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• W2040349912 abstract "Blood group P1/P2 is a glycolipid antigen system for which the genetic mechanism has not yet been clarified. We analyzed the potential of the cloned Gb3/CD77 synthase to synthesize P1 antigen, because Gb3/CD77 and P1 share a common structure, Galα1,4Galβ1,4Glc (NAc)–. L cell transfectants with Gb3/CD77 synthase cDNA expressed marginal levels of P1 on the cell surface but contained high levels of P1 in the cytoplasm. P2-type erythrocytes, which were serotyped as P2, also contained definite P1 antigen inside cells, although the amounts were lower than those of P1 cells. Only p erythrocytes lacked P1 antigen corresponding with function-losing mutations in the Gb3/CD77 synthase gene. Synthesis of P1 antigen from paragloboside in vitro was demonstrated using membrane fraction of the transfectants and a fusion enzyme with protein A. These results strongly suggested that P1 synthase is identical to Gb3/CD77 synthase and appear to propose a clue for the solution of the long-pending P1/P2/p puzzle. The P1/P2 difference might result from the difference in P1 quantity based on either different enzyme activity or the presence/absence of other enzyme modulators. Because P2 erythrocytes showed lower levels of Gb3/CD77 synthase mRNA than P1, 5′-upstream promoter regions were analyzed, resulting in the identification of two P2-specific homozygous mutations. Differences in the transcriptional regulation in erythrocytes might be a major factor determining P1/P2. Blood group P1/P2 is a glycolipid antigen system for which the genetic mechanism has not yet been clarified. We analyzed the potential of the cloned Gb3/CD77 synthase to synthesize P1 antigen, because Gb3/CD77 and P1 share a common structure, Galα1,4Galβ1,4Glc (NAc)–. L cell transfectants with Gb3/CD77 synthase cDNA expressed marginal levels of P1 on the cell surface but contained high levels of P1 in the cytoplasm. P2-type erythrocytes, which were serotyped as P2, also contained definite P1 antigen inside cells, although the amounts were lower than those of P1 cells. Only p erythrocytes lacked P1 antigen corresponding with function-losing mutations in the Gb3/CD77 synthase gene. Synthesis of P1 antigen from paragloboside in vitro was demonstrated using membrane fraction of the transfectants and a fusion enzyme with protein A. These results strongly suggested that P1 synthase is identical to Gb3/CD77 synthase and appear to propose a clue for the solution of the long-pending P1/P2/p puzzle. The P1/P2 difference might result from the difference in P1 quantity based on either different enzyme activity or the presence/absence of other enzyme modulators. Because P2 erythrocytes showed lower levels of Gb3/CD77 synthase mRNA than P1, 5′-upstream promoter regions were analyzed, resulting in the identification of two P2-specific homozygous mutations. Differences in the transcriptional regulation in erythrocytes might be a major factor determining P1/P2. The molecular basis of many histo-blood group antigen systems such as A/B/O, Lewis a/Lewis b, X/Y, or Se/se has recently been clarified (1Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (908) Google Scholar, 2Rajan V.P. Larsen R.D. Ajmera S. Ernst L.K. Lowe J.B. J. Biol. Chem. 1989; 264: 11158-11167Abstract Full Text PDF PubMed Google Scholar, 3Kukowska-Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (473) Google Scholar, 4Larsen R.D. Ernst L.K. Nair R.P. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6674-6678Crossref PubMed Scopus (303) Google Scholar, 5Nishihara S. Narimatsu H. Iwasaki H. Yazawa S. Akamatsu S. Ando T. Seno T. Narimatsu I. J. Biol. Chem. 1994; 269: 29271-29278Abstract Full Text PDF PubMed Google Scholar, 6Kudo T. Iwasaki H. Nishihara S. Shinya N. Ando T. Narimatsu I. Narimatsu H. J. Biol. Chem. 1996; 271: 9830-9837Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Kaneko M. Nishihara S. Shinya N. Kudo T. Iwasaki H. Seno T. Okubo Y. Narimatsu H. Blood. 1997; 90: 839-849Crossref PubMed Google Scholar). However, the genetic basis of the blood group P antigen system including P, P1/P2, Pk, and p has not yet been clearly disclosed. The genetic basis of P, Pk and p was recently elucidated with molecular cloning of the Pk synthase gene (α1,4-galactosyltransferase, α1,4Gal-T 1The abbreviations used are: α1,4Gal-T, α1,4-galactosyltransferase, Gb3/CD77 synthase, or Pk synthase; Gb3, globotriaosylceramide or Pk; Gb4, globotetraosylceramide, globoside, or P; paragloboside, neo-lactotetraosylceramide (nLc4) or Galβ1,4GlcNAcβ1,3Galβ1,4Glc-Cer; P1, Galα1,4-paragloboside; P2, P1 negative; mAb, monoclonal antibody; TLC, thin layer chromatography; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; RT, reverse transcription; PBS, phosphate-buffered saline; nt, nucleotides; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RACE, rapid amplification of cDNA ends.; Gb3/CD77 synthase) (8Kojima 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, 9Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Nilsson S.B. Bennett E.P. Jersild C. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 10Keusch J.J. Manzella S.M. Nyame K.A. Cummings R.D. Baenziger J.U. J. Biol. Chem. 2000; 275: 25315-25321Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and P synthase gene (β1,3-N-acetylgalactosaminyltransferase; Gb4 synthase) (11Okajima T. Nakamura Y. Uchikawa M. Haslam D.B. Numata S.I. Furukawa K. Urano T. Furukawa K. J. Biol. Chem. 2000; 275: 40498-40503Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Namely, Pk is globotriaosylceramide (Gb3) synthesized from lactosylceramide with the action of Gb3/CD77 synthase. Lack of Gb3 synthase results in p phenotype expressing neither Gb3 nor P (Gb4), because P is generated from Gb3 with Gb4 synthase (12Naiki M. Marcus D.M. Biochemistry. 1975; 14: 4837-4841Crossref PubMed Scopus (102) Google Scholar). In fact, multiple mutations in the Gb3/CD77 synthase gene leading to functional loss of the enzyme activity were identified in the individuals with p phenotype (9Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Nilsson S.B. Bennett E.P. Jersild C. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 13Furukawa K. Iwamura K. Uchikawa M. Sojka B.N. Wiels J. Okajima T. Urano T. Furukawa K. J. Biol. Chem. 2000; 275: 37752-37756Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). On the other hand, P1/P2 (P1 negative) is the last glycolipid antigen system for which the genetic mechanisms has not yet been clarified, because the P1 synthase gene has not been isolated to date. P1 is a member of the neolacto-series glycosphingolipids with α1,4-linked galactose at the non-reducing end (14Naiki M. Fong J. Ledeen R. Marcus D.M. Biochemistry. 1975; 14: 4831-4837Crossref PubMed Scopus (111) Google Scholar) (Table I), sharing a very similar structure with Pk, Gb3/CD77 antigen. This similarity of the antigen structures between P1 and Gb3/CD77 suggests that a single α1,4-galactosyltransferase gene is responsible for the synthesis of both antigens. The P1/P2 polymorphism is linked to 22q11.3-ter (15McAlpine P.J. Kaita H. Lewis M. Cytogenet. Cell Genet. 1978; 22: 629-632Crossref PubMed Scopus (10) Google Scholar), and the Gb3/CD77 synthase gene is assigned at 22q13.2, supporting the idea that these two synthases are identical or closely linked. However, the approximate frequencies of P1 and P2 are 80 and 20%, respectively, in Caucasians (16Landsteiner K. Levine P. Proc. Soc. Biol. Exp. Biol. N. Y. 1927; 24: 941-942Crossref Scopus (108) Google Scholar, 17Daniels G. Anstee D.J. Carton J.P. Dahr W. Garratty G. Henry S. Jorgensen J. Judd W.J. Kornstad L. Levene C. Lomas-Francis C. Lubenko A. Moulds J.J. Moulds J.M. Moulds M. Overbeeke M. Reid M.E. Rouger P. Scott M. Seidl S. Sistonen P. Tani Y. Wendel S. Zelinski T. Vox Sang. 1999; 77: 52-57Crossref PubMed Google Scholar), and the ratio in Asians is almost the reverse. On the other hand, the occurrence of the p phenotype is very rare in all nationalities (18Watkins W.M. Adv. Hum. Genet. 1980; 10: 1-136PubMed Google Scholar, 19Marcus D.M. Immunol. Ser. 1989; 43: 701-712PubMed Google Scholar), suggesting that the genetic basis of p and P2 phenotypes is not directly linked. Actually, no relevant polymorphic sequences in the coding region of the Gb3/CD77 synthase gene could be detected between P1 and P2 individuals, and no P1 synthase activity could be found in cell lysates transfected with a Gb3/CD77 synthase expression vector (8Kojima 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, 9Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Nilsson S.B. Bennett E.P. Jersild C. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Consequently, it appears difficult to accept that P1 synthase and Gb3/CD77 synthase are identical. One puzzling finding is that p individuals could never synthesize or express P1 antigen, suggesting the presence of a certain linkage between P1 and Gb3/CD77.Table IStructures and antibody specificities for the P1/P/Pk/p antigen systemGlycolipidsStructuresMAbsHIRO-59HIRO-3438-13H11Lactosylceramide (arrested here in p)Galβ1,4Glc-CerGlobotriaosylceramide (Gb3, Pk)Galα1,4Galβ1,4Glc-Cer+Globotetraosylceramide (Gb4, P)GalNAcβ1,3Galα1,4Galβ1,4Glc-Cer+Paragloboside (nLc4)Galβ1,4GlcNAcβ1,3Galβ1,4Glc-Cer+P1 (lacked in P2 ?)Galα1,4Galβ1,4GlcNAcβ1,3Galβ1,4Glc-Cer+ Open table in a new tab In the present study, we analyzed the biosynthesis of the P1 antigen in transfectant cells of Gb3/CD77 synthase cDNA. We demonstrated here that Gb3/CD77 synthase could generate P1 antigen, and P2 individuals except for p were also able to synthesize P1, although the levels of the product might be different. Furthermore, we analyzed 5′-upstream regulatory regions of Gb3/CD77 synthase gene and identified two P2-specific mutations that may determine the differential expression levels of the gene in P1/P2 individuals. Thus, the genetic basis of P1 synthesis has been clarified, whereas fine mechanisms for the transcriptional regulation need to be investigated. Materials—Anti-P1 monoclonal antibody (mAb) HIRO-59 (3D4) and anti-Gb4 mAb HIRO-34 (9H6) were established. 2M. Uchikawa, Y. Suzuki, T. Toyoda, and M. Satake, manuscript under preparation. Anti-paragloboside mAb H11 (20Myoga A. Taki T. Arai K. Sekiguchi K. Ikeda I. Kurata K. Matsumoto M. Cancer Res. 1988; 48: 1512-1516PubMed Google Scholar) was provided by Dr. T. Taki (Otsuka Pharmaceutical Company, Tokushima, Japan). Biotinylated anti-human IgM was from Vector Laboratories, Inc. (Burlingame, CA). Anti-Gb3/CD77 mAb 38–13 was as described previously (21Wiels J. Fellous M. Tursz T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6485-6488Crossref PubMed Scopus (150) Google Scholar). α-Galactosidase (from coffee beans) and papain were from Sigma. β-Galactosidase (from Jack beans) and Pronase were purchased from Seikagaku Corp. (Tokyo, Japan) and Calbiochem, respectively. The P1- and P2-type bloods were from healthy donors with their consent. The p (little p)-type bloods were obtained from the Department of Transfusion Medicine, Umeå University Hospital. All glycolipid structures and specificities of antibodies reactive with them used in this study are summarized in Table I. Cell Lines—A mouse fibroblast line L cell was provided by Dr. A. P. Albino (Sloan-Kettering Cancer Center, New York) and was maintained in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum. A stable transfectant of L cells (L-VTR) was established as described previously (9Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Nilsson S.B. Bennett E.P. Jersild C. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and maintained in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum and G418 (Invitrogen) (300 μg/ml). Flow Cytometry—Cell surface expression of Gb3/CD77 and P1 was analyzed by flow cytometry (BD Biosciences) as described previously (22Yamashiro S. Haraguchi M. Furukawa K. Takamiya K. Yamamoto A. Nagata Y. Lloyd K.O. Shiku H. Furukawa K. J. Biol. Chem. 1995; 270: 6149-6155Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). mAbs 38.13 or HIRO-59 were used with fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgM or anti-human IgM (ICN Pharmaceuticals, Aurora, OH), respectively. Glycolipid Extraction—Glycolipids were extracted as described previously (23Furukawa 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). For TLC immunostaining, a neutral glycolipid fraction derived from 0.25 ml of each blood sample was spotted on each lane. TLC and TLC Immunostaining—TLC was performed on high performance TLC plates (Merck) using the solvent system of chloroform/methanol/water (60:35:8) and sprayed by orcinol. The identity of P1 was confirmed by TLC immunostaining using an aluminum-backed silica plate (Merck) as described previously (24Ishikawa D. Taki T. Methods Enzymol. 2000; 312: 157-159Crossref PubMed Google Scholar). After TLC, the plate was heat-blotted onto polyvinylidene difluoride membrane. After blocking in 5% skim milk in PBS, the plate was incubated with mAb and then antibody binding was detected with an ABC kit (Vector Laboratories, Burlingame, CA) combined with an enhanced chemiluminescence system (PerkinElmer Life Sciences). Glycosidase Treatment—A neutral glycolipid fraction from erythrocytes was treated with α- or β-galactosidases according to the methods described by Bailly et al. (25Bailly P. Chevaleyre J. Sondag D. Francois-Gerard C. Piquet Y. Vezon G. Carton J.-P. Mol. Immunol. 1987; 24: 171-176Crossref PubMed Scopus (9) Google Scholar) with modification. Hydrolysis of neutral glycolipids was carried out with 0.05 units of α-galactosidase in 20 mm citrate-phosphate buffer, pH 5.5, containing 1 mg/ml of γ-galactonolactone with or without 1% BSA. Hydrolysis was carried out with 0.1 unit of β-galactosidase in 50 mm citrate-phosphate buffer, pH 3.5, with or without 1% BSA, 2 mm EDTA. After incubation for 20 h at 37 °C with shaking, the products were isolated using a C18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by TLC immunostaining using anti-P1 mAb. Then, the same membrane was immunostained using anti-Gb4 mAb HIRO-34. The intensity of the P1 bands was corrected with the intensity of the Gb4 bands using cricket graph software. Immunofluorescence Assay and Immunocytochemistry—For immunofluorescence assay, cells were plated in 60-well plates (Greiner) in Dulbecco's modified Eagle's minimal essential medium containing 7.5% fetal bovine serum. On the following day, cells were stained with mAbs and corresponding second antibodies as described in flow cytometry and observed under fluorescence microscopy (BX60; Olympus, Tokyo). For immunocytochemistry, cells were plated on cover glasses. On the following day, they were fixed with 90% acetone containing 10% PBS and then were stained with anti-P1 mAb HIRO-59 and FITC-conjugated goat anti-human IgM. The staining pattern was observed using a μ Radiance® confocal imaging system (Bio-Rad). Enzyme Treatment of Erythrocytes—Erythrocytes from P2- and p-type donors were washed with PBS and then treated with trypsin (0.25%) for 7 min at 37 °C, with papain (80 mg/ml) in 67 mm phosphate buffer, pH 5.4, for 7 min, or with Pronase (10 μg/ml) in PBS containing CaCl2 and MgCl2 at 37 °C for 30 min. After treatment, they were washed twice and then used for flow cytometry as described above. Enzyme Assay—Gb3/CD77 synthase activity was measured as described previously (8Kojima 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). The enzyme activity of α1,4Gal-T to generate P1 structure was measured as described previously (26Bailly P. Piller F. Gillard B. Veyrieres A. Marcus D. Cartron J.P. Carbohydr. Res. 1992; 228: 277-287Crossref PubMed Scopus (12) Google Scholar). Briefly, membrane fractions were prepared as described (8Kojima 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). The reaction mixture for the assay contained the following in a volume of 50 μl: 0.2 mm UDP-Gal (Sigma), UDP-[14C]Gal (2.5 × 105 dpm) (PerkinElmer Life Sciences), 2.5 μg of nLc4, 20 μm CDP-choline, and 100 μg of phosphatidylglycerol (Sigma), and after evaporation, 20 mm sodium cacodylate-HCl, pH 6.8, 10 mm MnCl2, 20 mm galactonolactone (Sigma), 0.3% Triton X-100 (Sigma), 250 μg of α-lactalbumin, and membrane fraction containing 100 μg of protein. The protein concentration was determined by the methods of Lowry et al. (35Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The products was isolated by a C18 Sep-Pak cartridge (Waters, Milford, MA) and analyzed by TLC and autofluorography using a Bio-Imaging Analyzer BAS2000 (Fuji Film, Tokyo). Reaction products was detected by TLC immunostaining using anti-P1 mAb. Construction and Generation of α1,4Gal-T Proteins Fused with Protein A—The putative catalytic domain of α1,4Gal-T was expressed as a secreted protein fused with protein A. DNA fragment encoding a C-terminal portion of α1,4Gal-T was amplified by PCR. The PCR was performed using 5′- and 3′-primer sequences flanked with EcoRI and XhoI sequences, respectively, forming a DNA fragment that codes 306 amino acids of α1,4Gal-T at the C′-terminal region. The primer sequences were as follows: forward primer, 5′-GGGAATTCCCCAAGGAGAAAGGGCAGCT-3′; reverse primer, 5′-GGCTCGAGGCGGGCCCCTCACAAGTACA-3′. The amplified fragment was first TA-cloned using pCR2.1-TOPO vector (Invitrogen). Subsequently, the insert DNA was excised by EcoRI and XhoI and was inserted between the EcoRI and XhoI sites of pCDSA (kindly provided by S. Tsuji at Riken Institute, Wako, Japan). This plasmid, pProt/α1,4Gal-T, was transfected into L cells with the DEAE-dextran method (8Kojima 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 the secreted fusion protein was collected and concentrated 20 times. Ten μl of the concentrated solution was used in the enzyme assay after dialysis as described above. Quantitative RT-PCR—Total RNA was isolated using Trizol reagent (Invitrogen) from human red blood cells (1 ml) prepared from healthy donors with their consent. cDNA was synthesized by avian myeloblastosis virus reverse transcriptase (Invitrogen) from 3 μg of total RNA as a template. For PCR amplification, the sense primer 5′-TGGAAGTTCGGCGGCATCTA-3′ corresponding to nucleotides (nt) +550 to +569 (5′-CTGGACTCACAACTGCAGTT-3′; position +1 = A of ATG of α1,4Gal-T cDNA) downstream of the ATG codon and the antisense primer 5′-CAGGGGGCAGGGTGGTGACG-3′ corresponding to nucleotides +825 to +844 were used, and PCR was carried out as follows: 94 °C for 1 min, 24–39 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min with a final extension of 72 °C for 5 min using Taq DNA polymerase (Promega, Madison, WI). For amplification of GAPDH cDNA, the sense primer 5′-CCACCCATGGCAAATTCCATGGCA-3′ and the antisense primer 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ were used. The reactions were performed using the following conditions: 94 °C for 1 min and then 24–36 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min, with a final extension of 72 °C for 5 min. The PCR products were electrophoresed in agarose gel and visualized using etidium bromide. The intensities of bands of products in RT-PCR were quantified by scanning the bands in pictures of gels using the public domain NIH Image program. Relative intensities of α1,4Gal-T gene amplified from P1- and P2-type erythrocytes were determined by comparing with the most intense band of P1-type individual and were plotted. For GAPDH, relative intensities of bands compared with the maximum in individual samples were determined and plotted. The cycle numbers of PCR with which band intensities reach 50% of the plateau were obtained from the graphs. 5′-Rapid Amplification of cDNA Ends (RACE) Analysis—A modified RACE analysis was performed to clone gene-specific 5′-ends using the SMART™ RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. Human prostate sample was obtained with informed consent. First strand cDNAs were synthesized by avian myeloblastosis virus reverse transcriptase from 0.7 μg of total RNA. oligo(dT) primer with two degenerate nucleotide positions at the 3′-end was used. Then, 5′-RACE was performed with primers consisting of a gene-specific primer and long or short 5′-primers as provided in the kit. The gene-specific antisense reverse transcription primer corresponding to nucleotides +71 to +95 (5′-AACGTGAACTTGAAGCCGATGATGA-3′; position +1 = A of ATG of α1,4Gal-T cDNA) (8Kojima 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) was used. The reactions were performed by the following conditions: five cycles of 94 °C for 5 s and 72 °C for 3 min, five cycles of 93 °C for 5 s, 70 °C for 10 s, and 72 °C for 3 min and then 25 cycles of 94 °C for 5 s, 68 °C for 10 s, and 72 °C for 3 min. The amplified product was TA-cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced. Sequence Analysis of the α1,4Gal-T Gene—Peripheral blood samples were obtained from healthy volunteers who had given informed consent. After isolation of mononuclear cells by density gradient centrifugation, genomic DNA was extracted with 100 mm EDTA, 0.5% SDS, and 500 μg of proteinase K in 50 mm Tris-HCl, pH 8.0, overnight at 55 °C. After treatment with RNase and phenol-chloroform extraction, DNA was precipitated with ethanol and dissolved in water. About 2 kb upstream genomic region from the transcriptional start site of α1,4Gal-T gene was amplified by PCR using LA Taq (Takara, Otsu, Japan). Two sets of primers used are as follows: 1) 5′-primer, 5′-TCTCGATCTCCTGCCCTTGT-3′ (nt –1991 to –1972) and 3′-primer, 5′-CCCAAGCCAATGAGAAAATG-3′ (nt –804 to –785); 2) 5′-primer, 5′-AGCCTGTGATGGGAATGACC-3′ (nt –1075 to –1056) and 3′-primer, 5′-ACAAATGTCGCCTCCAGAAC-3′ (nt +180 to +199); position +1 = G of the first nucleotide of exon 1, which was determined as the transcription initiation site by 5′-RACE. Subsequently they were subcloned into pCR2.1-TOPO vector (Invitrogen), and inserts were sequenced by the dideoxy termination method using an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Direct Sequence—The promoter region of α1,4Gal-T gene was amplified by PCR using genomic DNAs extracted from peripheral mononuclear cells and PfuTurbo™ DNA polymerase (Stratagene). Two sets of primers used are as follows: 1) 5′-primer, 5′-GAGGTTCCATTTTCTCAGTG-3′ (nt –811 to –792) and 3′-primer, 5′-GCGGAATTCCAGTTATTTGC-3′ (nt –481 to –462); 2) 5′-primer, 5′-ATGGGGAAAACGGGATGGTA-3′ (nt –306 to –287) and 3′-primer, 5′-ACAAATGTCGCCTCCAGAAC-3′ (nt +180 to +199). PCR was carried out as follows: 94 °C for 1 min, 35 cycles of 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 1 min, with final extension of 72 °C for 5 min. The amplified products were sequenced directly by the dideoxy termination method. For sequence of PCR products with primer set 1, another primer was used i.e. 3′-primer, 5′-TCCTGGTGCTTTTAGTTCTT-3′, corresponding to –687 to –668 to avoid the point with insertional mutation. Construction of Plasmids for Luciferase Assay—The upstream genomic region from the α1,4Gal-T gene transcriptional start site was amplified by PCR using PfuTurbo™ DNA polymerase (Stratagene) from genomic DNAs extracted from P1 and P2 peripheral mononuclear cells. Three sets of primers used are as follows: 1) 5′-primer, 5′-CCCTCGAGAGCCTGTGATGGGAATGACC-3′ (nt –1075 to –1056) and 3′-primer, 5′-CCAAGCTTTAGCTCCAGCGG-CGGCGGGC-3′ (nt +1 to +20); 2) 5′-primer, 5′-CCCTCGAGGAGGTT-CCATTTTCTCAGTG-3′ (nt –811 to –792) and 3′-primer, 5′-CCAAGCTT-ACAAATGTCGCCTCCAGAAC-3′ (nt +180 to +199); 3) 5′-primer, 5′-CCCTCGAGAGCCTGTGATGGGAATGACC-3′ (nt –1075 to –1056) and 3′-primer, 5′-CCAAGCTTACAAATGTCGCCTCCAGAAC-3′ (nt +180 to +199); position +1 = G of first nucleotide of exon 1, which was determined by 5′-RACE. PCR was carried out as follows: 94 °C for 1 min, 35 cycles of 94 °C for 30 s, annealing at 53 °C for 30 s, and then extension at 72 °C for 1 min, and 72 °C for 5 min. Subsequently PCR products were TA-cloned into pCR2.1-TOPO vector, and inserts were sequenced for confirmation. Inserts were digested with XhoI and HindIII restriction enzymes and cloned into the XhoI and HindIII sites of the pGL3-Basic vector (Promega). Luciferase Assay for Promoter Activity—The Dual Luciferase™ reporter assay (Promega, Madison, WI) was used to evaluate the promoter activity. Luciferase expression vectors described above were cotransfected with pRL-TK vector for normalization of transfection efficiency into NCC-IT cells using LipofectAMINE™ (Invitrogen). After 48 h of transfection, the medium was removed, the cells were washed twice with PBS, and lysates were prepared to independently measure luciferase activity. The luciferase assay was performed using Pica Gene™ (Toyo Ink Corp., Tokyo) according to the manufacturer's instructions. The ratio of firefly luciferase activity to Renilla luciferase activity was calculated. Statistical Analysis—Significance of the obtained luciferase activities was examined with Student's t test, taking the case of p < 0.05 as statistically significant. Expression of P1 Antigen on Gb3/CD77 Synthase Gene Transfectant Cells—L-VTR1 cells (transfectant cells with pMIKneo/VTR-1) expressed a high level of Gb3/CD77 antigen as expected (Fig. 1A, left). In these cells, P1 was also expressed, although the positive population was low (Fig. 1A, right). P1 Antigen Was Significantly Synthesized in Gb3/CD77 Gene Transfectant Cells—To analyze the presence of P1 antigen in the transfectant cells of Gb3/CD77 synthase cDNA, glycolipids were extracted from the parent L cells and from L-VTR and served for TLC immunostaining. L-VTR cells definitely showed a clear band stained with anti-P1 mAb as did the extracts from erythrocytes of P1-type individuals (Fig. 1B). P1 Antigen Was Present Mainly in Cytoplasm—To examine the localization of P1 antigen in the transfectant cells, immunocytostaining was performed for unfixed and fixed cells using anti-P1 mAb. In immunofluorescence assay, a few cells in the transfectants were stained, whereas L cells were completely negative (Fig. 1C, a and b). On the other hand, the majority of fixed L-VTR cells were clearly stained, and P1 antigen appeared to be localized more abundantly in cytoplasm rather than at the surface membrane (Fig. 1C, d). Expression of P1 Antigen in p/P1/P2 Group Erythrocytes— Serological analysis of P1 antigen expression on various erythrocytes was performed with flow cytometry. P1 erythrocytes showed fairly high levels of P1 expression, whereas P2 cells showed completely negative or marginal levels of P1 antigen (Fig. 2A). Erythrocytes from p individuals were also negative as expected. P1 Antigen in P2 Erythrocytes Is Cryptic—To analyze the crypticity of P1 antigen in erythrocytes, p and P2 erythrocytes were treated by three kind of proteases and then P1 antigen expression was analyzed by flow cytometry compared with untreated cells. Only P2 erythrocytes showed low levels of P1 antigen on the cell surface after treatment with papain or Pronase (Fig. 2B), suggesting that P2 erythrocytes contained cryptic P1 antigen, which might be masked with cell surface proteins. P2 Erythrocytes Contain P1 Antigen at Lower Levels Than P1 Erythrocytes—To examine the presence/absence of P1 antigen in p and P2 individuals, glycolipids of erythrocytes from two p donors, in whom function-losing mutations were found in the Gb3/CD77 synthase gene (13Furukawa K. Iwamura K. Uchikawa M. Sojka B.N. Wiels J. Okajima T. Urano T. Furukawa K. J. Biol. Chem. 2000; 275: 37752-37756Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and five P2 samples, as well as three P1 samples, were analyzed. In TLC, Gb3 and Gb4 were not found in the two p samples, whereas bands of lactosylceramide were increased in intensity as detected with orcinol (Fig. 3A). In TLC immunostaining, all five P2 individuals showed definite bands migrating at the same levels of the P1 bands detected in P1 individual erythrocytes with anti-P1 mAb (Fig. 2B). However, the intensity of the bands in P2 erythrocytes was generally weaker than that in P1 erythrocytes, suggesting the difference in the P1 quantity between P1 and P2 erythrocytes. Neutral glycolipids from p individuals showed no P1 band (Fig. 3B), confirming that p individuals really lack P1 antigen. The" @default.
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• W2040349912 title "The Blood Group P1 Synthase Gene Is Identical to the Gb3/CD77 Synthase Gene" @default.
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