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J. Biol. Chem., Vol. 275, Issue 48, 37752-37756, December 1, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/48/37752    most recent C000625200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Furukawa, K. Articles by Furukawa, K. Search for Related Content PubMed PubMed Citation Articles by Furukawa, K. Articles by Furukawa, K. Social Bookmarking                      What's this?

Molecular Basis for the p Phenotype

IDENTIFICATION OF DISTINCT AND MULTIPLE MUTATIONS IN THE 1,4-GALACTOSYLTRANSFERASE GENE IN SWEDISH AND JAPANESE INDIVIDUALS^*

Keiko Furukawa, Koichi Iwamura, Makoto Uchikawa^§, Birgitta Nilsson Sojka^¶, Joelle Wiels, Tetsuya Okajima, Takeshi Urano, and Koichi Furukawa^**

From the  Department of Biochemistry II, Nagoya University School of Medicine, Tsurumai, Nagoya 466-0065, Japan, ^§ Central Blood Center, Japanese Red Cross, Hiroo 4-1-31, Shibuya-Ku, Tokyo 150-0012, Japan, ^¶ Department of Transfusion Medicine, Umeå University Hospital, S-901 85 Umeå, Sweden, and  CNRS UMR 1598, Institut Gustave Roussy, Villejuif Cedex, 94805 France

Received for publication, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p phenotype individuals lack both P^k (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (1,4-galactosyltransferase; 1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated 1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of 1,4Gal-T revealed that three residues, Met¹⁸³, Gly¹⁸⁷, and Pro²⁵¹, at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of 1,4Gal-T.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The P blood group system (1) has two common phenotypes, i.e. P₁ and P₂ (with P) and three rare phenotypes (p, P₁^k, and P₂^k). p individuals are very rare, and the defective structure was determined to be a glycolipid globotriaosylceramide (Gb3¹), consisting of Gal1,4Gal1,4Glc-ceramide (2, 3). Gb3 is converted to P (globoside; Gb4), a major species of neutral glycolipids in human erythrocytes. Therefore, erythrocytes deficient in the synthesis of Gb3 lack both Gb3 and Gb4 (3), whereas erythrocytes lacking Gb4 synthase show absence of Gb4 (4) and accumulation of Gb3 (P^k phenotype). P1 antigen, in contrast, belongs to the neolacto-series glycolipids and has been identified as 1,4Gal-neolactotetraosylceramide (5). Individuals of the p phenotype lack P1 antigen, in addition to Gb3 and P (Gb4), presumably as a result of deficiency in the enzyme 1,4-galactosyltransferase (1,4Gal-T) responsible for the synthesis of Gb3 from lactosylceramide (3, 4) and also for reasons that are not fully understood.

Gb3 was detected not only in erythrocytes but in various epithelial cells and hematopoietic cells including immature B cells in the germinal center. Although Gb3 was defined as Burkitt lymphoma-associated antigen (6), it is now called Gb3/CD77 and is considered to be a differentiation antigen of B cell lineage (7, 8). Gb3/CD77 is also known as a receptor molecule for Shiga-like toxins from Escherichia coli O157, causing hemolytic uremic syndrome (9). The presence/absence of Gb3/CD77 may, therefore, have critical implications in long-term survival of mankind.

Recently, we have isolated cDNAs for 1,4Gal-T (9) and showed that the introduction of this cDNA could reconstitute the functional receptor for verotoxins leading to apoptosis (10). In the present study, we have analyzed the molecular basis of P^k/p antigen phenotypes by the investigation of abnormalities in the 1,4Gal-T gene in p phenotype individuals from Japan and Sweden. Although Steffensen et al. (11) reported that Swedish p individuals have a missense mutation at M183K, we show that Japanese p individuals have different and multiple missense or nonsense point mutations in the coding region. We also defined another missense mutation in a Swedish p individual. These mutations seem to have been generated after the divergence of the two ethnic groups. The importance of the defined mutation sites in the enzyme function is discussed based on the aligned genes from the BLAST data base.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Analysis of the 1,4Gal-T Gene-- The 1,4Gal-T gene was amplified by polymerase chain reaction (PCR) using PfuTurbo^TM DNA polymerase (Stratagene) from genomic DNAs extracted from peripheral mononuclear cells. The four sets of primers used are as follows: 1) 5'primer, 5'-TGGCGGGTGCTCATCTCTTG-3' (in the first intron) and 3'primer, 5'-TTGGTCCGG TCTGAAGTCTC-3'; 2) 5'primer, 5'-CCACTCCAGGCAACATCTTC-3' and 3'primer, 5'-AGGTAGATGCCGCCGAACTT-3'; 3) 5'primer, 5'-GACGCCTCCAGGATCGCACT-3' and 3'primer, 5'-CTCCTCCGGGTTGATGTCCT-3'; and 4) 5'primer, 5'-GCAGGACTGGAAGAA GTAC-3' and 3'primer, 5'-GCCCC ATCAGGAGCAGGTTG-3'. Conditions of PCR consisted of denaturing at 94 °C for 1 min, annealing at 55 °C for 1 min, and then extension at 74 °C for 1 min. The amplified products were directly sequenced by dideoxy termination method using an ABI PRISM® 310 genetic analyzer (PE Biosystems).

Construction of Expression Vectors-- The 1,4Gal-T expression vectors for individual mutants were constructed by PCR of the coding region using a 5'primer containing an EcoRI site, 5'-ACCATGTCCAAGCCCCCCGACCTC-3', and a 3'primer containing an XhoI site, 5'-CCCCTCACAAGTACATTTTCATG-3', and by subcloning into EcoRI and XhoI sites of pcDNA3.1 (+) vector (Invitrogen). Myc-tagged expression vectors were also constructed with PCR products of the coding region to form a fusion enzyme with the myc epitope at the N' terminus by subcloning into BamHI and XhoI sites of myc-tagged pcDNA3 vector. The primers for the construction of the myc fusion protein were as follows: 5'primer containing a BamHI site, 5'-TCCAAGCCCCCCGACCTC and 3'primer containing an XhoI site, 5'-CCCCTCACAAGTA CATTTTCATG-3'. These constructs were served to sequence analysis to confirm the correct junctions and the absence of artificial mutations.

Cell Culture-- A mouse fibroblast L cell line was kindly provided by Dr. A. P. Albino (Sloan-Kettring Cancer Center, New York, NY) and cultured in Dulbecco's modified Eagle's minimum essential medium containing 7.5% fetal bovine serum.

Transfection-- Constructs for the gene expression were introduced into cells by DEAE-dextran method as described previously (10). After culture for 48 h, cells were harvested to be used for further analyses.

Flow Cytometry-- Expression of Gb3/CD77 antigen was analyzed by flow cytometry (Becton Dickinson) as described (10).

Enzyme Assay-- 1,4Gal-T activity was measured as described previously (10). Briefly, membrane fractions were prepared with a nitrogen cavitation apparatus and centrifugation of the extracts at 105,000 × g for 1 h. The enzyme products were separated by Sep-Pak C₁₈ (Waters) and then applied to a thin layer chromatograph. Autofluorography was performed using a BioImaging Analyzer BAS2000 (Fuji Film, Tokyo, Japan).

Western Immunoblotting-- The transfectant cells with myc-tagged expression vectors were served for Western immunoblotting after lysis with lysis buffer consisting of 20 mM Tris-HCl (pH7.4), 1.0% Nonidet P-40, 150 mM NaCl, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Lysates containing 50 µg each of protein were separated by SDS polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride membrane. The membranes were incubated with anti-myc monoclonal antibody, and antibody binding was detected with an Vectastain ABC kit (Vector Laboratories, Inc.) according to the manufacturer's instruction.

Intracellular Localization-- Myc-tagged expression vectors were transfected into L cells and then stained by anti-myc monoclonal antibody and fluorescein isothiocyanate-anti-mouse IgG after fixation. The staining pattern was observed by confocal microscopy (MicroRadiance; Bio-Rad).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutations in the 1,4Gal-T Gene in p Individuals-- 1,4Gal-T is the enzyme that synthesizes Gb3/CD77 (P^k antigen) from lactosylceramide (Fig. 1A). BLAST analysis of the genomic structure of 1,4Gal-T revealed that the 1,4Gal-T gene consisted of at least 2 exons, and the entire coding region existed in exon 2 (Fig. 1B). Using primers designed as indicated in Fig. 1B, the entire coding region of 1,4Gal-T was amplified with the genomic DNA as a template and was directly sequenced. Sequence analyses of six p samples consisting of four Japanese (J1, J2, J3, and J4) and two Swedish (S1 and S2) individuals were performed. Four kinds of missense mutation nucleotides (nt.), nt.109AG (M37V), nt.548TA (M183K), nt.560GA (G187D), and nt.752CT (P251L), and one nonsense mutation nt.783GA (W261stop) were detected. Three of four Japanese individuals contained the P251L mutation, and one of them contained W261stop. Both of them were different from the mutations M183K and G187D found in two Swedish donors (Fig. 1C). Two silent mutations, nt.903GC (P301P) and nt.987GA (T329T), were also found (Fig. 1D). In contrast, mutations could not be detected in two P individuals (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Sequencing strategy and identification of 1,4Gal-T gene mutations. A, synthetic reaction of Gb3/Pk. B, genomic structure of the 1,4Gal-T gene and four sets of primers used for PCR. C, mutations found by PCR of genomic DNAs. PCR and sequencing were performed as described under "Experimental Procedures." The sequences of three missense mutations and one nonsense mutation were shown. J1-J4 are of p type Japanese individuals, and S1 and S2 are of p type Swedish individuals. D, summary of defined mutations in the 1,4Gal-T gene. Mutation P251L was detected in J1-J3, mutation W261stop was in J4, mutation G187D was in S1, and mutation M183K was in S2.

Function of the Enzymes from Mutant cDNAs-- To confirm whether the mutations G187D (S1 type), P251L (J1 type), W261stop (J4 type), and M183K (S2 type) were responsible for the defects of 1,4Gal-T activity, we examined the function of the expressed mutant cDNAs, as well as the wild type. The expression vectors simply containing the full-length of the coding region or myc-tagged expression vectors for each mutant, as well as wild type, were constructed and transfected into L cells. The M37V mutation is known not to affect the enzyme activity (11). In the flow cytometric analysis of L cell transfectants, the WT gene caused definite Gb3 expression. In contrast, transfectant cells with 1,4Gal-T/J1 (J1 type) and 1,4Gal-T/S2 did not express Gb3 at all, and those with 1,4Gal-T/J4 and 1,4Gal-T/S1 expressed minimal levels of Gb3 (Fig. 2A). Myc-tagged expression vectors and non-tagged vectors showed identical results (data not shown). The expression levels of the transfected gene products were almost equivalent between mutants and the wild type as detected by an anti-myc antibody (Fig. 2B). The 42-kDa bands seemed to be myc-tagged 1,4Gal-Ts, and a 32-kDa band seemed to be a truncated form of 1,4Gal-T (J4) because of the stop codon at the residue Trp²⁶¹.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Cell surface expression of Gb3/Pk antigen in L cells after transient transfection of 1,4Gal-Ts. A, Myc-tagged expression vectors (1,4Gal-T/WT, 1,4Gal-T/J1, 1,4Gal-T/J4, 1,4Gal-T/S1, and 1,4Gal-T/S2, as well as pcDNA3.1) were transfected into L cells, and Gb3/Pk expression was tested after 48 h using anti-Gb3 monoclonal antibody 38.13 and rabbit anti-rat IgM-fluorescein isothiocyanate (empty histgrams) or with the second antibody alone (gray histograms). B, Western blot analysis of myc-tagged 1,4Gal-Ts expressed in L cells. The transfected cells were lysed, and total cell lysate (50 µg protein) was separated and immunoblotted with mouse anti-myc-tagged antibody. Transfected plasmids were as follows: 1, pcDNA3 and 2-6, myc tag/pcDNA containing 1,4Gal-T/WT (lane 2); 1,4Gal-T/J1 (lane 3); 1,4Gal-T/J4 (lane 4); 1,4Gal-T/S1 (lane 5); and 1,4Gal-T/S2 (lane 6).

The 1,4Gal-T activities in the membrane fractions of the transfectant cells were examined using lactosylceramide or galactosylceramide as acceptors (10). All mutants exhibited loss of enzyme activity (Fig. 3A). Because weak Gb3 expression was detected in W261stop (J4 type) and G187D (S1 type) by fluorescence-activated cell sorter analysis we tried long exposure, resulting in the appearance of faint Gb3 bands in these mutants (Fig. 3B). The enzyme activities toward lactosylceramide were 8.46, 0.007, and 0.03 nmol/mg of protein/h for wild type, J4, and S1, respectively (Fig. 3C). The enzyme activities toward galactosylceramide as an acceptor were quite similar to those for lactosylceramide (Fig. 3, D and E), i.e. two of them showed no activity, and the other two showed minimal levels of activity only by long-exposed autofluorography (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   1,4Gal-T activity in the membrane from transfectant cells with 1,4Gal-T expression vectors. A, TLC of enzyme products using lactosylceramide (LacCer) as an acceptor. Membrane fractions were prepared from L cells transfected with the following: 1, pcDNA3.1 and 2-6, myc tag/pcDNA containing 1,4Gal-T/WT (lane 2); 1,4Gal-T/J1 (lane 3); 1,4Gal-T/J4 (lane 4); 1,4Gal-T/S1 (lane 5); and 1,4Gal-T/S2 (lane 6), and 50 µg of protein was used as a source of enzyme. Enzyme activity was determined as described under "Experimental Procedures." The TLC plate was exposed for 15 h (A) or for 5 days (B). C, enzyme activities calculated from the intensity of bands in A and B using Bio-image analizer (BAS) were presented as nmol/mg/h. D, TLC of enzyme products using galactosylceramide (GalCer) as an acceptor. The TLC plate was exposed for 15 h. E, enzyme activities were measured from the bands in D and a long-exposed plate (data not shown) and were presented as nmol/mg/h.

Multiple Sequence Alignment of 1,4Gal-Ts/1,4GlcNAc-Ts-- Based on BLAST analysis of data bases using the coding region of 1,4Gal-T, homologous sequences from human, other primates, rat, Drosophila melanogaster and Arabidopsis thaliana were identified and aligned. These genes seemed to belong to the 1,4Gal-T/1,4GlcNAc-T family. The residues Gly¹⁸⁷ and Pro²⁵¹, which were mutated in S1 and J1, J2, and J3 p individuals, respectively, were completely conserved among all species examined, indicating the importance of these residues for enzyme function. The residue Met¹⁸³ was also highly conserved.

Intracellular Localization-- To investigate the mechanisms responsible for the lack of enzyme activity in the mutant clones, intracellular localization of the expressed enzymes was compared. All mutants showed a similar staining pattern to the wild type (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we have found 3 novel mutations and one previously reported mutation (10) in the 1,4Gal-T gene. Among 4 mutations found in the coding region, W261stop and G187D showed minimal levels of enzyme activity, i.e. only 0.08-0.36% of the wild type. Consequently, they seemed not to be able to synthesize Gb3 in vivo, resulting in p phenotype. Our results indicated that mutations in 1,4Gal-T had occurred at multiple and distinct sites, at least in Japanese and Swedish individuals. The results described here indicated that gene mutations inducing p phenotype had been generated after the divergence of mankind into different ethnic groups, as had mutations of the 1,2-fucosyltransferase II (Se/se) gene (12, 13). The cluster of the gene mutation found in Vasterbotten County in Sweden may be one of the representative mutations found in Caucasian individuals (11). This situation is quite contrastive to the ABO blood group, in which the polymorphism based on commonly shared gene mutations in the allelic locus (14) is found all over the world.

The most controversial point in the P/p blood group antigen system has been whether P1 (15) and P^k are synthesized by a single enzyme or by distinct two enzymes (5). The ratio of P1⁺/P1 is 8:2 in Caucasian individuals (1, 16) and 3:7 in Japanese individuals (17), and incidence of p is very rare in all races, indicating that P1 synthase and P^k synthase are not identical. Furthermore, no significant mutation in the Gb3/CD77 synthase gene corresponding to P1⁺/P1 phenotypes could be found in the studies of our own (data not shown) and other groups (11), confirming that the Gb3/CD77 synthase gene is not responsible for the synthesis of P1. Actually, no incorporation of [¹⁴C]Gal onto paragloboside could be detected in the substrate specificity analysis of the cloned cDNA (10). The remaining mystery is that the p phenotype is always associated with a lack of P1 expression. One explanation is that Gb3/CD77 synthase acts exclusively toward lactosylceramide, and P1 synthase mainly utilizes neolacto-series acceptors but also, to some degree, lactosylceramide. In this case, p phenotype can be generated only when inactivation of both of these two enzyme genes occurs, although other reasons are possible (5).

In contrast to the 1,3-galactosyltransferase or 1,4-galactosyltransferase families (18), the 1,4Gal-T/1,4GlcNAc-T gene family has only two members in mammals. No other homologous cDNA was detectable in any data base a year ago (10). Surprisingly, the same Gb3/CD77 synthase cDNA was subsequently cloned using 1,4GlcNAc-T cDNA (19) as a probe for PC cloning (11). Furthermore, from sequences that have been deposited within the last year, many homologous cDNAs to 1,4Gal-T can now be found in the data base of many other species from primates to insects or plants (Fig. 4). These genes share several highly homologous regions conserved through all species examined, and it is significant that all missense mutations found in human p phenotype occurred in these conserved regions (Fig. 4).

View larger version (88K): [in this window] [in a new window]   Fig. 4.   Alignment of 1,4Gal-T/1,4GlcNAc-T genes. Multiple sequence analysis (ClustalW) of 1,4Gal-T homologues was performed. The arrowheads indicate the mutated residues. Residues highlighted by a black or gray background are identical or highly conserved, respectively. Note that Gly187 and Pro251 were completely, and Met183 was highly, conserved among species examined. The numbers in the parentheses indicate GenBankTM accession numbers.

These results indicate that this gene family has been conserved because of its importance in the survival or protection of the host against exogenous invasions, although the real substrates for these gene products in individual species remain to be identified. Recent studies on the substrate preferentiality of an 1,4Gal-T in insect cells suggest that the activity may be toward Gal1,3GalNAc1-R and not for Gal1,4Glc- structures (20). Consequently, enzyme products of these sequence-conserved genes may not necessarily be identical, and utilized nucleotide sugars may also be diverse, as shown in the analysis of the 1,3Gal-T family (18, 21).

p individuals produce natural anti-P^k, anti-P, and anti-P1 IgG antibodies (22, 23), resulting in the high incidence of spontaneous abortion (23). Gb3/CD77 has been considered to mediate apoptosis of B cells (8). Furthermore, it was recently reported that Gb3/CD77 might be involved in the fusion and entry of human immunodeficiency virus, type I (24, 25) or in the initiation of signal transduction (26). Thus, the presense or absence of the antigen might strongly affect the cellular phenotype and the health of humans. Precise roles of Gb3/CD77 and its derivatives will be investigated using the obtained cDNA and/or targeted disruption of the 1,4Gal-T gene.

	ACKNOWLEDGEMENTS

We thank Dr. Anna Pirkola at Finnish Red Cross Blood Transfusion Service for helpful discussions. We also thank Dr. Kenneth O. Lloyd at Memorial Sloan-Kettring Cancer Center for carefully reading the manuscript.

	FOOTNOTES

^* This work was supported by Grants-in-Aid for the Center of Excellence Research (10CE2006) and Scientific Research of Priority Areas (12215058, 12204055, and 10178104) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant-in-aid from the Mizutani Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi@med.nagoya-u.ac.jp.

Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.C000625200

	ABBREVIATIONS

The abbreviations used are: Gb3, globotriaosylceramide, (P^k, CD77, Gal1,4Gal1,4Glc-ceramide); Gb4, globotetraosylceramide, (P, GalNAc1,3Gal 1,4Gal1,4Glc-Cer); 1, 4Gal-T, 1,4-galactosyltransferase; PCR, polymerase chain reaction; nt., nucleotide; WT, wild type; 1, 4GlcNAc-T, 1,4N-acetylglucosaminyltransferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/48/37752    most recent C000625200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Furukawa, K. Articles by Furukawa, K. Search for Related Content PubMed PubMed Citation Articles by Furukawa, K. Articles by Furukawa, K. Social Bookmarking                      What's this?