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• W2034124003 abstract "Differences in glycolipid expression between species contribute to the tropism of many infectious pathogens for their hosts. For example, we demonstrate that cultured human and monkey urinary epithelial cells fail to bind a canine Escherichia coli uropathogenic isolate; however, transfection of these cells with the canine Forssman synthetase (FS) cDNA enables abundant adherence by the same pathogen, indicating that addition of a single sugar residue to a glycolipid receptor has marked effects on microbial attachment. Given the contribution of glycolipids to host-microbial interactions, we sought to determine why human tissues do not express Forssman glycolipid. Query of the GenBankTM data base yielded a human sequence with high identity to the canine FS cDNA. Reverse transcription polymerase chain reaction and Northern blotting demonstrated the presence of FS mRNA in all tissues examined. A human FS cDNA was characterized, revealing identities with the canine FS gene of 86 and 83% at the nucleotide and predicted amino acid sequences, respectively. In contrast to the canine FS cDNA, transfection of COS-1 cells with the human FS cDNA resulted in no detectable FS enzyme activity. These results suggest that variability in glycolipid synthesis between species is an important determinant of microbial tropism. Evolutionary pressure from pathogenic organisms may have contributed to diversity in glycolipid expression among species. Differences in glycolipid expression between species contribute to the tropism of many infectious pathogens for their hosts. For example, we demonstrate that cultured human and monkey urinary epithelial cells fail to bind a canine Escherichia coli uropathogenic isolate; however, transfection of these cells with the canine Forssman synthetase (FS) cDNA enables abundant adherence by the same pathogen, indicating that addition of a single sugar residue to a glycolipid receptor has marked effects on microbial attachment. Given the contribution of glycolipids to host-microbial interactions, we sought to determine why human tissues do not express Forssman glycolipid. Query of the GenBankTM data base yielded a human sequence with high identity to the canine FS cDNA. Reverse transcription polymerase chain reaction and Northern blotting demonstrated the presence of FS mRNA in all tissues examined. A human FS cDNA was characterized, revealing identities with the canine FS gene of 86 and 83% at the nucleotide and predicted amino acid sequences, respectively. In contrast to the canine FS cDNA, transfection of COS-1 cells with the human FS cDNA resulted in no detectable FS enzyme activity. These results suggest that variability in glycolipid synthesis between species is an important determinant of microbial tropism. Evolutionary pressure from pathogenic organisms may have contributed to diversity in glycolipid expression among species. Co-evolution of microbial pathogens with their eucaryotic hosts has resulted in a remarkable degree of specificity in their interaction. As a consequence, pathogenic organisms are typically restricted in their ability to cause disease to a limited number of species. An early stage in the pathogenesis of most infectious diseases is microbial adherence to host cells (1Beachey E.H. Giampapa C.S. Abraham S.N. Am. Rev. Resp. Dis. 1988; 138: 45-48Crossref PubMed Google Scholar). As a consequence, the capacity for adherence is a major determinant of the host range available to a given pathogen. Interestingly, glycolipids are the initial attachment site for several pathogenic organisms (2Hansson G.C. Karlsson K.-A. Larson G. Strömberg N. Thurin J. Anal. Biochem. 1985; 146: 158-163Crossref PubMed Scopus (118) Google Scholar, 3Karlsson K.-A. Strömberg N. Methods Enzymol. 1987; 138: 220-231Crossref PubMed Scopus (120) Google Scholar). Illness caused by such diverse agents as bacteria, viruses, and bacterial toxins are known to recognize host glycolipids as their initial attachment site (2Hansson G.C. Karlsson K.-A. Larson G. Strömberg N. Thurin J. Anal. Biochem. 1985; 146: 158-163Crossref PubMed Scopus (118) Google Scholar, 4Leffler H. Svanborg-Eden C. Hansson G. Larson G. Karlsson K.-A. Dahmen J. Frejd T. Magnusson G. Noori G. Chester M.A. Heinegård D. Lundblad A. Svensson S. Proceeding of the 7th International Symposium on Glycoconjugates. Rahms, Lund, Sweden1983: 643-644Google Scholar, 5Hansson G.C. Karlsson K.-A. Larson G. Strömberg N. Thurin J. Orvell C. Norrby E. FEBS Lett. 1984; 170: 15-18Crossref PubMed Scopus (54) Google Scholar, 6Strömberg N. Ryd M. Lindberg A.A. Karlsson K.-A. FEBS Lett. 1988; 232: 193-198Crossref PubMed Scopus (44) Google Scholar, 7Strömberg N. Deal C. Nyberg G. Normark S. So M. Karlsson K.-A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4902-4906Crossref PubMed Scopus (57) Google Scholar, 8Karlsson K.A. Angstrom J. Bergstrom J. Lanne B. Apmis Suppl. 1992; 27: 71-83PubMed Google Scholar). Viewed from the host perspective it appears that susceptibility to infectious diseases is impacted greatly by factors regulating glycolipid synthesis.Glycolipids consist of a variable carbohydrate moiety attached to a ceramide backbone and are a component of virtually all eucaryotic cells. Hundreds of distinct glycolipids have thus far been described (9Basu M. De T. Das K.K. Kyle J.W. Chon H.C. Schaeper R.J. Basu S. Methods Enzymol. 1987; 138: 575-607Crossref PubMed Scopus (112) Google Scholar). Variability in glycolipid structure occurs primarily in the carbohydrate moiety due to differences in the number, type, or anomeric linkage between sugar residues (10Stults C.L. Sweeley C.C. Macher B.A. Methods Enzymol. 1989; 179: 167-214Crossref PubMed Scopus (233) Google Scholar). Each cell type, however, expresses glycolipids containing a limited repertoire of all possible carbohydrate structures. This cell lineage-specific pattern of glycolipid expression is tightly regulated during cellular differentiation and development and varies between species (11Hakomori S. Prog. Clin. Biol. Res. 1980; 41: 873-886PubMed Google Scholar, 12Monner D.A. Muhlradt P.F. Immunobiology. 1993; 188: 82-98Crossref PubMed Scopus (8) Google Scholar, 13Sadahira Y. Yasuda T. Kimoto T. Immunology. 1991; 73: 498-504PubMed Google Scholar, 14Sandhoff K. van Echten G. Adv. Lipid Res. 1993; 26: 119-142PubMed Google Scholar).Forssman glycolipid (FG 1The abbreviations used are:FGForssman glycolipidFSForssman glycolipid synthetasePCRpolymerase chain reactionGbO4globosideHAhemagglutininPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidkbkilobase pairbpbase pair1The abbreviations used are:FGForssman glycolipidFSForssman glycolipid synthetasePCRpolymerase chain reactionGbO4globosideHAhemagglutininPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidkbkilobase pairbpbase pair; Forssman antigen) is a member of the globoseries glycolipid family, all of which have in common a core galactosyl-(α1,3)galactose moiety. Unlike many other mammalian species, human cells do not normally produce Forssman glycolipid but produce the precursor glycolipids globotriaosylceramide and globoside (GbO4). In some species, including humans, these precursors serve as an attachment site for bacteria, viruses, and toxins (15Keusch G.T. Jacewicz M. Mobassaleh M. Donohue-Rolfe A. Rev. Inf. Dis. 1991; 13 Suppl. 4: 304-310Crossref Scopus (31) Google Scholar, 16Sandvig K. van Deurs B. Physiol. Rev. 1996; 76: 949-966Crossref PubMed Scopus (263) Google Scholar, 17Jacewicz M.S. Mobassaleh M. Gross S.K. Balasubramanian K.A. Daniel P.F. Raghavan S. McCluer R.H. Keusch G.T. J. Inf. Dis. 1994; 169: 538-546Crossref PubMed Scopus (45) Google Scholar, 18Lingwood C.A. Adv. Lipid Res. 1993; 25: 189-211PubMed Google Scholar, 19Lingwood C.A. Trends Microbiol. 1996; 4: 147-153Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 20Brown K.E. Anderson S.M. Young N.S. Science. 1993; 262: 114-117Crossref PubMed Scopus (713) Google Scholar, 21Cooling L.L. Koerner T.A. Naides S.J. J. Infect. Dis. 1995; 172: 1198-1205Crossref PubMed Scopus (123) Google Scholar). In other species it is likely that modification of these glycolipids (such as by the addition of an N-acetylgalactosamine to create Forssman glycolipid) alters adherence of pathogenic organisms, directly affects microbial ecology, and modifies host susceptibility to infectious diseases. It is in this context that we sought to elucidate the mechanisms controlling Forssman glycolipid expression in human cells.EXPERIMENTAL PROCEDURESMaterialsT24 (human bladder epithelium) and Vero (monkey kidney) cells were obtained from ATCC and passaged in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and following stable transfection were maintained in geneticin (Life Technologies, Inc.) at 600 (Vero) or 200 μg/ml (T24). Escherichia coliHB101 containing plasmid pRS-1 (encoding the class III P-pilus operon) was kindly provided by Staffan Normark. Anti-Forssman antibody was concentrated from hybridoma M1/22.21 (ATCC) tissue culture supernatant. A human substantia nigra λgt10 cDNA library and a panel of human tissue λgt10 cDNA libraries (CLONTECH, Palo Alto CA) were kindly provided by Jonathan Gitlin. Human multiple tissue RNA blots were purchased from CLONTECH. Double-stranded DNA probes were radiolabeled with [32P]dCTP (3000 Ci/mmol) by random priming using reagents from Roche Molecular Biochemicals. Taq DNA polymerase and dNTPs were from Life Technologies, Inc. Glycolipid substrates and standards were purchased from Sigma. UDP-[3H]galactose and UDP-[3H]N-acetylgalactosamine were purchased from American Radiochemicals.Stable Transfection of Human and Monkey Uroepithelial Cells with Canine FS cDNAThe canine FS cDNA from pFS-10 (22Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar) was ligated into neomycin-selectable plasmid pCR3.1 to create pFS-35. A truncated (non-functional) FS cDNA was created by deleting a 400-bpApaI fragment from pFS-35 to create plasmid pFS-36, used as a transfection control. T24 and Vero cells were transfected with plasmids pFS-35 and pFS-36 using LipofectAMINE reagent as described previously (22Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar). Forty eight hours after transfection neomycin was added to the media, and resistant clones were allowed to proliferate. After 10 days, the clones were pooled, and the 5% of pFS-35-transfected cells reacting most strongly with anti-Forssman antibody were selected by fluorescence-activated cell sorter and further expanded in the presence of neomycin. As we found that serial passage resulted in a decrease in the percentage of FS-expressing cells over time, the cells were resorted by fluorescence-activated cell sorter for anti-Forssman reactivity prior to performing bacterial adherence assays.Bacterial Adherence Assays and Immuno-Thin Layer ChromatographyIn order to examine the binding of E. coli pRS-1 to monolayers of FS-transfected cells, each cell line was seeded onto slide chambers, allowed to attach overnight, and then were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and overlaid with a suspension of E. coli:pRS-1 at approximately 1 × 109 bacteria/ml. The slides were incubated for 30 min at 37 °C and then washed 5 times with cold PBS. Following another incubation in 4% paraformaldehyde for 15 min at room temperature, cells were stained with Geimsa (Diff. Quick, Roche Molecular Biochemicals) and visualized by light microscopy.For immunologic detection of Forssman glycolipid and examination of bacterial adherence to cellular glycolipids, a crude lipid extract was prepared from 1 × 107 pFS-36- or pFS-35-transfected cells by resuspending cell pellets in an equal volume of methanol (approximately 100 μl) and vortexing vigorously. An equal volume of chloroform was added; the cells were vortexed, and then methanol was added dropwise until organic and aqueous phases resolved into a single phase. Cellular debris was pelleted by centrifugation (10,000 ×g), and the organic supernatant was removed and evaporated under reduced pressure. The lipid residue was resuspended in chloroform:methanol:water and spotted onto TLC plates. Glycolipids were separated in organic solvent (chloroform:methanol:water, 65:35:8); the plates were air-dried and then blocked with bovine serum albumin (5% in PBS). Plates were then overlaid either with monoclonal anti-Forssman antibody (M1/M22.21) or bacteria that had been metabolically radiolabeled as described previously (23Haslam D. Borén T. Falk P. Ilver D. Chou A. Xu Z. Normark S. Mol. Microbiol. 1994; 14: 399-410Crossref PubMed Scopus (31) Google Scholar). The plates were washed and then subjected either to secondary antibody and enhanced chemiluminescence (anti-Forssman glycolipid) or directly to autoradiography (radiolabeled bacteria).PCR Amplification of Human FS cDNATwo oligonucleotide primers (MFS-4, 5′-CCCCACCATAATAGAAGTCCC-3′, and MFS-7, 5′-GCACCCATCGTCTCCGAGGGAACC-3′) that span intron VI in the human FS gene were used to PCR-amplify this region from individual λgt10 human tissue cDNA libraries. PCR was performed in a Perkin-Elmer 2400 thermal cycler using 1 μl (approximately 1 × 109plaque-forming units) of λgt10 library lysate, 200 μmeach dNTP, 25 pmol of each primer, and 1 unit of Taq DNA polymerase. Thirty cycles of PCR were performed using the following conditions: 30 s at 94 °C, 30 s at 60 °C, and 90 s at 72 °C. The PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light.Northern Blotting of Human RNAA 589-bp fragment from exon VII of the human FS gene was PCR-amplified from 1 μg of human genomic DNA using primers FS-26 (5′-TTCRTCCAGYMCTTCCTGGAGTC-3′) and FS-28 (5′-CAGAGGTA-CTCRGGGGACAGC-3′) under the conditions described above. The PCR product was subcloned into plasmid pCR3.1 (Invitrogen) to create plasmid pHFS. The plasmid was digested with EcoRI, and the insert was gel-purified, labeled with [32P]dCTP, and used as probe for Northern blots. 50 ng of purified β-actin DNA (CLONTECH) was similarly labeled. Human multiple tissue RNA blots were prehybridized for 30 min at 68 °C in RapidHyb solution (CLONTECH) and then hybridized for 1 h at 68 °C in the same solution containing 1 million cpm per ml of probe. Filters were washed at 68 °C in 0.2× SSC, 0.1% SDS and exposed to Kodak Biomax MS film at −70 °C.Isolation and Characterization of FS cDNAA human λgt10 substantia nigra cDNA library was plated on NZCY media, transferred to nitrocellulose, and hybridized overnight with radiolabeled pHFS insert at 65 °C in 5× SSC, 5× Denhardt's, 1% SDS, and 10 μg/ml herring sperm DNA. Filters were washed in 0.2× SSC, 0.1% SDS at 65 °C and then exposed to autoradiography film. Several hybridizing plaques were identified. Three were purified to homogeneity. One such clone, λHFS-3, contained the entire FS coding region and an unspliced intervening sequence between exons VI and VII.Nucleotide SequencingTwo EcoRI restriction fragments obtained from λHFS-3 were subcloned into plasmid pCR3.1 to create plasmids pHFS-1.2 and pHFS-1.8. Cycle sequencing was performed using RC and T7 (Invitrogen) or gene-specific primers, fluorescently labeled dideoxynucleotide dye terminators (BigDye), andTaq polymerase using conditions recommended by the manufacturer (Applied Biosystems). The sequence of both inserts was determined in forward and reverse directions. Additional sequencing reactions were performed to resolve ambiguous nucleotide designations. Nucleotide sequencing of the inserts in the FS expression constructs was performed in a similar manner.Generation of Human, Chimeric, and Epitope-tagged FS Expression VectorsTo generate a eucaryotic expression vector containing the entire human FS coding region and intron VI, plasmid pHFS-1.2 was digested with XbaI and BamHI and ligated intoBglII/XbaI-digested plasmid pHFS-1.8 to create plasmid pHFS. A chimeric human:dog cDNA expression vector was generated by PCR amplifying nucleotides −41 to 357 (encoding amino acid residues 1–119) of the human FS cDNA from plasmid pHFS-1.8 using primers MFS-20 (5′-TTCCCTGCGAAGGGACAGCC-3′) and MFS-8 (5′-CCCCACGGCAAACAC-SGTGACSCC). Nucleotides 334–1273 (encoding residues 111–347, the stop codon, and 3′-untranslated DNA) from the canine FS cDNA were amplified from plasmid pFS-10 with primers MFS-31 (5′- GGGGTCACGGTGTTTGCCGTGGG-3′) and FS-9 (5′-CTCTACAGTGTACGAAGGCC-3′). Five microliters of each PCR product were mixed and brought to 100 μl in 1× PCR buffer containing primers MFS-20 and FS-9, 200 μm dNTPs, and 1 unit ofTaq DNA polymerase. Twenty five additional cycles of PCR were performed, and the 1.2-kb product was directly subcloned into pCR3.1 to create plasmid pHD. Primer MFS-31 changed residue 113 in the canine FS from leucine to valine to match the human FS at residue 113, and since residues 120–122 are identical between human and canine FS proteins, this chimera matches the human FS from residues 1 to 122 and matches the canine FS from residues 123 to 347.A dog:human chimeric cDNA was similarly generated by PCR amplification from the plasmid polylinker to nucleotide 431 of the canine FS cDNA (encoding residues 1–136 of the canine FS except that residue 134 was changed from glutamine to glutamate to match the human FS) using primers T7 and MFS-32 (5′-GAAGAACTCCTC-GGCTGACTCCAGG-3′). Nucleotides 1298–1966 of the human cDNA (encoding residues 129–347 and the stop codon) were amplified with primers MFS-33 (5′-CCTGGAGTCAGCCGAGGAGTTCTTC-3′) and MFS-27 (5′-CCGTGGTCAGCTCC-TCAGGC-3′). The PCR products were mixed and subjected to a further 25 cycles of PCR using primers T7 and MFS-27. The chimeric PCR product was subcloned directly into pCR3.1 to create plasmid pDH, which matches the canine FS from residues 1 to 127 and the human FS from residues 128 to 347 (Fig. 8 A).In order to remove exon VI from the human FS expression plasmid, overlapping PCR was performed in a similar manner to that described above. Nucleotides −41 to 359 plus nucleotides 5′ to the splice site of the human FS cDNA were amplified from plasmid pHFS-1.8 using primers MFS-20 and MFS-40 (5′-GGACTGGATGAA-ATGAGTGTACTTCCCCACGGCAAACACCGTG-3′). Nucleotides spanning the splice site of the human FS were amplified from plasmid pHFS-1.2 using primers MFS-39 (5′-CACGGTGTTTGCCGTGGGGAAGTACACTCATTTCATCCAGTCC-3′) and MFS-27. Primers MFS-39 and MFS-40 were designed to contain complementary and overlapping regions that flank intron VI, such that these ends prime extension in both directions when the two products anneal and delete intron VI. Following five rounds of amplification without flanking primers, the entire coding region was amplified with primers MFS-20 and MFS-27. The resulting product was ligated into plasmid pCR3.1 and its sequence fidelity verified by nucleotide sequence determination.Addition of an amino-terminal HA epitope tag (consisting of amino acids MGYPYDVPDYASG) to the human and dog FS cDNAs was accomplished by amplification of either the human or dog FS cDNA, respectively, with primers HA-2 (5′-ATGGGCTATCCTTATGACGTGCCTGACTATGCCTCAGGCATGCGCTGCCGCAGACTG-3′) with FS-9 or HA-3 (5′-ATGGGCTATCCTTATGACGTGCCTGACTATGCCTCAGCCATGCATCGCCGGAGACTGGCC-3′) with MFS-27. The resulting products were ligated into plasmid pCR3.1, and fidelity of the inserts was determined by nucleotide sequencing.FS Enzyme AssaysCOS-1 cells transfected with plasmid DNA were harvested by trypsinization, resuspended in 1 ml of ice-cold 100 mm MES, pH 6.7, and disrupted by sonication. Nuclei were removed by sedimentation for 5 min at 6000 × g. The supernatants were brought to a final concentration of 20 mmMnCl2 and incubated on ice for 30 min. The aggregated membranes were pelleted by centrifugation for 5 min at 6000 ×g and then were resuspended in 250 μl of 1% Triton X-100 in 100 mm MES and incubated for 1 h at 4 °C with gentle rocking. Insoluble material was removed by sedimentation for 5 min at 6000 × g. Enzyme reactions were performed in a 150-μl volume containing 100 mm MES, pH 6.7, 10 mm MnCl2, 50 μl of membrane extract (approximately 0.5 mg/ml protein), 5 μm3H-labeled UDP-N-acetylgalactosamine (1000 cpm per pmol; American Radiochemicals), and 20 μmol of glycolipid substrate (globoside; Sigma). After incubating at 37 °C for 2 h, reactions were stopped by the addition of 1 ml of ice- cold water. Glycolipid products were separated from unincorporated nucleotide sugar by passing over a reverse phase C18 column, washing with 5 ml of water, 3 ml of 20% methanol in water, and then eluting with 1.5 ml of 100% methanol. The eluted samples were evaporated under reduced pressure, resuspended in 30 μl of chloroform:methanol (2:1), and then spotted onto TLC plates along with a lane of glycolipid standards (5 μg each). After developing in chloroform:methanol:water (65:35:8), the plates were air-dried, the lane of glycolipid standards was cut off, and the plates were sprayed with En3Hance and exposed to Kodak XAR film. Glycolipid standards were visualized with orcinol.Control reactions detecting UDP-galactose:globoside galactosyltransferase activity endogenous to COS-1 cells (unaffected by transfection with the FS cDNA) were performed to ensure approximately equal amounts of cellular extract in each reaction (22Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar). The control reactions were identical to FS enzyme assays except that3H-labeled UDP-galactose (5 μm, 1000 cpm per pmol; American Radiochemicals) was substituted for UDP-N-acetylgalactosamine as the sugar nucleotide donor.Detection of Epitope-tagged FS Proteins by Western BlottingCOS cells transfected with epitope-tagged canine or human FS, or untransfected cells, were harvested by scraping into ice-cold PBS. Half of the cells (approximately 4 × 105) were utilized for enzyme assays as described above. The remaining cells were harvested by centrifugation and resuspended directly in 100 μl of SDS-polyacrylamide gel electrophoresis buffer. After heating to 94 °C for 5 min, the samples were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose. After blocking with 5% skim milk in PBS for 30 min, primary antibody (HA-11, Babco) was added at 1:1000 dilution and incubated for 2 h at room temperature. After washing and incubating in a 1:5000 dilution of secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG; Roche Molecular Biochemicals), Western blots were developed by enhanced chemiluminescence.RESULTSBacterial Adherence to Cells Expressing Forssman SynthetaseCanine uropathogenic bacteria commonly produce adhesive fibers terminating with the class III PapG adhesin (24Strömberg N. Marklund B.-I. Lund B. Ilver D. Hamers A. Gaastra W. Karlsson K.-A. Normark S. EMBO J. 1990; 9: 2001-2010Crossref PubMed Scopus (218) Google Scholar). We examined the ability of E. coli HB101 harboring plasmid pRS-1, encoding the class III adhesin, to adhere to wild-type and FS-transfected uroepithelial cells. Bacteria were overlaid on monolayers of human or monkey kidney cells transfected with pFS-36 (control; WT) or pFS-35 (canine FS; + FS). Adherence was examined by light microscopy. Whereas no bacterial adherence was seen to cells transfected with the truncated FS cDNA (wild-type cells), numerous bacteria bound to cells expressing the full-length FS cDNA (Fig. 1).Figure 1Adherence of E. coli:pRS-1 to primate epithelial cells. Cell lines T24 (Human) or Vero (Monkey) stably transfected with the canine expression plasmid pFS-35 (+ FS) or a control plasmid pFS-36 (WT) were overlaid with E. coli expressing the class III P-pilus adhesin. After washing, cell monolayers were fixed, stained, and visualized by light microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bacterial Adherence to Glycolipids Extracted from Uroepithelial CellsIn order to demonstrate that bacterial adherence was to Forssman glycolipid in transfected cells, extracts were prepared from pFS-36- (WT) and pFS-35 (+ FS)-transfected human and monkey uroepithelial cells and examined for the presence of Forssman glycolipid and the ability to mediate bacterial adherence. Immuno-thin layer chromatography demonstrated an intense band of anti-FG reactivity comigrating with authentic FG in lipid extracts of human and monkey cells transfected with plasmid pFS-35, whereas no reactivity is seen to extracts of cells transfected with a truncated FS cDNA (Fig. 2 A). Bacterial adherence to glycolipid extracts demonstrated a faint band of adherence to the precursor glycolipid (GbO4, globoside) in extracts from all four cell lines (23Haslam D. Borén T. Falk P. Ilver D. Chou A. Xu Z. Normark S. Mol. Microbiol. 1994; 14: 399-410Crossref PubMed Scopus (31) Google Scholar, 24Strömberg N. Marklund B.-I. Lund B. Ilver D. Hamers A. Gaastra W. Karlsson K.-A. Normark S. EMBO J. 1990; 9: 2001-2010Crossref PubMed Scopus (218) Google Scholar). An intense band of bacterial binding to FG was seen in extracts of FS-transfected cells (Fig.2 B).Figure 2Detection of Forssman glycolipid in extracts of transfected cells by anti-FG antibody (A) and adherence by FG-specific bacteria (B). Lipid extracts were prepared from T24 (Human) and Vero (Monkey) cells stably transfected with pFS-36 (WT) and pFS-35 (+ FS). Lipid extracts and a lane of purified Forssman glycolipid were spotted onto TLC plates and developed in organic solvent. Forssman glycolipid was detected using anti-Forssman glycolipid antibody (A) or radiolabeledE. coli HB101 transformed with plasmid pRS-1 (B) as described in the text. The migration of FG and GbO4standards on an adjacent lane are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of the Human FS Genomic LocusDifferences in Forssman glycolipid expression between humans and other species contribute to variable host susceptibility to microbial pathogens. In order to determine whether absent Forssman glycolipid synthesis in humans had a genetic basis, we characterized the human FS gene. GenBankTM and dEST data bases were queried with the canine FS nucleotide sequence using the program BLAST, yielding human genomic cosmid sequences from chromosome 9q34 (GenBankTM accession numbers AC002319 and AC001643). Overall, the nucleotide sequence identity was 86% to the canine FS cDNA. Highest sequence identity was seen between nucleotides 725 and 1050 of the canine cDNA which resides in the putative catalytic domain and is located in a single large exon in the human cosmid sequence. The human FS gene is separated into at least seven exons spanning more than 8 kb of DNA (Fig.3). Genomic organization of the human FS gene is very similar to the highly homologous α(1,3)-galactosyltransferase and ABO transferase loci (25Joziasse D.H. Shaper N.L. Kim D. Van den Eijnden D.H. Shaper J.H. J. Biol. Chem. 1992; 267: 5534-5541Abstract Full Text PDF PubMed Google Scholar, 26Yamamoto F.-I. McNeill P.D. Hakomori S.-I. Glycobiology. 1995; 5: 51-58Crossref PubMed Scopus (174) Google Scholar). In humans each of these related glycosyltransferase genes are located on chromosome 9q34, further supporting the suggestion that this family of glycosyltransferases arose by gene duplication and subsequent divergence (22Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar, 25Joziasse D.H. Shaper N.L. Kim D. Van den Eijnden D.H. Shaper J.H. J. Biol. Chem. 1992; 267: 5534-5541Abstract Full Text PDF PubMed Google Scholar).Figure 3Genomic organization of the human Forssman synthetase gene. Query of the GenBankTM data base with the canine FS cDNA yielded high sequence identity to cosmid clones derived from human chromosome 9q34 (GenBankTM accession numbers AC002319 and AC001643). Filled boxes indicate putative coding exons, and shaded boxes indicate 5′ and 3′ non-coding sequence. Subsequent analysis of a human FS cDNA confirmed the assignment of exon boundaries. Numbers belowthe boxes indicate the corresponding nucleotides in the published genomic clone AC002319.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Detection of Human FS in cDNA Libraries by PCRIn order to determine whether the FS gene was transcribed in human tissues, PCR was carried out on lysates from cDNA libraries prepared from a panel of human tissues. Primers were designed to amplify specifically a 590-bp product from within exon VII of the FS cDNA. A single band of the expected molecular weight was amplified from cDNA prepared from all tissues tested, suggesting that the FS gene was transcribed in each of these tissues (Fig.4 A). In order to exclude the possibility that the PCR product represented amplification of contaminating genomic DNA, primers spanning intron VI were utilized. Genomic DNA or incompletely processed cDNA was expected to yield a product of approximately 1.5 kb, whereas processed message was expected to yield a product of 528 bp. Brain and kidney cDNA libraries were subjected to PCR with these primers and yielded the expected ≈530-bp product, indicating the presence of spliced transcripts in these cDNA libraries (Fig. 4 B). Genomic DNA yielded the expected 1.5-kb product and failed to yield the s" @default.
• W2034124003 created "2016-06-24" @default.
• W2034124003 creator A5029449795 @default.
• W2034124003 creator A5043117396 @default.
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• W2034124003 creator A5063647963 @default.
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• W2034124003 date "1999-10-01" @default.
• W2034124003 modified "2023-09-26" @default.
• W2034124003 title "Characterization of the Human Forssman Synthetase Gene" @default.
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