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From www.bloodjournal.org by guest on July 12, 2017. For personal use only. Analysis of Lewis Fucosyltransferase Genes From the Human Gastric Mucosa of Lewis-Positive and -Negative Individuals By Yoshiro Koda, Hiroshi Kimura, and Eisuke Mekada The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le( )] and t w o Lewis-negative [Le( )] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le( ) and Le( ) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le( ) and Le( - ) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le( ) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in t w o amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le( ) gene, w e constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G t o A at position 508 is responsible for the Le( ) gene. The G to A transition at position 508 created a new site for h u l l endonuclease. The digestion by h u l l endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le( ) individuals proved t o be homozygous for the h u l l site. However, the other Le( ) individual was heterozygous for the h u l l site, suggesting the presence of other Le( ) allele(s). Thus, w e isolated one of the silent Lewis genes (le). 0 1993 by The American Society of Hematology. L gawa, Department of Surgery, Kurume University School of Medicine. RNA preparation and cDNA construction. Total RNA of gastric mucosa was prepared by the guanidinium thiocyanate method.'* The poly(A)+fraction was selected by oligo(dT) cellulose column chromatography. Double-strand cDNA of gastric mucosa was constructed using an Amersham cDNA synthesis kit (Amersham Japan, Tokyo). Single-strand cDNA was constructed by Superscript reverse transcriptase (BRL, Gaithersburg, MD). Northern blot hybridization. Poly(A)+ RNAs (2 pg) were electrophoresed through a denaturing formaldehyde agarose gel and transferred onto nylon membrane (Hybond N+; Amersham, Japan). According to the manufacturer's recommended method, the filter was prehybridized and then hybridized with a 32Prandom primed-labeled probetgfrom a 1.7-kb XholI-XbaI fragment of the insert in pCDM7-a(1,3/1,4)FT (kindly donated by Dr J.B. Lowe, Howard Hughes Medical Institute, University of Michigan)." The membrane was then washed and subjected to an Image Analyzer (Fuji film, Tokyo, Japan). PCR amplification of Lewis gene product. The polymerase chain reaction (PCR)*Owas performed using two pairs of synthetic oligonucleotides as primers (-23 to -4 sense and 1 153 to I 174 antisense nucleotides of Lewis FT cDNA in the case of amplification of cDNA containing the protein-coding region (1.2 kb) of the Lewis gene for DNA sequencing and expression study, and 386 to 407 sense and 592 to 612 antisense nucleotides of the catalytic domain of Lewis gene cDNA for restriction fragment analysis (230 + - + - + - - EWIS ANTIGENS are oligosaccharides and are composed of Le" and Leb antigens. Lea antigen is formed by the action of Lewis gene-encoded a( I ,4)fucosyltransferase (IT from ) type 1 precursor, while Leb antigen is formed by the action of Lewis gene-encoded a( 1,4)FT and Se geneencoded a( 1,2)FT from type 1 precursor in tissues such as salivary gland, digestive mucosa, and respiratory mucosa.''* Lewis F T also contains a( 1,3)FT activity, which catalyzes the formation of Le" and LeYantigens from type 2 precursor and H type 2, respectively, in ~ i t r o .Unlike ~ - ~ AB0 antigens, which are synthesized in red blood cells (RBC), Lewis antigens on RBC are secondarily acquired from plasma.6 In addition, the Lewis phenotype on RBC has been reported to change with various conditions, such as pregnancy,' alcoholic pancreatitis and liver cirrhosis,' hydatid cyst,' and various carcinomas.'0,'' It has been generally believed that the Lewis-positive [Le(+)] phenotype results from the action of a( 1,3/1,4)FT encoded by an active allele Le, while the Lewis-negative [Le(-)] phenotype results from the homozygous presence of the silent allele le." However, Lewis antigens have been detected in the normal small inte~tine,'~ colonic mucosa,14 urotheliuml* and various cancer tisof Le(-) individuals. Recently, Kukowska-Latallo et all' isolated cDNA of the Lewis gene-encoded a( 1,3/ 1,4)FT from the cDNA library of A43 1 cells by a gene transfer technique. However, the Le-) mechanism has not yet been examined. An analysis of the silent allele le from Le(-) individuals is important for understanding the Lewis gene and aberrant tissue expression of these antigens in Le-) individuals. In the present study, we examined levels of Lewis gene mRNA obtained from gastric mucosa from Le(+) and Le(-) individuals. Because the mRNA levels were found to be similar, we isolated and analyzed Lewis gene cDNA of Le(+) and Le(-) individuals. MATERIALS AND METHODS Distant uninvolved gastric mucosa from gastrectomy samples of patients with gastric cancer were kindly donated by Dr T. Kake- Blood, Vol82, No 9 (November 1). 1993: pp 2915-2919 - - - - ~~ From the Department of Legal Medicine, School of Medicine, and Division of Cell Biology, Institute of Life Science, Kurume University, Kurume, Japan. Submitted May 4, 1993; accepted July 2, 1993. Address reprint requests to Hiroshi Kimura, MD, PhD, Department of Legal Medicine, Kurume University School of Medicine, Kurume, Fukuoka 830, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1993 by The American Society of Hemutology. 0006-4971/93/8209-0033$3.00/0 2915 From www.bloodjournal.org by guest on July 12, 2017. For personal use only. 2916 KODA, KIMURA, AND MEKADA bp)" with Tth DNA polymerase (Toyobo, Osaka, Japan). Thirty cycles of denaturation (94OC, I minute), annealing (55 to 60°C, I minute), and DNA polymerization (72"C, 1 to 2 minutes) were performed. Double-strand cDNAs from Le(+) and Le(-) gastric mucosa were separately subjected to PCR, and then 1.2-kb Lewis gene cDNAs were ligated to a synthetic BstXl adapter and constructed into P C D M ~ . PCR ~ ' products of the catalytic domain of Lewis FT cDNA (230 bp) amplified from single-strand cDNAs were digested with 20 U of Awl1 endonucleaseand analyzed by 2% agarose gel. Construction of Le(-)/Le(+) chimeras. pCDM8 containing Lewis gene cDNA from Lewis-positive [Le(+) cDNA] or -negative [Le(-) cDNA] gastric mucosa was digested with EcoRV and Mliil, resulting in the formation of I .O-kband 4.5-kb fragments. Ligations of a 1.0-kb fragment from Le(-) cDNA with a 4.5-kb fragment from Le(+)cDNA and ofa I .O-kb fragment from Le(+) cDNA with a 4.5-kb fragment from Le(-) cDNA produced chimera cDNAs containing mutations of the 59th and 508th nucleotides, respectively. DNA sequencing. Dideoxynucleotide termination sequencing reactionz2 was performed with a double-strand plasmid DNA '7 C L R C 5' 3 3' Fig 2. Nucleotide sequence of portions of PCR-products from Le( ) and Le( ) gastric mucosa. The coding sequence is shown aligned with the sequence ladders. 'Sies of nucleotide substitutions in the Le( - ) gene. The encoded amino acid residues are shown. + Fig 1. Northern blot analysis of gastric mucosa mRNA. mRNAs (2 pg) from gastric mucosa of Le( ) and Le( ) individuals were subjected to Northern blot analysis. The blots were probed with a radiolabeledXhol-Xhl fragment of the insert in pCDMTI-a(1,3/1,4)FT. The positions of 28s and 18s ribosomal RNAs are indicated by arrows. + - - (pCDM8 and pBluescript; Stratagene, San Diego, CA) insert. T7 promoter primer, MI3 universal primer, and several synthetic primers were used. DNA sequencing was performed using Sequenase DNA polymerase (United States Biochemical,Cleveland, OH). Detection of Lewis antigen. Formalin-fixed, paraffin-embedded 3-pm sections of human gastric mucosa were deparaffinized, rehydrated, and stained with anti-Le' and -Leb antibodies using a DAKO quick staining kit (DAKO, Carpinteria, CA). COS cells were transfected with plasmid DNAs using a DEAEdextran method as described previously?' The cells were harvested after an expression period of48 hours and incubated for 30 minutes on ice with either anti-Le', anti-Leb (diluted 1:IOO in phosphatebuffered saline [PBS]; Immucor, Norcross, GA), or anti-Le" monoclonal antibody (diluted 1: 100 in PBS; Signet, Dedham, MA). The cells were then stained with fluorexein-conjugated goat antimouse IgM antibody (20 pg/mL). RESULTS We obtained gastric mucosa from four unrelated individuals. Two of the four individuals were k(a-b-) as judged by a hemagglutination test and an immunohistochemical study of gastric mucosa using anti-le" and -Lebantibodies (not shown). From www.bloodjournal.org by guest on July 12, 2017. For personal use only. 2917 LEWIS-NEGATIVE GENE I - Fig 3. Lewis antigen expression in COS cells after transfection of Lewis gene cDNA. Plasmid DNA (10 rg) was transfected into 5 X 1Oe COS cells by a DEAE-dextran method. After transfection of plasmids for 48 hours, the cells were harvested and subjected to indirect immunofluorescence staining. COS cells were transfected with pCDM8 containing Lewis gene cDNA from (A) Le(a b ) gastric mucosa, (B) Le(a b - ) gastric mucosa, (C) chimera cDNA substituted G for T at position 59, and (D) chimera cDNA substituted A for G at position 508. - Northern blot hybridization. To detect the Lewis fl mRNA, Northern blot hybridization was performed. As shown in Fig 1, the hybridization with Lewis FT cDNA demonstrated 2.4-kb bands in poly(A)+ RNAs from both Le(+) and Le(-) gastric mucosa. The intensity of the bands normalized by the amount of actin mRNA was similar (not shown). Therefore, the structural, rather than expression, failure of the Lewis FT gene is responsible for its inability to express Lewis antigen. Direct cloning of Lewis gene cDNA by PCR amplification. To investigate the detailed molecular mechanism, we isolated the protein-coding region of Lewis FT cDNA by PCR amplification from both Le(+) and Le(-) individuals. The 1.2-kb specific bands were gel-purified and added to a BsfXI adapter and then ligated into pCDM8 for DNA sequencing and for an expression study. DNA sequencing. To identify the structural failure of the Le(-) gene, DNA sequencing of the protein-coding regions of Le(+) cDNA and Le(-) cDNA was performed. The sequence of the Le(+) cDNA was identical to that reported - + by Kukowska-Latallo et al.'7 However, the sequence of one of the Le(-) cDNAs contained two base changes from T to G at position 59 and from G to A at position 508 (Fig 2). These nucleotide substitutions may result in the replacement of leucine by arginine at position 20 and of glycine by serine at position 170 from the N-terminal of Lewis FT. Expression of Lewis gene cDNA in COS cells. Kukowska-Latallo et a1 isolated Lewis FT cDNA from A431 cells by a gene transfer method using pCDM7 as mammalian expression vector and COS cells.'7 We used the same systems, but used pCDM8 instead of pCDM7. The only difference between pCDM7 and pCDM8 is that pCDM7 lacks the polyoma sequences present in pCDM8F4 Those COS cells transfected with Le(+) cDNA expressed Le" (Fig 3A), but not Leb antigen. Neither Lea nor Leb antigen was expressed in those COS cells transfected with Le(-) cDNA (Fig 3B). Lewis FT contains two enzymatic activities; one is a( 1,4)FT activity that produces Lewis antigens and the other is a(1,3)FT activity that produces Le" and Ley antigens. Those COS cells transfected with Le(+) cDNA ex- From www.bloodjournal.org by guest on July 12, 2017. For personal use only. KODA, KIMURA, AND MEKADA 2918 1 2 4 3 5 6 7 8 9 found in one of the alleles by sequencing of the 230-bp PCR product of heterozygote for the PvuII site. To identify any other mutation(s) responsible for Le(-), we attempted to amplify the protein-coding region (1.2 kb) of Lewis FT cDNA from heterozygote for the PvuII site; however, we failed to amplify it, probably due to degradation of mRNA as judged by Northern blot analysis. DISCUSSION Fig 4. Restriction endonuclease digestion of Lewis gene PCR product. A PCR product of Lewis FT cDNA between the 386th and 612th nucleotideswas digested with 20 U of h u l l restrictionendonucleasefor 1 hour and subjected to 2%agarose gel. Lane 1 : molecular weight marker (Hpell-digested pUCl8); lanes 2 and 6: PCR products from Le( ) gastric mucosa cDNA; lanes 4 and 8: PCR products from Le( - ) gastric mucosa cDNA; lanes 3, 5, 7, and 9: Arull-digested PCR products of lanes 2,4, 6, and 8. + pressed Le" antigen, whereas those COS cells transfected with Le(-) cDNA did not (not shown). To determine whether either or both of the nucleotide substitutions (of G for T at position 59, and of A for G at position 508) contributes to the failure of the expression of Lewis antigen in COS cells, we constructed chimera cDNAs. The chimera cDNA containing a base change from T to G at position 59 (Leu to Arg) was able to express Le" antigen (Fig 3C), whereas the chimera cDNA containing a base change from G to A at position 508 (Gly to Ser) failed to express Le" antigen (Fig 3D). Our results suggest that the replacement of Gly by Ser at position 170 is responsible for the inability to express Lewis antigen in COS cells. Pvull endonuclease digestion. As shown in Fig 2, the G to A transition at position 508 creates a new site for endonuclease PvuII. To investigate the presence of a mutation at position 508 in other individuals, the endonuclease digestion of 230-bp PCR products between the 386th and 612th nucleotides of Lewis FT cDNA was performed (Fig 4). While the PCR products of the two Le(+) individuals (lanes 3 and 7) were not cleaved by PvuII as expected, the PCR product of one of the Le(-) individuals was cleaved by PvuII (lane 5). However, the PCR product of the other Le(-) individual was demonstrated to be heterozygous for the Pvull site (lane 9). A mutation at position 508 was not In this report, we determined two individuals to be Le(-) by RBC and immunostaining of gastric mucosa and isolated mRNA from gastric mucosa of these two individuals. Those COS cells transfected with Le(-) cDNA did not express Le antigens, suggesting the presence of mutation($ in Le(-) cDNA making Lewis FT activity extremely low or absent. We found two single-base changes at positions 59 and 508 from one of the Le(-) cDNAs. From studies on transfections of chimera cDNAs, a single-base change from G to A at position 508, which results in an amino acid substitution of Ser for Gly at position 170 from the N-terminal of Lewis FT,was found to be responsible for the defective Lewis FT. That only a single-base change found in the Lewis gene causes it to be Le(-) may not be incompatible with the results of 0mtoft et who found 5% to 16% of a(I ,4)FT activity in Le(-) colonal mucosa compared with that of Le(+) mucosa, since a single amino acid substitution would result not in the complete absence of the activity, but rather in a low activity of the enzyme. We need to characterize the nature of the enzyme (activity, stability, affinity for substrates, etc) produced by the Le(-) cDNA after overexpression. Recently, Lowe et a12s27 isolated three a(1,3)FT genes that were different from Lewis FT using Lewis FT cDNA as a probe. Another group also isolated cDNA for ELAM-I ligand FT?* Its sequence showed that 133 of 231 amino acids are identical at corresponding positions within the catalytic domain of the Lewis FT.25The other two a( I ,3)FT genes showed approximately 90% homology with Lewis FT.26,27 It is of interest that the 170th Gly of the Lewis gene, which was found to be replaced by Ser in the Le(-) FT, resulting in its inability to express Lewis antigen (Fig 3B), is conserved between Lewis FT and these three a(1,3)FTs, and located within the catalytic domains ofthese FTs,'~*~'*~ suggesting the importance of the 170th Gly for the activity of the four different a(I,3)FTs. In the present study, we found another Le(-) allele that did not contain a substitution of G for A at position 508 by PvuII digestion ofthe catalytic domain (230 bp) ofthe Lewis FT gene. Unfortunately, we failed to amplify the proteincoding region (1.2 kb) of cDNA by PCR, probably due to degradation of mRNA. Therefore, other functional or expression failure may be the cause of the Le(-) gene. However, this is the first report in which we have identified a mutation of a nucleotide at position 508 in the Le(-) gene. ACKNOWLEDGMENT We thank Edward Moran, Fordham University, for linguistic advice. From www.bloodjournal.org by guest on July 12, 2017. For personal use only. 2919 LEWIS-NEGATIVE GENE REFERENCES 1. Watkins WM: Blood group substances; in the AB0 system the genes control the arrangement of sugar residues that determines blood-group specificity. Science 152: 172, 1966 2. Ono1 R, Le Pendu J, Mollicone R: Genetics of ABO, H, Lewis, X and related antigens. Vox Sang 5 1: 16I , 1986 3. Prieels J-P, Monnom D, Dolmans M, Beyer TA, Hill R L Copurification of the Lewis blood group N-acetylglucosaminide a 1-4fucosyltransferaseand an N-acetylglucosaminide a 1-3fucosyltransferase from human milk. J Biol Chem 256:10456, 1981 4. Johnson PH, Yates AD, Watkins WM: Human salivary fucosyltransferase: Evidence for two different a-3-L-fucosyltransferase activities, one of which is associated with the Lewis blood group Le gene. Biochem Biophys Res Commun 100:16 1 1, 1981 5. Johnson PH, Watkins WM: Separation of an a-3-Gfucosyltransferase from the blood group Le gene-specified a-3/4-L-fucosyltransferase in human milk. Biochem SOCTrans 10:445, 1982 6. Watkins WM: Biochemistry and genetics of the ABO, Lewis, and P blood group systems, in Hams H, Hirschhorn K (eds): Advances in Human Genetics, vol 10. New York, NY, Plenum, 1980, Pl 7. Hammer L, Mansson S, Rohr T, Chester T, Ginsburg V, Lundblad A, Zopf D: Lewis phenotype of erythrocytes and Leb active glycolipid in serum of pregnant women. Vox Sang 40:27, 1981 8. Stigendal L, Olsson R, Rydberg L, Samuelsson BE: Blood group Lewis phenotype on erythrocytes and saliva in alcoholic pancreatitis and chronic liver disease. J Clin Pathol37:778, 1984 9. Makni S, Dalix AM, Caillard T, Compagnon B, Le Pendu J, Ayed K, Ono1 R: Discordance between red cell and saliva Lewis phenotypes in patients with hydatid cysts. Exp Clin Immunogenet 4:136, 1987 IO. Hirano K, Kawa S, Oguchi H, Kobayashi T, Yonekuni H, Ogata H, Homma T: Loss of Lewis antigen expression on erythrocytes in some cancer patients with high serum CA 19-9 levels. J Natl Cancer Inst 79:1261, 1987 1 I . Yazawa S, Asao T, Izawa H, Miyamoto Y, Matta KL: The presence of CA 19-9 in serum and saliva from Lewis blood group negative cancer patients. Jap J Cancer Res (Gann) 79538, 1988 12. Grollman EF, Kobata A, Ginsburg V: An enzymatic basis for Lewis blood types in man. J Clin Invest 48:1489, 1969 13. Bjork S, Breimer ME, Hansson GC,Karlsson KA, Leffler H: Structures of blood group glycosphingolipidsof human small intestine: A relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotypes of the donor. J Biol Chem 262:6758, 1987 14. 0mtoft TF, Holmes EH, Johnson P, Hakomori S, Clausen H: Differential tissue expression of the Lewis blood group antigens: Enzymatic, immunohistologic, and immunochemical evidence for Lewis a and b antigen expression in Le(a-b-) individuals. Blood 77:1389, 1991 15. Limas C Detection of urothelial Lewis antigens with monoclonal antibodies. Am J Pathol 125515, 1986 16. Ernst C, Atkinson B, Wysocka M, Blaszczyk M, Herlyn M, Sears H, Steplewski Z, Korprowski H: Monoclonal antibody localization of Lewis antigen in fixed tissue. Lab Invest 50:394, 1984 17. Kukowska-Latallo JF, Larsen RD, Nair RP, Lowe JB: A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and Lewis blood group a( I ,3/ I ,4)fucosyltransferase. Gene Develop 4: 1288, 1990 18. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989 19. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6, 1983 20. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487, 1988 21. Seed B: An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 3292340, 1987 22. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977 23. Tsuneoka M, Mekada E: Degradation of nuclear-localized protein in mammalian COS cells, using Escherichia coli p-galactosidase as a model protein. J Biol Chem 267:9107, 1992 24. Larsen RD, Rajan VP, Ruff MM, Kukowska-Latallo J, Cummings RD, Lowe JB: Isolation of a cDNA encoding a murine UDP-galactose: @-D-galctosyl1,4-N-acetyl-~-glucosaminide a-1,3galactosyltransferase: Expression cloning by gene transfer. Proc Natl Acad Sci USA 869227, 1989 25. Lowe JB, Kukowska-Latallo J, Nair RP, Larsen RD, Marks RM, Macher BA, Kelly RJ, Emst LK: Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion. J Biol Chem 266: 17467, 199 I 26. Weston BW, Nair RP, Larsen RD, Lowe JB: Isolation of a novel human a( 1,3)fucosyltransferasegene and molecular comparison to the human Lewis blood group a( 1,3/ 1,4)fucosyltransferase gene. J Biol Chem 267:4152, 1992 27. Weston BW, Smith PL, Kelly RJ, Lowe JB: Molecular cloning of a fourth member of a human a( 1,3)fucosyltransferasegene family. J Biol Chem 267:24575, 1992 28. Goelz SE, Hession C, Goff D, Griffiths B, Tizard R, Newman B, Chio-Rosso G, Lobb R ELFT: A gene that directs the expression of an ELAM-I ligand. Cell 63: 1349, 1990 From www.bloodjournal.org by guest on July 12, 2017. For personal use only. 1993 82: 2915-2919 Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals Y Koda, H Kimura and E Mekada Updated information and services can be found at: http://www.bloodjournal.org/content/82/9/2915.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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