Molecular Genetic Analysis of the Human Lewis Histo-blood Group System

doi:10.1074/jbc.271.16.9830

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Volume 271, Number 16, Issue of April 19, 1996 pp. 9830-9837
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Genetic Analysis of the Human Lewis Histo-blood Group System
II. SECRETOR GENE INACTIVATION BY A NOVEL SINGLE MISSENSE MUTATION A385T IN JAPANESE NONSECRETOR INDIVIDUALS (*)

(Received for publication, September 26, 1995; and in revised form, January 2, 1996)

Takashi Kudo (1), (4), Hiroko Iwasaki (1), Shoko Nishihara (1), Naoko Shinya (1), Takao Ando (2), Ikuyo Narimatsu (3), Hisashi Narimatsu (1)(§)

From the (1)Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192, Japan, (2)Basic Research Division, BML Inc., Kawagoe, Saitama 350-11, Japan, (3)Department of Medicine, Saitama Central Hospital, Miyoshi-cho, Saitama 354, Japan, and (4)Department of Microbiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

The Lewis histo-blood group system comprises two major antigens, Lewis a and Lewis b. The Lewis b antigen is a product of two fucosyltransferases, the alpha (1,3/1,4)fucosyltransferase (Lewis enzyme; Fuc-TIII) encoded by the Lewis gene and an alpha (1,2)fucosyltransferase which is not required for synthesis of Lewis a antigen. An enzyme responsible for secreting ABH antigens into body secretions (secretor enzyme) is also one of alpha (1,2)fucosyltransferases. A candidate gene encoding secretor enzyme Sec2 gene was recently cloned by Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D. ((1995) J. Biol. Chem. 270, 4632-4639) and Kelly, R. J., Rouquier, S., Giorgi, D., Lennon, G. G., and Lowe, J. B. ((1995) J. Biol. Chem. 270, 4640-4649) who demonstrated a G428A nonsense mutation (Trp to terminal codon) in Sec2 of nonsecretors. However, the G428A nonsense mutation discovered in the Sec2 gene of nonsecretors in an ethnic group other than Japanese was not found in any of 45 Japanese nonsecretors, whereas one Filipino who had been erroneously registered as a Japanese possessed the G428A mutation heterozygously.

In order to explore the Sec2 gene of a Japanese population, we performed a molecular genetic analysis of the Sec2 gene on 226 Japanese individuals, 21 in a family study and 205 in a random sampling study. We discovered two novel mutations in the Sec2 gene, an A385T missense mutation (Ile to Phe) that results in inactivation of Sec2-encoded alpha (1,2)fucosyltransferase and a C357T silent mutation which is irrelevant to amino acid substitution, in Japanese nonsecretors.

The analysis of Japanese individuals using the polymerase chain reaction-restriction fragment length polymorphism method found three alleles in the Sec2 gene, the first having no mutation, the second having a C357T mutation, and the third having both C357T and A385T mutations, which we designated as Se1, Se2, and sej, respectively. Among 226 Japanese individuals, 40 having a Le(a+b-) phenotype and 5 having a Le(a-b-) nonsecretor phenotype were homozygous for sej/sej, whereas 149 having a Le(a-b+) phenotype and 32 having a Le(a-b-)-secretor phenotype possessed at least one Se1 or Se2.

The frequencies of occurrence of Se1, Se2, and sej among 410 alleles examined in a random sample of 205 Japanese individuals were 15, 46, and 39%, respectively, indicating a rather wide distribution of the sej allele in the Japanese population. The results show that the Sec2 gene really encodes the secretor enzyme alpha (1,2)fucosyltransferase and indicate that a ethnic group-specific nonsense or missense point mutation in the Sec2 gene determines nonsecretor status. The phylogenic aspect and biological significance of the Se and Le genes are discussed.

INTRODUCTION

The biosynthetic pathways of the Lewis histo-blood group system in correlation with ABH antigen synthesis shown in Fig. 1are considered to function in the epithelial cells of digestive organs, although some of the pathways involved have not yet clearly been proven. Individuals are divided by their erythrocyte Lewis antigen phenotypes into three types, Le(a+b-) which has Le ()antigen but not Le antigen, Le(a-b+) which has Le but not Le, and Le(a-b-) having neither Le nor Le. Besides these three phenotypes, Le(a+b+) which possesses both Le and Le, has occasionally been found in some ethnic groups(1) .

Figure 1: Biosynthetic pathways involved in synthesis of Lewis blood group antigens and secretion of ABH antigens into saliva.

Based on the biosynthetic pathways in Fig. 1, both antigens, Le and Le require involvement of the Lewis enzyme, that has alpha (1,4)fucosyltransferase activity, for their synthesis. It was verified by our and others' previous studies(2, 3, 4) that the Lewis gene (Le gene) encoding the Lewis enzyme is the Fuc-TIII gene (5) since the FucT-III gene of Le(a-b-) individuals was found to be inactivated by missense mutations(2, 3, 4) . Le and Le antigens are thought to be synthesized in digestive organ epithelial cells and to be secreted into blood and body secretions such as saliva(6, 7) . Red blood cells adsorb the glycolipids carrying Lewis antigens in plasma(8) , and their phenotypes are typed by a hemagglutination test. ABH antigens of the ABO blood group system are synthesized not only in digestive organ epithelial cells but also in red blood cell precursor cells(9) .

Individuals have been divided into two groups, secretors and nonsecretors, depending on the presence and absence of ABH antigens in their saliva, respectively. The ABH antigens of erythrocytes are the product of H gene-encoded enzyme which synthesizes both H type-1 and H type-2 structures by alpha (1,2)fucosyltransferase activity. Bombay individuals who lack H antigens of erythrocytes were shown to be mutants whose H gene is completely inactivated by the nonsense mutation(10) . Some para-Bombay individuals who lack H antigens of erythrocytes, but can secrete ABH antigens into saliva (secretor), were also found to be H gene mutants, with the nonsense mutation causing complete inactivation of the H enzyme(10) . Some para-Bombay individual's ability to secrete ABH antigen into saliva was therefore thought to be determined not by the H enzyme, but by the other alpha (1,2)fucosyltransferase that should be encoded by the secretor gene (Se gene).

The Le(a-b+) phenotype of erythrocytes is usually in accord with secretor status in saliva, whereas the Le(a+b-) phenotype is in accord with nonsecretor status. Although inconsistency between the Le(a-b+) and secretor phenotype or between the Le(a+b-) and nonsecretor phenotype is occasionally observed, this could be due to an ambiguous hemagglutination of erythrocytes in the test or a failure in detection of ABH antigens in saliva. If the pathways in Fig. 1actually operate in digestive organ epithelial cells, individuals lacking the Se enzyme cannot synthesize the H type-1 structure that is a precursor of A, B, and Le antigens, and this should reveal their nonsecretor status. Also according to the pathways in Fig. 1, the erythrocyte phenotype of individuals who lack the Se enzyme should be Le(a+b-) or Le(a-b-), and not Le(a-b+).

A candidate for the Se gene, termed Sec2, having a highly homologous sequence to the H gene(11) , has recently been cloned from a rat(12) , human(13, 14) , and rabbit(15) . The recombinant enzyme directed by the Sec2 gene apparently had alpha (1,2)fucosyltransferase activity, transferring fucose preferentially to type-1 chain acceptor oligosaccharides rather than type-2 chain acceptors. Substrate specificities of the Sec2-directed enzyme (12, 13, 14, 15) were also very similar to those of the Se enzyme previously purified by biochemical methods(16) . It was proven that the Sec2 gene is the Se gene by these molecular analyses(13, 14) . Due to recent successes in cloning(13) , three homologous genes, H, Sec1, and Sec2, were found to be in close physical linkage to each other within 100 kb on human chromosome 19. One of them, termed the Sec1 gene, seemed to be a pseudogene in which the open reading frame is interrupted by translational frameshifts and termination codons(14) . The three genes, H, Sec1, and Sec2, form a gene cluster and are mapped on 19q13.3(13) , suggesting that they evolved by gene duplication, as is the case in the genes of some members of the alpha (1,3)fucosyltransferase family, i.e. Fuc-TIII, -TV, and -TVI, which cluster on the short arm of chromosome 19p13.3(17, 18) . The human Sec2 gene, a candidate for the human Se gene, has been analyzed by Kelly et al.(14) on a molecular genetic basis. They found a nonsense mutation G428A which changed Trp into a termination codon in the Sec2 gene of a nonsecretor individual and completely inactivated the Sec2 gene-directed enzyme. It was also demonstrated that all six nonsecretors among 52 individuals randomly sampled had the G428A nonsense mutation homozygously. This strongly suggests that the Sec2 gene encodes the Se enzyme(14) .

In this study, we performed a molecular genetic analysis of the Sec2 gene on 226 Japanese individuals comprising 21 individuals belonging to four pedigrees and 205 randomly sampled individuals and found the novel mutations, C357T and A385T, differing from G428A, in the Sec2 gene of the Japanese nonsecretors. The allele inactivated by a novel single missense mutation A385T, termed sej, was distributed homozygously in all Japanese nonsecretors examined in this study, whereas no G428A mutation in the Sec2 gene was found in the Japanese nonsecretors.

In this report, we present these results together with the finding that Se and Le genotypings performed in parallel distinguished three Lewis phenotypes, Le(a+b-), Le(a-b+), and Le(a-b-), as well as secretors and nonsecretors with 100% accuracy, and discuss the phylogenic aspect and biological significance of the Se and Le genes.

MATERIALS AND METHODS

Peripheral Blood and Saliva Samples

Five ml of peripheral blood were bled with heparin from volunteers who also donated saliva samples for detection of ABH antigens. Erythrocytes were subjected for Lewis blood group typing, and genomic DNAs extracted from white blood cells were applied for DNA sequencing and genotyping.

Typing of Lewis Blood Group Phenotypes of Erythrocytes and of the Secretor Satus in Saliva

Lewis blood group phenotypes of erythrocytes were determined as described previously(3) . In brief, Ortho bioclone anti-Le

and anti-Le

monoclonal antibodies (Ortho Diagnostic Systems, Raritan NJ) were used for hemagglutination tests according to the manufacturer's instructions. The 0.5% bromelin solution treatment was done at 37 °C for 15 min prior to observation of hemagglutination. The presence or absence of ABH antigens in saliva was determined by a hemagglutination inhibition using standard serological techniques(19) .

Primers and the First PCR Amplification

PCR primers used in this study are listed in Table 1. To amplify the full-length Sec2 gene, the 5`- and 3`-flanking sequences of the Sec2 gene reported by Kelly et al.(14) were employed for sense tk1 and antisense tk2 primer sequences. The tk1 and tk2 primers (1 µM) were added to 1 µg of genomic DNA in total volume of 50 µl containing 200 µM of each dNTP, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 2.5 mM MgCl (2)

, and 0.1 mg/ml gelatin. Thirty cycles (1 min at 94 °C, 2 min at 65 °C, 2 min at 72 °C) were run, and the products were isolated from electrophoresed agarose gel for subcloning into pBluescript SK(-) (pBS). The tk1 and tk2 primers were flanked by EcoRI and HindIII sequences, respectively, for the convenience of subcloning into a pBS vector.

Sequencing of Sec2 Genes and Their Expression in COS-1 Cells

The PCR products of the full-length Sec2 gene amplified by tk1 and tk2 primers were first cloned into a pBS vector having EcoRI and HindIII sites. Eight subclones in the pBS from YoN in Fig. 4, Family 2, who has a Le(a+b-) phenotype of erythrocytes and nonsecretor status, were sequenced by the dideoxy chain termination method. Eight subclones from SN in Fig. 4, Family 1, who has a Le(a-b+)-secretor phenotype, were also sequenced by the same methods. We searched for the best PCR condition by which we could avoid artificial misincorporations during PCR amplification. The PCR buffer and Taq DNA polymerase obtained from Perkin-Elmer were found to be the most optimal for this purpose. It was, however, inevitable to have some misincorporations. The mutation found in the sole clone and not shared by the other seven clones was regarded as a nongenuine mutation due to misincorporation by Taq DNA polymerase during PCR amplification. Sequencing of the 16 clones obtained from YoN and SN under these conditions resulted in only three misincorporations within about 18 kb sequences. Inserts having no artificial misincorporations were excised by EcoRI and XhoI from the pBS vector and subcloned into the expression vector pCDM8 (20) , having EcoRI and SalI sites. Modification of the pCDM8 vector into having an EcoRI site was done in order to facilitate subcloning. Transfection of the subcloned DNA into COS-1 cells was done as described previously(3) . COS-1 cells were transfected with 15 µg of each subcloned DNA in combination with 1 µg of the beta

-actin promotor-driven luciferase expression vector as an indicator of transfection efficiency.

Figure 4: Family studies on the Sec2 gene. a, erythrocyte phenotypes, secretor status, and Sec2 genotypes determined on 21 individuals of four pedigrees are shown. Square, male; circle, female; filled symbol, homozygote of sej allele (sej/sej); half-filled symbol, heterozygote of Se and sej alleles (Se1 or Se2/sej); open symbol, homozygote of Se allele (Se1 or Se2/Se1 or Se2). Closed triangles and open circles in schematic alleles represent C357T and A385T mutations, respectively. b, electrophoresis patterns on PCR-RFLP. C357T, AseI digestion for detection of the C357T mutation; A385T, AluI digestion for detection of the A385T mutation. Control PCR-RFLPs were done using the cloned plasmid DNA having each allele as templates. +/+, PCR-RFLPs using the cloned relevant mutant allele into pBS as a template; -/-, PCR-RFLPs using the cloned allele having no relevant mutation as a template; +/-, PCR-RFLPs using the mixed templates of the cloned wild-type allele and the cloned mutant allele.

Assays of (1,2)Fucosyltransferase and Luciferase Activities

Luciferase activity was measured as described previously(3) . For measurement of alpha

(1,2)fucosyltransferase activity, phenyl- beta

-D-galactose (Phe-Gal) was used as an acceptor substrate. Assays were performed in a total volume of 100 µl containing 50 mM Tris-HCl (pH 7.2), 0.3% Triton X-100, 10 mM NaN (3)

, 10 mM MnCl (2)

, 5 mM ATP, 25 µM GDP-Fuc, 0.2 µM [

H]GDP-Fuc, 30 mM Phe-Gal as an acceptor, and 10 µg of protein of soluble fraction of homogenate of the transfected COS-1 cells as enzyme source. After incubation at 37 °C for 1 h, the reaction mixture supernatant was diluted with 5 ml of water and applied to Sep-Pak plus C (18)

(Waters, Millford, MA) for binding of Phe-Gal. [

H]Fuc-incorporated Phe-Gal was eluted from the Sep-Pak plus C (18)

with 5 ml of methanol, and the radioactivity in the eluate was counted in a liquid scintillation counter.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Methods for Detection of Mutations in Sec2 Gene and Le Gene

One µl of the first PCR product containing the full-length gene was used as the template in the second PCRs in order to detect point mutations. The second PCRs were carried out under the same conditions as in the first PCR except for the annealing temperatures indicated in Table 1.

For detection of the G428A nonsense mutation, the tk3 primer was designed as a mismatched primer to create an XbaI site, TCTAGA, in the PCR product from the mutant allele having the G428A. In case of the product having the G428A mutation, the second PCR product amplified by the primer set of tk3 and tk4 was separated into two fragments, 84 and 23 bp, by XbaI digestion.

For detection of the C357T and A385T mutations in the Sec2 gene that had been discovered by complete sequencing of the Sec2 gene of YoN (a Le(a+b-) nonsecretor phenotype), the second PCRs were performed by the primer sets, tk5 and tk6, and tk7 and tk8, respectively. The tk5 primer was designed as a primer mismatched at two positions of the Sec2 gene sequence by changing C to A at position 352 and A to T at position 354. These artificially changed bases are indicated by boldface letters in the sequence of primers in Table 1. The tk5 primer created an AseI site, ATTAAT, in the second PCR product from the mutant allele having C357T. The 98-bp PCR product was cleaved into two fragments, 70 and 28 bp, by AseI digestion. For detection of the A385T, the tk7 primer was designed as a primer mismatched at two positions, 382 and 383, to create an AluI site, AGCT, in the second PCR product from the mutant allele having the A385T mutation. The PCR product with this mutation was cleaved into two fragments, 51 and 29 bp, by AluI digestion.

For detection of the three missense mutations, T59G, G508A, and T1067A, in Le genes, the same primer sets as reported in the previous study (3, 21) were used except for the detection of G508A where a new primer set, sn8 and sn9, was designed in this study to improve the PCR condition. The methods for detection of the three mutations in Le genes using these primer sets are described in detail in our previous studies(3, 21) .

The second PCR products digested with the enzymes shown in Table 1were electrophoresed in a 4% agarose (NuSieve GTG agarose, FMC Corp. BioProducts, Rockland, ME), and the separated bands were observed after ethidium bromide staining.

RESULTS

The G428A Nonsense Mutation (Trp to Terminal Codon) Was Not Found in the Japanese Population

The nonsense mutation, G428A (Trp

to terminal codon), that terminates the Sec2 gene translation, in the Sec2 gene of nonsecretors has been reported by Kelly et al.(14) . We first determined how widely the G428 mutation is distributed in the Japanese population. We designed a primer set, tk3 and tk4, for detection of this nonsense mutation by PCR-RFLP. The tk3 primer was designed to create an XbaI site in the PCR product from the allele having the G428A mutation. The PCR product having this mutation was cleaved into two fragments, 84 and 23 bp, by XbaI digestion. As seen in the PCR-RFLP experiment in Fig. 2, no Japanese nonsecretors were found to have this mutation, except for EA, who showed a heterozygous pattern for this mutation with three bands of 107, 84, and 23 bp. EA was later identified as a Filipino living in Tokyo. None of 45 nonsecretor Japanese individuals examined in this study had the G428A mutation.

Figure 2: Absence of G428A mutation in Japanese nonsecretors. From left to right, 10 Japanese nonsecretors showing no G428A mutation; EA, a Filipino, showing a heterozygous pattern for G428A mutation; M, molecular size markers (bp).

Discovery of C357T and A385T Mutations by Sequencing of the Full-length Sec2 Gene from a Le(a+b-) Nonsecretor Individual

The Sec2 gene of a Le(a+b-) nonsecretor individual, YoN in the Family 2 in Fig. 4, was amplified by PCR using the primer set, tk1 and tk2. The amplified fragment was then subcloned in the pBS vector for sequencing. Eight clones inserted into the vector were fully sequenced and the results revealed that all eight clones possessed the two point mutations, C357T and A385T. Although two other mutations were found in one clone in addition to the C357T and the A385T, they were not shared with the other clones. These were considered nongenuine mutations due to misincorporation by Taq DNA polymerase. As all eight clones from YoN shared the C357T and A385T mutations, YoN was considered to be homozygous for these mutations.

We also sequenced the full-length Sec2 gene of SN, in Family 1 in Fig. 4, who has a Le(a-b-) secretor phenotype. The results revealed that three of eight clones shared the C357T mutation without any other mutation, but the other five clones had no mutation at all.

We designated the three alleles in Sec2 gene, the first having no mutation, the second having C357T alone, and the third having both C357T and A385T, as Se1, Se2, and sej, respectively. In Fig. 3, we show the schematic primary structure of the enzyme and these three alleles together with the positions of primer sets for detection of the mutations by PCR-RFLP methods to be described in the following section.

Figure 3: Schematic diagrams of PCRs employed for Sec2 genotyping by PCR-RFLP, three Sec2 alleles having point mutations and PCR-RFLP methods for detection of C357T and A385T. a, the positions of four primer sets for PCRs. The tk1 and tk2 set was employed for full-length Sec2 gene amplification. The primer sets, tk3 and tk4, tk5 and tk6, and tk7 and tk8, were employed for detecting G428A, C357T, and A385T, respectively. Schematic primary protein structure of the Sec2-directed enzyme: C, cytoplasmic tail; TM, transmembrane domain; CR, catalytic region. Three alleles found in the Japanese population are Se1, Se2, and sej. The Se1 allele has the same sequence as originally defined (10) without any mutations. The Se2 allele has a silent mutation, C357T, and the sej allele has C357T and a missense mutation, A385T (Ile to Phe). b, schematic diagram of PCR-RFLP methods for detection of point mutations, C357T () and A385T ( circle ). For detection of the C357T mutation, the 98-bp PCR products from Se2 and sej alleles using the primer set of tk5 and tk6 are cleaved into two fragments by AseI digestion, whereas the product from the Se1 allele is not cleaved by AseI digestion. For detection of A385T mutation, the 80-bp product from the sej allele using the primer set of tk7 and tk8 is cleaved into two fragments by AluI digestion, whereas the products from Se1 and Se2 alleles are not cleaved.

Detection of the C357T and A385T Mutations in the Sec2 Gene by PCR-RFLP Methods

We first performed a family study to confirm that the Sec2 alleles were inherited according to genetic inheritance rules. 21 individuals of four pedigrees whose Le genotypes had been determined in a previous study (3) were analyzed for their Sec2 gene. Before PCR-RFLP experiments for detection of Sec2 gene mutations, we first determined secretor status of the six Le(a-b-) individuals by hemagglutination inhibition testing: SN in Family 1, HN in Family 2, AK and TK in Family 3, and HT and YT in Family 4. HN in Family 2 was the only nonsecretor among the six Le(a-b-) individuals. We also determined the erythrocyte Lewis phenotypes of all members by hemagglutination. The secretor status and the erythrocyte phenotypes determined are indicated adjacent to their initials in Fig. 4.

The primer sets mismatched to the original sequence of the Sec2 gene for detection of the two mutations, C357T and A385T, were designed for PCR-RFLP as described under ``Materials and Methods.'' As seen in the electrophoresed patterns of PCR-RFLP in Fig. 4, the alleles having the C357T mutation, Se2 and sej, were separated into two fragments, 70 and 28 bp, by AseI digestion, and the allele having the A385T mutation, sej, was separated into two fragments, 51 and 29 bp by AluI digestion. Although the faint residual bands, 98 bp by AseI digestion and 80 bp by AluI digestion, were sometimes observed depending on digestion conditions, we could clearly determine their zygosity by this experiment. As seen in PCR-RFLP of Family 1 in Fig. 4, TO, HN, and AN possessed the C357T mutation homozygously, whereas MO, SN, and TN were heterozygotes for this mutation. With regard to the A385T mutation, PCR-RFLP revealed that HN was a homozygote having this mutation in both alleles, and TO, TN, and AN were heterozygotes for this mutation. MO and SN did not have the A385T mutation in either allele.

All of the members, except for HN, in Family 1 were secretors with Le(a-b+) or Le(a-b-) phenotype. PCR-RFLP results revealed that all, except for HN, possessed at least one Se1 or Se2 allele. Only HN, a Le(a+b-) nonsecretor, in Family 1 was determined to be homozygous with sej/sej. The Sec2 genotype determined by PCR-RFLP is shown with initials of each individual in Fig. 4a. All the members of Family 2, except for HN who has a Le(a-b-) nonsecretor phenotype, were typed as Le(a+b-) nonsecretors. The results of PCR-RFLP on Family 2 as seen in the electrophoresed patterns clearly demonstrated that all the members of Family 2, including HN, were homozygous for the two mutations, C357T and A385T. Thus, all including YoN whose Sec2 gene was sequenced in the previous section, were determined to be homozygous for sej/sej. The PCR-RFLP methods that detected the two mutations revealed that the alleles were inherited according to genetic rules in all four families examined in this study. In conclusion, all individuals with the Le(a-b+) or Le(a-b-) secretor phenotypes had at least one of the Se1 or Se2 alleles, whereas all Le(a+b-) and Le(a-b-) nonsecretor individuals were homozygous for sej/sej.

Inactivation of Sec2 Gene-directed (1,2)Fucosyltransferase by the Missense Mutation, A385T, which results in Ile to Phe

Each of the three alleles, Se1, Se2, and sej, was subcloned into a pCDM8 vector for transient expression in COS-1 cells. The recombined constructs were transiently transfected to COS-1 cells, and lysates of the transfected cells were subjected to alpha

(1,2)fucosyltransferase activity assay using Phe-Gal as an acceptor. The luciferase expression vector, driven by the beta

-actin promotor, was co-transfected to COS-1 cells as an indicator of transfection efficiency, and the alpha

(1,2)fucosyltransferase activity in each lysate was normalized by transfection efficiency.

As shown in Fig. 5, lysates of the COS-1 cells transfected with the Se1 allele and those transfected with the Se2 allele showed full alpha (1,2)fucosyltransferase activity, indicating that the Se1 and Se2 alleles encode a wild-type enzyme, while the sej-transfected COS-1 cell lysate showed quite low activity, i.e. about 2-3% of the activity of Se1- or Se2-transfected cell lysate. These results demonstrated that a single amino acid substitution, Ile to Phe, in the enzyme directed by the sej allele causes almost complete inactivation of the enzyme. Individuals with the sej/sej genotype are, therefore, mutants who lack alpha (1,2)fucosyltransferase activity directed by this gene.

Figure 5: alpha (1,2)Fucosyltransferase activity directed by each of three alleles, Se1, Se2, and sej. From left to right, pCDM8, activity of lysate of COS-1 cells mock-transfected with pCDM8 vector; pCDM8/Se1, activity of Se1-transfected COS-1 cell lysate; pCDM8/Se2, activity of Se2-transfected COS-1 cell lysate; pCDM8/sej, activity of sej-transfected COS-1 cell lysate. The activity in each lysate was normalized by transfection efficiency which was determined by expression of co-transfected luciferase expression vector. Data represent means ± S.D. of three independent assays.

Complete Consistency between Sec2, Le Genotypes, and Lewis Phenotypes, and Frequency of Occurrence of Se1, Se2, and sej Alleles in the Japanese Population Determined by Random Sampling Study

In a random sampling study, we determined erythrocyte Lewis phenotype, secretor status, and genotypes of Sec2 gene and Le gene in 205 Japanese volunteers in order to determine how widely the sej allele is distributed in the Japanese population and to look for mutations other than the A385T mutation in the Sec2 gene of Japanese nonsecretors. Among 205 individuals, 50 individuals whose Le genotypes had already been determined in the previous study (2) were included, and Le genotypes of the remaining 155 individuals were determined in this study.

All of the results in this random sampling study are summarized in Table 2. The number of individuals with erythrocyte phenotype Le(a+b-), Le(a-b+) and Le(a-b-) were 31, 143, and 31, respectively among the 205 individuals randomly sampled. 31 Le(a-b-) individuals were divided into secretors (27 individuals) and nonsecretors (4 individuals) by the presence or absence of ABH antigens in saliva. The frequency of the four phenotypes, Le(a+b-), Le(a-b+), Le(a-b-) secretor and Le(a-b-) nonsecretor, were 15, 70, 13, and 2%, respectively. All 31 individuals with Le(a+b-) phenotype were determined to be homozygous for the sej/sej genotype by PCR-RFLP. All 143 individuals with Le(a-b+) phenotype were determined to have a genotype possessing at least one of the Se1 or Se2 alleles. 27 secretors among 31 Le(a-b-) individuals were also determined to have the Sec2 genotype possessing at least one of the Se1 or Se2 alleles, whereas all 4 non-secretors with Le(a-b-) phenotype were determined to be sej/sej homozygous.

The results showed that 31 individuals with the Le(a+b-) phenotype and 4 individuals with the Le(a-b-) nonsecretor phenotype among 205 randomly sampled individuals possessed the sej/sej genotype. This indicates a rather wide distribution of the sej allele having C357T and A385T mutations in the Sec2 gene.

With regard to the Le genotype determined by PCR-RFLP, 174 individuals with the Le(a+b-) or Le(a-b+) phenotype were determined to possess at least one Le allele that encodes an active Le enzyme, whereas 31 individuals with the Le(a-b-) phenotype were confirmed to be homozygous for the inactive mutant Le genes, i.e. le1 and le2. The results of Le genotyping on 205 individuals were completely consistent with previous results (3) in which we demonstrated that the Le alleles of the Japanese population could be classified into three alleles, Le, le1, and le2, without exception.

Even though an extensive molecular genetic analysis of the Sec2 gene was performed on the 45 nonsecretor individuals with the Le(a+b-) phenotype or the Le(a-b-) nonsecretor phenotype, including 10 individuals in the family study and 35 individuals in the random sampling study, we could not find any mutant allele other than sej.

The results described in this section demonstrated that combined genotyping of the Le and Sec2 genes can clearly distinguish the three Lewis blood group phenotypes, Le(a+b-), Le(a-b+), and Le(a-b-), with 100% accuracy. The results of this random sampling study (Table 2) indicate that the frequencies of occurrence of Le, le1, le2, Se1, Se2, and sej among 410 alleles examined in the Japanese population are 66, 27, 7, 15, 46, and 39%, respectively (Table 3). The frequencies of occurrence of Le, le1 and le2 were slightly different from the figures in the previous study (3) due to the increase in number of alleles examined in the present study.

DISCUSSION

In a previous study(3) , we demonstrated that the Le gene (Fuc-TIII gene) is solely responsible for the expression of the Lewis blood group antigens, Le and Le, of erythrocytes. The Le alleles in a Japanese sample were found to be divided in the three kinds, Le, le1, and le2. The latter two alleles possess different point mutations and encode nonfunctional Lewis enzymes which are inactivated by each single missense mutation in the catalytic domain.

As proposed schematically in Fig. 1, the Le antigen on erythrocytes is a cooperative product of two fucosyltransferases, an alpha (1,2)fucosyltransferase, which catalyses the transfer of fucose to galactose with an alpha (1,2)-linkage and synthesizes an H type-1 structure (O-type antigen), and the Lewis enzyme with alpha (1,4)fucosyltransferase activity. Lewis antigens, Le and Le, are known to be distributed not only on erythrocytes but also in plasma, saliva, and other secretions(9) . It has been reported that the Lewis antigens of erythrocytes are not synthesized by the erythrocytes themselves but are acquired from plasma by adsorption(8) . The major cells that synthesize Lewis antigens are considered to be digestive organ epithelial cells, where the two fucosyltransferases should be localized. We recently established a monoclonal antibody that reacts specifically with the human Lewis enzyme. An immunohistochemical study using this antibody clearly demonstrated that the Lewis enzyme is localized in digestive organ epithelial cells, but not in the cells of myeloid lineage (results will be published elsewhere).

According to the pathways proposed in Fig. 1which are presumed to function in digestive organ epithelial cells, nonsecretor individuals who lack Se enzyme activity would not synthesize the H type-1 structure that is also a precursor structure of A, B, and Le antigens, resulting in lack of secretion of ABH antigens into saliva or no expression of Le antigen in plasma or of erythrocytes. If this is the case, Le(a+b-) and Le(a-b-) nonsecretor individuals are likely to be mutants whose Se gene is probably inactivated by point mutation.

It was proven by Kelly et al.(14) that the Sec2 gene is the the Se gene based on the finding of the G428A nonsense mutation in Sec2 gene in all six nonsecretors among 52 individuals randomly sampled. This was confirmed in this study by discovery of the A385T (Ile to Phe) missense mutation in the Sec2 gene of Japanese nonsecretors. This mutation is rather widely distributed in the Japanese population, and this amino acid substitution inactivated enzyme activity almost completely. It remains to be elucidated whether the amino acid substitution is located at the active sites of the enzyme, i.e. the acceptor- or the donor-binding site. There is also the possibility that a single amino acid substitution causes alteration of the tertiary structure of the protein, resulting in marked susceptibility to proteolytic degradation in the cells.

All Le(a+b-) and Le(a-b-) nonsecretors examined were determined to be homozygous for sej/sej, whereas all Le(a-b+) and Le(a-b-) secretors possessed active genes, Se1 or Se2, at least. Complete consistency between phenotypes and genotypes in this extensive analysis of more than 200 individuals may be enough to assert that the Sec2 gene is the Se gene. It was strongly suggested that the Se enzyme in the pathways proposed in Fig. 1functions in digestive organ epithelial cells in order to determine not only the secretion of ABH antigens into saliva but also the expression of Le on erythrocytes.

The number of Lewis antigens on erythrocytes are far fewer than the ABH antigens. This is why we sometimes observe very weak hemagglutination by anti-Lewis antibodies, especially in certain conditions such as pregnancy and intestinal cancer. The typing of secretor status is also often ambiguous due to the low sensitivity of the assay method. The combination genotyping of Le and Se genes described in this study can be applied to ambiguous samples as a definitive typing method. Because of the higher frequency of occurrence of the Se2 allele compared with the Se1 allele, and because the C357T mutation found in the sej allele exists in the Se2 allele, we assume that the Se2 allele is the antecedent wild-type allele that gave rise to the Se1 and sej alleles afterward.

It was a quite interesting observation that all the Japanese nonsecretors examined had sej/sej homozygosity, making up more than 15% of the population. Similarly, a previous study by Kelly et al.(14) demonstrated that the Se enzyme-inactivating G428A mutation, which differs from the A385T mutation we found, is also rather widely distributed in another ethnic group, although they did not mention what ethnic group they analyzed. We never found the G428A mutation in Japanese individuals. Thus, these results suggest that Se gene inactivation must have occurred after the ethnic groups diverged.

An interesting question is why a sole inactivated allele spread in each ethnic group during a relatively short period. Kelly et al.(14) conjectured that the inactivated Se allele might confer some specific selective advantage upon the mutant individuals. Ethnic-specific alleles are not found in ABO genes, and these allelic mutations are conserved in all ethnic groups. The single inactivated sej allele in the Japanese population, even though it arose more recently than did the ABO alleles, already occupied a frequency of 39% among Se alleles. This situation might be similar to that of Le gene inactivation. This extensive study on 205 Japanese individuals (410 alleles) determined the frequencies of occurrence of two inactivated le alleles, le1 and le2, as 27 and 7%, respectively. Although the le2 allele has been found in Indonesian individuals(4) , the le1 allele having the G508A mutation, which occupies a much higher frequency than le2 in Japanese people, has not been found in other ethnic groups yet. This suggests that the le1 allele might be specific to the Japanese.

If the absence or the presence of Lewis antigens, now proven to be the product of Le and Se genes, confers some selective pressure upon the human species, it is conceivable that antigen expression in digestive organs is biologically much more important than the expression in erythrocytes. Recent investigation on Helicobacter pylori, a causative agent of gastric disorders (22, 23) , suggested that it resides in the stomach by attaching to gastric epithelium via an H type-1 structure and a Le antigen expressed thereon. Transgenic mice with the human Le gene have been recently developed(23) , and it was demonstrated that H. pylori obviously attached to gastric epithelial cells in the transgenic mice but not in their normal littermates. This implies that le/le individuals might have an advantage in avoiding H. pylori infection.

Another possible biological significance of Lewis antigens in the digestive system concerns tumor-metastasizing capacity. The level of expression of sialylated Lewis antigens, sialyl Lewis a and sialyl Lewis x, on tumor cells is thought to correlate with metastasizing capacity(24, 25, 26) , since these antigens have been shown to be the ligands to E-selectins(27) . Some statistical results (26) have indicated that the more sialylated Lewis antigens tumor cells have, the more malignant the cells are. If the Le enzyme and the Se enzyme are also involved in the pathway of sialylated Lewis antigen synthesis in intestinal cancer cells, the genotypes of the Le and Se genes of cancer patients would affect their prognosis. Further study might analyze statistical correlations of the Le and Se genotypes with H. pylori infection and with the metastasizing capacity of cancer cells.

FOOTNOTES

*: This is Paper II in the series, ``Molecular Genetic Analysis of the Human Lewis Histo-blood Group System.'' (3) is Paper I in this series. This work was supported in part by Special Coordination Funds from the Science and Technology Agency of the Japanese Government and by a Grant-in-Aid for Scientific Research on Priority Areas No. 05274103 from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§: To whom all correspondence should be addressed. Tel.: 81-426-91-9466 or 9470; Fax: 81-426-91-9315; hisashi{at}t.soka.ac.jp.
(): The abbreviations used are: Le, Lewis a; Le, Lewis b; Fuc-TIII or Le, (1,3/1,4)fucosyltransferase gene; le, Lewis negative individual's Fuc-TIII gene; Se, secretor gene; Se enzyme, secretor enzyme; Sec1, secretor candidate 1 gene; Sec2, secretor candidate 2 gene; Fuc-T, -L-fucosyltransferase; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; Fuc, L-fucose; GlcNAc, N-acetyl-D-glucosamine; Phe-Gal, phenyl--D-galactose; pBS, pBluescript SK(-); bp, base pair(s); kb, kilobase pair(s).

ACKNOWLEDGEMENTS

We thank Drs. K. Saito and N. Saito for critical reading of the manuscript and valuable comments.

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