lactosylceramide by α1,4-galactosyltransferase (Gb3/CD77 synthase, α1,4Gal-T). This glycolipid has been characterized as Pk blood group antigen (1Marcus D.M. Kundu S.K. Suzuki A. Semin. Hematol. 1981; 18: 63-71PubMed Google Scholar) and has also been referred to as Burkitt lymphoma-associated antigen (2Wiels J. Fellous M. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6485-6488Crossref PubMed Scopus (150) Google Scholar). In addition, its role in the apoptosis of immature B cells has been reported previously (3Gregory C.D. Dive C. Henderson S. Smith C.A. Williams G.T. Gordon J. Rickinson A.B. Nature. 1991; 349: 612-614Crossref PubMed Scopus (462) Google Scholar). A unique aspect of Gb3/CD77 is its role as a receptor for bacterial toxins produced by Escherichia coli O157 strain (4Jacewicz M. Clausen H. Nudelman E. Donohue-Rolfe A. Keusch G.T. J. Exp. Med. 1986; 163: 1391-1404Crossref PubMed Scopus (312) Google Scholar, 5Lingwood C.A. Law H. Richardson S. Petric M. Brunton J.L. De Grandis S. Karmali M. J. Biol. Chem. 1987; 262: 8834-8839Abstract Full Text PDF PubMed Google Scholar), also referred to as the Shiga-like toxin(s) or verotoxin(s) (VTs) (6Konowalchuk J. Speirs J.I. Stavric S. Infect. Immun. 1977; 18: 775-779Crossref PubMed Google Scholar). VT-producing E. coli are considered to be causative agents of hemorrhagic colitis (7Riley L.W. Remis R.S. Helgerson S.D. McGee H.B. Wells J.G. Davis B.R. Hebert R.J. Olcott E.S. Johnson L.M. Hargrett N.T. Blake P.A. Cohen M.L. N. Engl. J. Med. 1983; 308: 681-685Crossref PubMed Scopus (2223) Google Scholar), and infections have often been associated with hemolytic uremic syndrome (HUS), which has been associated with acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia (8Karmali M.A. Petric M. Lim C. Fleming P.C. Arbus G.S. Lior H. J. Infect. Dis. 1985; 151: 775-782Crossref PubMed Scopus (1106) Google Scholar). HUS is also frequently associated with disruption of the central nervous system (9Tzipori S. Chow C.W. Powell H.R. J. Clin. Pathol. 1988; 41: 1099-1103Crossref PubMed Scopus (70) Google Scholar) and can be lethal. Gb3/CD77 was reported to bind specifically to the β-subunit of VTs. Consequently, tissue damage by VTs is considered to depend on Gb3 expression in individual tissues (10Boyd B. Lingwood C. Nephron. 1989; 51: 207-210Crossref PubMed Scopus (163) Google Scholar). However, the following issues remain to be clarified, specifically, whether Gb3 is the only specific receptor for VTs in vivo and whether clinical signs of HUS are dependent upon Gb3 expression. In the present study, we established knock-out mice for the Gb3/CD77 synthase gene (11Kojima Y. Fukumoto S. Furukawa K. Okajima T. Wiels J. Yokoyama K. Suzuki Y. Urano T. Ohta M. Furukawa K. J. Biol. Chem. 2000; 275: 15152-15156Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and analyzed their sensitivity to VTs as well as Gb3/CD77 expression in mouse tissues. We clearly demonstrated that Gb3/CD77 and/or its derivatives are exclusive receptors in vivo and that they mediate the tissue damage and pathological features caused by VTs. These results suggest that therapeutic approaches directed at disrupting the function of Gb3/CD77 could be an effective method for protecting against disorders related to pathogenic E. coli infection. Generation of Knock-out Mice—A targeting vector was constructed using a 10.5-kb mouse genomic fragment including the α1,4Gal-T gene. Part of the α1,4Gal-T gene (nucleotides 68-615 in the open reading frame) was replaced with a neo-resistant gene (Fig. 1A). The targeting vector was linearized and transfected into embryonic stem cells, and G418-resistant clones were isolated. Homologous recombination was confirmed by dual Southern blotting. Chimeric mice were generated by microinjection of the embryonic stem clones into embryos at the 8-cell stage and were mated with C57BL/6 mice to generate heterozygotes. Genotyping was performed by PCR using genomic DNA isolated from mouse tails and amplified with primers: P1, 5′-ACG ACC TCT GGA CTT GCA AGA ACT GTT TGA-3′; P2, 5′-AAG GCA CCG TTG AGG ACA TAG CGG GAT-3′; and P3, 5′-GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT-3′ (Fig. 1C). The Nagoya University Committee on Animal Research approved all experimental procedures. Southern Blotting—Southern blotting was performed using a current method. Briefly, genomic DNA was prepared from embryonic stem cells or mouse tails with a lysis buffer (10 mm Tris-HCl, pH 7.2, 150 mm NaCl, 10 mm EDTA, 0.1% SDS) or Tail DNA extraction solution (0.2 mg/ml of proteinase K, 50 mm Tris-HCl, pH 8.0, 100 mm EDTA, 0.5% SDS), respectively. Isolated DNA was digested with restriction enzymes, electrophoresed on agarose gels, and hybridized with 32P-labeled probes. First, EcoRV digests were blotted with 32P-labeled probe-1 prepared by PCR using primers 5′-CGG CCT GAT GTC CTT AAT TCA CCA ACA-3′ and 5′-GAT ATC TGC CTT GGA CTC TAG TGT CAC-3′. Second, BglII digests were probed with 32P-labeled probe-2 prepared by PCR using primers 5′-AGG GTA CAC ACC TAG AGG CCA CA-3′ and 5′-TCC TGA CCC CAC CTC TAA CCA G-3′ as depicted in Fig. 1A. Northern Blotting—Total RNA was prepared from mouse tissues using TRIzol™ (Invitrogen). Total isolated RNAs were then separated using 1.25% agarose gels and hybridized with 32P-labeled probes. The cDNA fragment corresponding to nucleotides 90-637 in the open reading frame of the mouse α1,4Gal-T gene (GenBank accession number AA682117) was used as a probe. Enzyme Assay—The enzyme activity of α1,4Gal-T was measured as described previously (11Kojima Y. Fukumoto S. Furukawa K. Okajima T. Wiels J. Yokoyama K. Suzuki Y. Urano T. Ohta M. Furukawa K. J. Biol. Chem. 2000; 275: 15152-15156Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and membrane fractions were prepared from kidneys. Glycolipid Extraction, TLC, and TLC Immunostaining—Glycolipid extraction and TLC immunostaining were performed as described using kidney from 10-week-old mice (12Furukawa K. Clausen H. Hakomori S. Sakamoto J. Look K. Lundblad A. Mattes M.J. Lloyd K.O. Biochemistry. 1985; 24: 7820-7826Crossref PubMed Scopus (72) Google Scholar). Antibody binding was detected using an ABC kit™ (Vector Laboratories, Burlingame, CA) and 4-chloro-1-naphthol (Wako, Osaka, Japan) as a substrate. Flow Cytometry and Measurement of Serum Antibody—Spleen cell and thymocyte subsets were analyzed with fluorescein isothiocyanate (FITC)-conjugated anti-CD45R (B220), FITC anti-CD4, phycoerythrin (PE)-conjugated anti-CD3, and PE-anti-CD8 mAbs (eBioscience, Kobe) by flow cytometry (BD Biosciences) as described previously (13Okada M. Itoh M. Haraguchi M. Okajima T. Inoue M. Oishi H. Matsuda Y. Iwamoto T. Kawano T. Fukumoto S. Miyazaki H. Furukawa K. Aizawa S. Furukawa K. J. Biol. Chem. 2002; 277: 1633-1636Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). IgG, IgM, and IgA levels in mouse serum were measured using a Mouse Immunoglobulin ELISA Quantitation Kit™ (Bethyl Laboratories, Montgomery, TX). VT-1 and VT-2 Preparation—A clinically isolated E. coli O157 H7 strain was used for production of VTs as reported (14Zhao Y.L. Cen X.B. Ito M. Yokoyama K. Takagi K. Kitaichi K. Nadai M. Ohta M. Takagi K. Hasegawa T. Antimicrob. Agents Chemother. 2002; 46: 1522-1528Crossref PubMed Scopus (15) Google Scholar). The Luria broth culture supernatant was precipitated with 60% saturated ammonium sulfate at 4 °C. The precipitate was dissolved in phosphate-buffered saline and used after dialysis against phosphate-buffered saline. Contaminating lipopolysaccharides (LPS) in this preparation (10.62 mg/ml) were eliminated by Detoxi-Gel™ (Pierce); the LPS concentration was < 0.001% (final 99.52 ng/ml), but VT-2 concentration was >93% after the purification. VT-2 concentration was determined by the VTEC-RPLA™ kit (Denka Seiken, Tokyo). LPS concentration in the crude VT-2 preparation was determined by the ENDOSPESY™ kit (Seikagaku Corp., Tokyo). Histology and Immunohistochemistry—For pathological analysis, tissues from 10-15-week-old mice before and after treatment with VT-2 were fixed with 3.7% formalin in phosphate-buffered saline and embedded in paraffin. The sections were stained with hematoxylin-eosin. For immunohistochemical analysis, tissues were frozen in liquid nitrogen and 7-μm sections were prepared on a cryostat (Leica) and fixed with ice-cold acetone for 15 min at -20 °C. After blocking with 0.05% H2O2 and 10% normal goat serum, cryosections were incubated with mAb-38.13 (1:50) or an anti-CD31 mAb-390 (1:50) (eBioscience), and the antibody bindings were detected with Histofine Simple Stain Mouse MAX PO™ (Rat) (Nichirei, Tokyo) and 3,3′-diaminobenzidine-tetrahydrochloride (Dojin, Kumamoto, Japan) as a substrate. Nuclei were stained with hematoxylin. For negative controls, immunohistochemistry was carried out using non-relevant antibodies with the same isotypes. To determine the expression sites of Gb3 in tubules, we carried out immunohistochemistry using fluorescence-conjugated second antibodies and performed our analysis with a Fluoview FV500™ confocal laser microscope (Olympus, Tokyo). Antibody binding to Gb3 and CD31 was detected with anti-rat-IgM fluorescein isothiocyanate (ICN Pharmaceuticals, Aurora, OH) and anti-rat-IgG-Alexa-488 (Molecular Probe, Invitrogen), respectively. GM1 expression in tissues was detected with CTB-Alexa-555 (1:100) (Molecular Probe, Invitrogen). Real-time PCR—Real-time PCR was carried out using the DNA Engine Opticon2™ (Bio-Rad Laboratories) and DyNAmo™ kit (Finnzymes, Espoo, Finland). For α1,4Gal-T, amplification of a 264-bp fragment (nucleotides 800-1063) in the cDNA (AI647273) was performed. For glyceraldehyde-3-phosphate dehydrogenase, amplification of a 224-bp fragment (nucleotides 471-694) in the cDNA (BC083065) was performed. Measurement of TNF-α and IL-1β—After injection of VT-2, mouse serum was obtained and used for enzyme-linked immunosorbent assay using Mouse TNF-α or IL-1β immunoassay™ kits (Biosource International, Camarillo, CA). Generation of α1,4 Galactosyltransferase Knock-out Mice—After transfection of the targeting vector (Fig. 1A) and G418 selection, the obtained clones were Southern blotted. Of the 12 candidate homologous recombinants selected from the first screening, 9 clones were identified in the second screening (data not shown). Chimeric mice were generated by microinjection of three isolated clones, and heterozygotes were mated with each other to generate homozygotes. The results of Southern blotting and PCR genotyping are shown in Fig. 1, B and C. Northern blotting revealed that α1,4Gal-T mRNA was completely absent (Fig. 1D). Loss of Gb3 Synthase Activity and Globo-series Glycolipids in α1,4Gal-T-/- Mice—We examined Gb3 synthase activity and globo-series glycolipids (Fig. 1F). Gb3 synthase activity in membrane fractions from kidney was reduced by ∼50% in α1,4Gal-T+/- mice compared with the wild type, with no activity observed in α1,4Gal-T-/- mice. TLC analysis revealed that the globo-series glycolipids were completely deleted in α1,4Gal-T-/- mice; and were observed to glycolipids in mouse kidney were similar to reported previously M. T. Suzuki A. J. 1987; PubMed Scopus Google Scholar), and TLC immunostaining confirmed that α1,4Gal-T-/- mice no These mutant mice were at that and a revealed no serum, or disorders (data not shown). and Antibody in Gb3 Mice—A number of reported that a of immature expressed Gb3/CD77 and that they apoptosis (3Gregory C.D. Dive C. Henderson S. Smith C.A. Williams G.T. Gordon J. Rickinson A.B. Nature. 1991; 349: 612-614Crossref PubMed Scopus (462) Google Scholar). These that this antigen is a in In this study, as the of cells and of and of or were performed and no were and serum antibody levels in Gb3 null mutant mice were carried out with mice wild-type and Gb3 null mutant mice were of cells of cells in a of to a number of that Gb3/CD77 was a receptor for VTs C.A. Full Text PDF PubMed Scopus Google Scholar) and its in HUS, for Gb3/CD77 a for VTs in vivo has been To whether tissue damage on Gb3 expression in vivo, of mice to VTs were wild-type mice and and died after injection of 0.02 μg of VT-2 or μg of α1,4Gal-T-/- mice no and after the injection of μg of VT-2 or VT-1 analysis of mouse tissues revealed tissue damage in the brain tubules, and in wild-type mice after VT-2 changes in the brain cortex were observed from after VT-2 In the of the endothelial cells (Fig. and changes in were observed at (Fig. In the changes were observed in and A number of were observed to have cells with and (Fig. and indicating cell to necrosis as reported previously Gordon Infect. Immun. PubMed Google Scholar, Smith Lingwood C.A. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). were associated with and and of endothelial cells (Fig. and These changes were observed after VT-2 in to all at In the of was observed after injection (data not shown). changes were observed in the α1,4Gal-T-/- of mice with VTs to μg of VT-2 or to μg of VT-1 were dissolved in 100 of phosphate-buffered saline and into 10-15-week-old mice were a mouse mouse number in a Gb3 Expression in the of Gb3 in tissue we used immunohistochemistry to Gb3 expression in the tissues of wild-type mice in which damage In the Gb3 was expressed on the endothelial cells in the cortex (Fig. and and the pia mater (Fig. with the of antibody after VT-2 treatment (Fig. These were similar to that observed for CD31 (Fig. and a of endothelial cells, indicating that Gb3 was expressed in of the endothelial cells of the In the Gb3 was expressed in the of the (Fig. and with the of antibody after VT-2 treatment (Fig. However, no expression was observed in the at all (Fig. and The that were stained in the were considered to be they with the expression sites of CD31 (Fig. and Gb3 expression was in after the VT-2 (data not shown). The of of has been reported that LPS Gb3 expression the of Lingwood C.A. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we analyzed serum levels of TNF-α and IL-1β after VT-2 administration (Fig. A and showed serum Real-time PCR also showed of α1,4Gal-T mRNA in tissues (Fig. as well as an in antibody However, no changes were observed in the times of mice administered μg of VT-2 with or μg of (Fig. times of mice with To the effects of LPS in the crude VT-2 preparation, mice were with crude and VT-2 and open crude or VT-2 (0.2 respectively. The crude 10 μg of 12 or were analyzed with the could be in the results crude and VT-2 disruption of the Gb3 synthase gene in of the Gb3 synthase and all glycolipids to the globo-series in the tissues These results that the gene is the only gene Gb3 This mutant mouse for analysis of globo-series glycolipids in vivo and also for the expression analysis of glycolipids that are synthesized the of Gb3 in tissues and shown in 1, are no in and subsets in and Immunoglobulin levels were also wild and the null However, of Gb3/CD77 in the to to be Although gene is in the of glycolipids in the that null mutant mice showed no or changes their was S. H. U. Cohen Clausen H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and is that the glycolipid the of globo-series A number of have that Gb3 and its derivatives be in as and of B bacterial and other we that mutant mice in the they are not at this However, and are to the of Gb3 and its and we are mutant mice for that Gb3 is a receptor for VTs have been using cells, and has been that other as (4Jacewicz M. Clausen H. Nudelman E. Donohue-Rolfe A. Keusch G.T. J. Exp. Med. 1986; 163: 1391-1404Crossref PubMed Scopus (312) Google Scholar) or other receptors J. C. J. J. PubMed Google Scholar) also be to VT The of this revealed that mice Gb3 synthase gene to be completely to with clinical signs pathological changes in the null mutant mice. These results clearly that Gb3/CD77 its derivatives) is the only that a role as a receptor for VTs. was reported that the antigen also be a receptor for VTs (4Jacewicz M. Clausen H. Nudelman E. Donohue-Rolfe A. Keusch G.T. J. Exp. Med. 1986; 163: 1391-1404Crossref PubMed Scopus (312) Google Scholar). was shown that the antigen is also a of Gb3/CD77 synthase K. Furukawa K. M. Y. Wiels J. H. Urano T. Furukawa K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the null mutant mouse is to the However, no cells only the antigen are to that as a receptor for VTs. can be that only Gb3/CD77 synthase as receptors for VTs. for is well that are receptors for toxins and/or toxins S. K. C. Full Text Full Text PDF PubMed Scopus Google Scholar), and this has been confirmed using mice M. K. Aizawa S. Furukawa K. Furukawa K. PubMed Scopus Google Scholar), which were to the However, the that the mice died that for individual toxins were present S. K. C. Full Text Full Text PDF PubMed Scopus Google Scholar). null mutant mice for Consequently, the that Gb3/CD77 as the receptor for VTs was mutant mice were observed to be negative in was to expression of Gb3/CD77 from the and that the of VTs in tissue damage were Gb3/CD77 on endothelial cells in (10Boyd B. Lingwood C. Nephron. 1989; 51: 207-210Crossref PubMed Scopus (163) Google Scholar, C. C. Lingwood C. M. J. Clin. Med. Google Scholar). have been on the expression of Gb3/CD77 in C.A. Full Text PDF PubMed Scopus Google Scholar, C. C. Lingwood C. M. J. Clin. Med. Google Scholar, C.A. Nephron. PubMed Scopus Google Scholar, K. J. A. M. T. Hakomori J. 1985; PubMed Scopus Google Scholar, E. A. J. 1991; PubMed Scopus Google Scholar) and mouse Gordon Infect. Immun. PubMed Google Scholar, Smith Lingwood C.A. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) with observed in the In our study, expression of Gb3/CD77 was observed in the microvascular endothelial cells of the brain cortex and pia mater and the endothelial cells renal and as well as of the pathological changes observed after VT administration in suggesting that VT sites are endothelial cells changes detected at in the and to be of caused by disruption of the endothelial damage in was observed before suggesting that the damage was not a of VT-2 but that to of endothelial cells in the brain damage HUS has been also been considered (9Tzipori S. Chow C.W. Powell H.R. J. Clin. Pathol. 1988; 41: 1099-1103Crossref PubMed Scopus (70) Google Scholar, J. K. T. T. Y. T. PubMed Scopus Google Scholar). of Gb3/CD77 and CD31 and of their expression sites and pathological changes in brain cortex in mice the that endothelial cells are the targeting sites of VTs in the These the signs and observed in was reported that LPS and Gb3/CD77 expression on endothelial cells Lingwood C.A. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar), be that they VT Karmali M.A. PubMed Google Scholar). However, the effects of LPS on VT depend on is administered Infect. Immun. 1989; PubMed Google Scholar). In addition, the mouse used and tissue of endothelial cells are also for the of LPS effects (10Boyd B. Lingwood C. Nephron. 1989; 51: 207-210Crossref PubMed Scopus (163) Google Scholar, Infect. Immun. 1989; PubMed Google Scholar). In our the α1,4Gal-T gene be by LPS but the sensitivity to VTs. the of in Gb3/CD77 expression not be a tissue damage in vivo. Consequently, basal levels of Gb3/CD77 expressed on endothelial cells in mice be sufficient to tissue damage and lethal for the of crude and VT-2 in our was that the have been from the blood before sufficient Gb3/CD77 was by The obtained with the null mutant mice are as they clearly the of for against the effects of VTs. Although by Gb3/CD77 be C. T. K. Y. J. Clausen H. Wiels J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), to HUS on the VTs and We M. and T. for We also K. Lloyd for reading the