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J. Biol. Chem., Vol. 275, Issue 22, 16723-16729, June 2, 2000

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Cloning and Expression of the Histo-blood Group P^k UDP-galactose:Gal1-4Glc1-Cer 1,4-Galactosyltransferase

MOLECULAR GENETIC BASIS OF THE p PHENOTYPE^*

Rudi Steffensen^§, Karine Carlier^¶, Joelle Wiels^¶, Steven B. Levery, Mark Stroud^**, Bertil Cedergren, Birgitta Nilsson Sojka, Eric P. Bennett, Casper Jersild^§, and Henrik Clausen^§§

From the  School of Dentistry, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N, Denmark, the ^§ Regional Center for Blood Transfusion and Clinical Immunology, Aalborg Hospital, 9000 Aalborg, Denmark, ^¶ CNRS UMR 1598, Institut Gustave Roussy, 94805 Villejuif Cedex, France, the  Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, the ^** Department of Cell Surface Biochemistry, Northwest Hospital, Seattle, Washington 98125, and the  Department of Transfusion Medicine, Umeå University Hospital, S-901 85 Umeå, Sweden

Received for publication, January 31, 2000, and in revised form, March 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P₁, P, and P^k glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Gal1-R 4--galactosyltransferases catalyze the syntheses of the structurally related P₁ and P^k antigens. The P₁ polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an 4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated 4Gal-T1. Expression of full coding constructs of 4Gal-T1 in insect cells revealed it encoded P^k but not P₁ synthase activity. Northern analysis showed expression of the transcript correlating with P^k synthase activity and antigen expression in human B cell lines. Transfection of P^k-negative Namalwa cells with 4Gal-T1 resulted in strong P^k expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that 4Gal-T1 represented the P^k gene. Sequence analysis of the coding region of 4Gal-T1 in P₁+/ individuals did not reveal polymorphisms correlating with P₁P₂ typing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The P histo-blood group system is the last of the known carbohydrate defined blood group systems for which the molecular genetic basis has not yet been clarified. The P blood group system involves two major blood group phenotypes, P₁+ and P₁, with approximate frequencies of 80% and 20%, respectively (1, 2). P₁ individuals normally express the P antigen (P₁ is designated P₂ when P antigen expression is demonstrated), but the rare P^k phenotype lacks the P antigen, while the rare p phenotype lack both P and P^k antigens (for reviews, see Refs. 3-7). The P₁+ phenotype is defined by expression of the neolacto-series glycosphingolipid P₁ (for structures, see Table I) (8). In contrast, the P, P^k, and p antigens constitute intermediate steps in biosynthesis of globo-series glycolipids and give rise to P₁^k, P₂^k, and p phenotypes (9). Although the rare P^k phenotype shows the same frequency of P₁ antigen expression as individuals expressing the P antigen, the p phenotype is always associated with lack of P₁ antigen expression. Extensive studies of the chemistry, biosynthesis, and genetics of the P blood group system identified the antigens as being exclusively found on glycolipids, with the blood group specificity being synthesized by at least two distinct glycosyltransferase activities; UDP-galactose:-D-galactosyl-1-R 4--D-galactosyltransferase (4Gal-T)¹ activity(ies) for P^k and P₁ syntheses and UDP-GalNAc:Gb₃ 3--N-acetylgalactosaminyltransferase activity (EC 2.4.1.79) for P synthesis (for reviews, see Refs. 6 and 7). At least two independent gene loci, P and P₁P^k, are involved in defining these antigens. The P blood group-associated LKE antigen, shown to be the extended sialylated Gal-globoside structure (10), may involve polymorphism in an 2,3-sialyltransferase activity.

A long-standing controversy has been whether a single or two independent 1,4-galactosyltransferases catalyze the synthesis of the P₁ neolacto-series glycolipid antigen and the P^k globo-series structure (3-7). Several hypotheses have been proposed, including: (i) a model with two distinct functional genes being allelic or non-allelic, where the P₁ gene encodes a broadly active 4Gal-T, the P^k gene encodes a restricted 4Gal-T, and a null allele encodes a non-functional protein; (ii) a model with two distinct non-allelic genes, where P₁ encodes an 4Gal-T that can only synthesize P₁ structures and the P^k encodes an 4Gal-T that only synthesize the P^k structure; and (iii) a model where one gene locus encodes an 4Gal-T that is modulated by an independent polymorphic gene product to synthesize both P₁ and P^k structures. Bailly et al. (11) reported that kidney microsomal 4Gal-T activity from P₁ individuals does not compete for the two substrates used by P₁ and P^k 4Gal-T activities, and no accumulative effect in P₁ synthase activity was observed when mixing microsomal fractions from individuals of P₁ and P^k groups. Based on this, Bailly and colleagues suggested the existence of two distinct genes, coding for one P₁ 4Gal-T with exclusive activity for neolacto-series substrates and one P^k 4Gal-T with exclusive activity for the globo-series substrate. Since p individuals lack the P₁ antigen, this model inferred that two independent genetic events inactivating both genes was responsible for the p phenotype.

Several approaches to gain insight into the P blood group 4Gal-T gene(s) have been attempted. Purification of the mammalian enzymes has not been successful, but identification and cloning of a bacterial 4Gal-T involved in lipopolysaccharide biosynthesis (12, 13) potentially provided a strategy to clone the mammalian genes using sequence similarity. Previously, a bacterial 3-fucosyltransferase was identified in Helicobacter pylori using a short sequence motif conserved among mammalian 3-fucosyltransferases (14). BLAST analysis of gene data bases with the coding region of the 4Gal-T gene from Neisseria meningococcae resulted in identification of two human genes encoding putative type II transmembrane proteins with low sequence similarity to the bacterial gene.² The genes have open reading frames encoding 349 (EST cluster Hs.251809) and 371 (EST cluster Hs.82837) amino acid residues and are located at 8q24 and 3p21.1, respectively. Previously, we established Epstein-Barr virus (EBV)-transformed B cells from two p individuals (15). Only the gene at 3p21.1 was found to be expressed in the EBV-transformed p cells, as well as in Ramos cells known to have high P^k 4Gal-T activity. Sequencing of the coding region of the gene showed no mutations in p cells. Finally, expression of full coding or truncated, secreted constructs of either gene in insect cells failed to demonstrate glycosyltransferase activity with a large panel of substrates, including lactosylceramide, for P^k 4Gal-T activity.

The P₁ polymorphism is linked to 22q11.3-ter (16), whereas no information is available for the p phenotype. In the present report, we used BLAST searches of gene data bases with the coding region of a recently cloned human 4GlcNAc-transferase located at 3p14.3 (17) to identify a novel homologous gene located at 22q13.2. Expression of full coding constructs of the gene, designated 4Gal-T1, in insect cells revealed P^k synthase activity, but no P₁ activity was demonstrated. Sequence analysis of the coding region of 4Gal-T1 in P₁ and P₂ individuals did not reveal polymorphisms correlating with the P₁P₂ blood group phenotypes. In contrast, a single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p type.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Cloning of 4Gal-T1-- tBLASTn analysis of the human genome survey sequences, unfinished high throughput genomic sequences, and dbEST data bases at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) with the coding sequence of a human 4GlcNAc-transferase recently cloned by Nakayama et al. (17), produced a novel open reading frame of 1059 bp with significant similarity. The full coding sequence was available from BAC clone SC22CB-33B7 on chromosome 22 (GenBank^TM accession no. Z82176) in a single exon. With the release of the sequence of chromosome 22, the mapping data are cB33B7.1 at 2.65055 × 10⁷ to 2.65044 × 10⁷ flanked by diaphorase (NADH) and an unknown protein. Linkage analysis of the P1 polymorphism was originally performed with NADH-cytochrome b₅ reductase (16). Few ESTs cover the coding region (e.g. R45869), but the 3'-UTR is covered by EST Unigene cluster Hs.105956. Available ESTs are mainly derived from tonsil, prostate, and germ cell tumors.

Identification of Sequence Polymorphisms in the Coding Region of 4Gal-T1-- The sequence analysis was performed in three steps. Initially, the coding region of 4Gal-T1 from seven P₁+, five P₁, and six p phenotype individuals were sequenced in full by direct sequencing of a genomic fragment of 1295 bp derived by PCR with primer pair HCRS122 (5'-CCAGCCTTGGCTCTGGCTGATG) and HCRS126 (5'-CCCGGTGGCAGCTCGGGCCTC) located downstream and upstream of the translational start and stop sites, respectively. The PCR products were sequenced in both directions using the primers HCRS122, HCRS126, HCRS1 (5'-ATCTCACTTCTGAGCTGC), and HCRS4 (5'-GTTGTAGTGGTCCACGAAGTC). Subsequently, the products from two individuals (194 and 321) homozygous for G109 and two (183 and 300) homozygous for A109, randomly selected were subjected to cloning into pBluescript KS+ (Stratagene) followed by sequencing of clones. Finally, a genotyping assay based on RcaI restriction enzyme digestion of a PCR product was developed for the identified A109G missense mutation allele. PCR was performed using primer pair HCRS133 (5'-AAGCTCCTGGTCTGATCTGG) and HCRS6 (5'-ACCGAGCACATGAACAGGAAGTT) (30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s), and a total of 31 P₁+ and 51 P₁ phenotyped individuals was typed. RcaI digestion cleaves the expected product (319 bp) of A109 in two fragments of 182 and 137 bp. The RcaI digestion of PCR products was confirmed by Southern analysis on 3 P₁+ and 2 P₁ individuals.

Expression of 4Gal-T1 in Insect Cells-- Full coding constructs were prepared by genomic PCR using primer pair HCRS131 (5'-ACCATGTCCAAGCCCCCCGACCTC) and HCRS125 (5'-CATGAAAATGTACTTGTGAGGGG) and genomic DNA from phenotyped individuals with phenotypes P₁+ (165) and p (4) (see Table II for sequence). Three different full coding constructs were selected for expression: 67 (A109, T548, G903, G987), 45 (G109, T548, G903, A987), and p5 (A109, A548, G903, G987). The products were cloned into BamHI and EcoRI sites of pBluescript KS+, and subsequently into the insect cell expression vector pVL1393 (PharMingen), and sequenced in full. A truncated, secreted construct (amino acid residues 46-353) was prepared using primer pair HCRS124 (5'-CCCAAGGAGAAAGGGCAGCTC) and HCRS125 from a P₁+ phenotype individual (165), and the sequence confirmed as described above. The products were cloned into the expression vector pAcGP67A (PharMingen). The variants of plasmids pVL-4Gal-T1-full and pAcGP67-4Gal-T1-sol were co-transfected with Baculo-Gold^TM DNA (PharMingen), and virus amplified as described previously (18). Standard assays were performed in 50-µl reaction mixtures containing 25 mM cacodylate (pH 6.5), 10 mM MnCl₂, 0.25% Triton X-100, 100 µM UDP-[¹⁴C]Gal (10,000 cpm/nmol) (Amersham Pharmacia Biotech), and the indicated concentrations of acceptor substrates (Sigma and Dextra Laboratories Ltd.) (see Table I for structures). The full coding constructs were assayed with 1% Triton X-100 homogenates of cells twice washed in phosphate-buffered saline or resuspended microsomal fractions.

Expression of 4Gal-T1 in P^k-negative Namalwa Cells-- The three full coding constructs, 67, 45, and p5, were cloned into pDR2 (CLONTECH). Insert was excised from pBKs with BamHI/XhoI and inserted into the BamHI/SalI sites of pDR2. Transient transfection of 5 × 10⁶ Namalwa cells with 20 µg of cDNA was done by double-pulse electroporation using an Easy-cell ject+ (Eurogentec). Expression of CD77/P^k antigen was evaluated by fluorescence-activated cell sorting analysis on a FACSCalibur (Beckton-Dickinson) using 1A4 monoclonal antibody (19).

Characterization of the Product Formed with 4Gal-T1-- For product characterization, 2 mg of CDH was glycosylated with a microsomal fraction of High Five cells infected with pVL-4Gal-T1-full (67) using thin layer chromatography to monitor reaction progress. The reaction products were purified on an octadecyl-silica cartridge (Bakerbond, J.T. Baker), deuterium exchanged by repeated addition of CDCl₃-CD₃OD 2:1, sonication, and evaporation under dry nitrogen, and then dissolved in 0.5 ml of Me₂SO-d₆, 2% D₂O (20) (containing 0.03% tetramethylsilane as chemical shift reference) for NMR analysis. One-dimensional ¹H NMR spectra were acquired at 35 °C on a Varian Inova 600 MHz spectrometer; 1200 free induction decays were accumulated, with solvent suppression by presaturation pulse during the relaxation delay. Spectra were interpreted by comparison to those of relevant glycosphingolipid standards acquired under virtually identical conditions, as well as to previously published data for which a somewhat different temperature (65 °C) was employed (20, 21).

Northern Analysis-- The cDNA fragment of full coding 4Gal-T1 (67) was used as probe. The probe was random primer-labeled using [³²P]dCTP and a Strip-EZ DNA labeling kit (Ambion). Multiple tissue northern (MTN-H12) blot was obtained from CLONTECH. Eight human cell lines (Ramos, MutuI, BL2, Namalwa, Remb₁, 8866, T51, and K562) were analyzed because P^k synthase activity and antigen expression have been characterized previously (22, 23). Total cellular RNA was extracted from cell lines using the RNeasy Midi kit (Qiagen SA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Cloning of Human P^k 4Gal-T1-- Nakayama et al. (17) recently reported the cloning of a novel human 4GlcNAc-transferase responsible for the synthesis of the structures GlcNAc1-4Gal1-4GlcNAc1-R and GlcNAc1-4Gal1-3GalNAc1-R. The gene was mapped to chromosome 3p14.3. Since this is the first mammalian glycosyltransferase gene available that forms an 1-4 linkage, we hypothesized that it would represent one member of a family of homologous glycosyltransferase genes. A characteristic feature of homologous glycosyltransferase genes is that different members may encode enzymes which have different donor or acceptor sugar specificities, but the nature of the linkage formed is often retained (24). BLAST analysis of data bases using the coding region of the 4GlcNAc-transferase identified a sequenced BAC clone containing an open reading frame of 1059 bp. The coding region depicts a type II transmembrane protein of 353 amino acids with 35% overall sequence similarity to human 4GlcNAc-T (Figs. 1 and 2). The two genes show conservation of a DXD motif (25), and spacings of five cysteine residues. The predicted coding region of 4Gal-T1 has a single initiation codon in agreement with Kozak's rule (26), which precedes a sequence encoding a potential hydrophobic transmembrane segment (Fig. 1).

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence and predicted amino acid sequence of human 4Gal-T1. The amino acid sequence is shown in single-letter codes. The hydrophobic segment representing the putative transmembrane domain is underlined with a double line (Kyte and Doolittle, window of 8 (Ref. 51)). One consensus motif for N-glycosylation is indicated by asterisks. The locations of the primers used for preparation of the expression constructs are indicated by single underlining. A potential polyadenylation signal is indicated in boldface underlined type.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Multiple sequence analysis (ClustalW) of human 4Gal-T1 and 4GlcNAc-T. Introduced gaps are shown as dashes, and aligned identical residues are black boxed. The two amino acid substitutions (M37V and M183K) are indicated above the 4Gal-T1 sequence. Conserved cysteine residues are shown by asterisks.

Genetic Polymorphism of P^k 4Gal-T1-- Sequence analysis of the 4Gal-T1 gene from six p phenotype individuals from northern Sweden revealed only one single homozygous missense mutation T548A leading to the change of residue 183 from methionine to lysine. This substitution is a few amino acid residues from the functionally important DXD motif (25). Although residue 183 is not invariant among 4Gal-T1 and the 4GlcNAc-T (M/I), the non-conservative substitution to a charged lysine residue may be expected to affect the function of the gene product. The finding that all six p individuals only revealed one missense homozygous mutation and this was not found in 12 P₁+/ individuals strongly indicated that the gene identified was the P^k gene. Because 4Gal-T1 was located to the same chromosomal region (22q13.2) where the P₁ polymorphism has been linked (22q12-ter), it was likely that it also represented the P₁ synthase. Analysis of the 4Gal-T1 gene in P₁+ and P₁ individuals, revealed two silent and one missense mutation; however, none of these showed association with the P blood group phenotype (Table II). This was confirmed by genotyping of 82 individuals, 31 P₁+ and 51 P₁, where no significant correlation of the 109A and the 109G allele was observed (Table III). The PCR-based RcaI restriction enzyme analysis was confirmed by Southern blot analysis of P₁+/ individuals (Fig. 3). The more common allele of the missense mutation at A109G encodes a methionine at residue 37 in the C-terminal part of the putative hydrophobic signal sequence (Figs. 1 and 2). The conservative substitution of residue 37 to valine is not predicted to change the catalytic activity or affect retention in the Golgi.

View this table: [in this window] [in a new window]   Table II Sequence polymorphisms identified in the coding region of 4Gal-T1 in P1+, P1, and p blood group individuals

View this table: [in this window] [in a new window]   Table III Correlation of the missense polymorphism with P1+/ blood group phenotype Genotyping was performed by RcaI restriction analysis of PCR products.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   RcaI genotyping of position A109G by Southern analysis. DNA from five phenotyped donors was digested with restriction enzymes as indicated, and the blot probed with the full coding 4Gal-T1 (67) construct. The RcaI digestion confirmed the PCR-based genotyping presented in Table II. The EcoRI polymorphism found in individuals 165 and 183 is outside the coding region of 4Gal-T1 and is unrelated to the P1 phenotype.

4Gal-T1 Encodes Exclusive P^k 4Gal-T Activity-- Expression of full coding constructs of 4Gal-T1^37M and 4Gal-T1^37V in insect cells resulted in marked increase in galactosyltransferase activity with CDH, compared with uninfected cells or cells infected with a control construct (Fig. 4). In contrast, no activity was found with the 4Gal-T1^183K gene from p individuals. Importantly, neither 4Gal-T1^37M or 4Gal-T1^37V constructs conferred 4Gal-T activity with the neolacto-series (paragloboside) glycolipid acceptor for P₁ synthase activity (Fig. 4). The assay conditions for measuring P^k and P₁ synthase activity was the same except substitution of the acceptor substrate, and these conditions were previously used to demonstrate both activities in kidney extracts from P₁+ and P₁ individuals (11). The soluble, secreted construct encoding residues 47-353 did not result in active 4Gal-T activity (data not shown). Attempts to obtain complete conversion of CDH to CTH were unsuccessful, but a one-dimensional ¹H NMR spectrum of the purified reaction mixture (data not shown) clearly exhibited H-1 resonances diagnostic for CTH at levels approximately 30% of those of the CDH acceptor substrate. Thus, in addition to major resonances at 4.205 ppm (³J_{1, 2} = 7.2 Hz) and 4.165 ppm (³J_{1, 2} = 7.9 Hz), corresponding to H-1 of Gal4 and Glc1of CDH, minor resonances were observed at 4.794 ppm (³J_{1, 2} = 3.7 Hz) and 4.258 ppm (³J_{1, 2} = 6.9 Hz), corresponding to H-1 of Gal4 and Gal4 of CTH (the chemical shift of Glc1 H-1 is not affected by the addition of the terminal Gal4 residue). The chemical shift and ³J_1,2 coupling of the downfield H-1 resonance are particularly characteristic for Gal4 of CTH and other globo-series glycosphingolipids (20, 21). Analysis with a number of saccharide acceptors including lactose, lactosamine, and benzyl -lactoside revealed no significant activity over background values (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Expression of full coding 4Gal-T1 variants in High Five cells. Assays were performed with microsomal fractions, and controls included constructs encoding polypeptide GalNAc-T3 and -T4 (52), as well as a 3GlcNAc-T (24). Autoradiography of high performance thin layer chromatography of reaction products (4 h) purified by SepPack C-18 columns. Panel A, pk assay using 25 µg of CDH as substrate. Plate was run in chloroform-methanol-water (60/35/8, v/v/v). Constructs from the two different alleles identified from P1+/ individuals (45 and 67) resulted in 4Gal-T activity toward CDH, while the construct derived from p (5) showed no activity above background found with control constructs. Panel B, P1 assay using 20 µg of paragloboside (PG) as substrate. Plate was run in chloroform-methanol-water (60/40/10, v/v/v). No specific product was formed with UDP-Gal donor substrate, whereas the 3GlcNAc-T transferred GlcNAc into paragloboside with UDP-GlcNAc. Considerable GlcNAc-T activity was observed in both 67 and 3GnT microsomal fractions, yielding a GlcNAc-CTH-related product.

Expression Pattern of 4Gal-T1-- Northern analysis with mRNA from 12 human organs revealed a ubiquitous expression pattern with high expression in kidney and heart and low expression in other organs (Fig. 5). Kidney primarily synthesize globo-series glycosphingolipids (27). Analysis of eight human cell lines revealed an expression pattern correlating with 4Gal-T1 activity and cell surface expression of P^k antigen (Fig. 6) (22, 23). Ramos cells have the highest antigen expression and 4Gal-T activity, and strong expression of 4Gal-T1. In contrast, Namalwa cells, which do not produce P^k antigens and have no measurable 4Gal-T activity, showed no expression of 4Gal-T1. However, transient transfection of Namalwa cells with the full coding constructs of 4Gal-T1 (67 and 45) resulted in P^k/CD77 expression as revealed by fluorescence-activated cell sorting analysis (Fig. 7).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Northern blot analysis with human organs. Multiple human Northern blot (MTN-H12) was probed with 32P-labeled 4Gal-T1 probe. kb, kilobases.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Northern blot analysis with eight human B cell lines. Transcript sizes are approximately 2 and 3 kilobases (kb).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Cell surface expression of Pk/CD77 antigen in Namalwa cells after transient transfection of 4Gal-T1. Constructs p5, 45, and 67 as well as empty pDR2 vector were electroporated in Namalwa cells, and expression of Pk/CD77 antigen was tested after 48 h. Cells were labeled with 1A4 monoclonal antibody and goat anti-mouse-fluorescein isothiocyanate (gray histograms) or with goat anti-mouse-fluorescein isothiocyanate alone (empty histograms) and analyzed with a FACSCalibur flow cytometer. Strong labeling with 1A4 was found only with constructs 45 and 67.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 4Gal-T1 gene characterized in this report provides a molecular genetic basis for the rare p histo-blood group phenotype found in Västerbotten County, in the northern part of Sweden (28). A single inactivating homozygous missense mutation in the catalytic domain of the enzyme was found in all six p phenotype individuals studied. We have previously characterized erythrocyte PP^k antigen expression and 4Gal-T activity in EBV-transformed cells from two of these individuals (15), and found a complete deficiency of P^k antigen and 4Gal-T activity. Iizuka et al. (29), reporting essentially the same experiment, suggested that a catalytically active P^k transferase was indeed expressed in p individuals, as evidenced by P^k synthase activity in EBV-transformed cells; however, in accordance with the proposed p phenotype of the individual studied, the transformed cells did not express P^k antigen. This led Iizuka et al. (29) to suggest that p phenotype individuals carry a functionally active P^k 4Gal-T gene, and that the p phenotype was a result of an yet unknown epigenetic mechanism. The data presented here are not in agreement with this, and support a simple allelic model with an active P^k and an inactive p allele. It is, however, possible that the p phenotype in different populations has a different molecular genetic basis. The molecular genetics of all other characterized histo-blood group systems defined by carbohydrate antigens, i.e. ABO (30), Hh (31), Sese (32), and Lewis (33, 34), have been shown to adhere to a model with simple inactivating mutations of glycosyltransferase genes.

The presented data, however, do not explain the molecular genetic basis of the P₁ blood group polymorphism. Although, the P₁ polymorphism is linked to the same chromosomal localization as 4Gal-T1, we found no genetic polymorphisms in the 4Gal-T1 gene associated with the P₁+/ phenotypes, and recombinant 4Gal-T1 variants did not express P₁ synthase activity in vitro (Tables II and III, Fig. 4). Searching the available chromosome 22 sequence did not reveal additional homologous genes. Therefore, the following possibilities exist: (i) 4Gal-T1 can be activated by another non-homologous polymorphic gene or gene product and function as a P₁ synthase; (ii) a second polymorphic 4Gal-T gene, which is non-homologous to 4Gal-T1, exists; or (iii) an alternatively spliced version of 4Gal-T1 encodes a form capable of functioning as a P₁ synthase. The first possibility has a precedent in two members of the 4Gal-T gene family, 4Gal-T1 and -T2, both of which are modulated by -lactalbumin to change their function from N-acetyllactosamine synthases to lactose synthases (35-37). Binding of -lactalbumin to these galactosyltransferases changes the acceptor substrate specificity from GlcNAc to Glc, but also to some degree affects the donor substrate specificity to include UDP-GalNAc (38). The induction of 4Gal-T1 by -lactalbumin to enable it to function as a lactose synthase is combined with a complex regulatory mechanism by which the 4Gal-T1 synthase is 100-fold up-regulated in mammary glands (39). As lactose is the major nutrient in milk, this complex model for its synthesis appears to be in accordance with the biological function. The P₁ antigen has only been detected as a minor glycosphingolipid component, and no biological function for this polymorphic antigen has been identified. At present, therefore, it may seem less likely that a unique modulator of the 4Gal-T1 gene has evolved. The second possibility of the existence of another polymorphic non-homologous 4Gal-T gene located in the same chromosomal region implies that the encoded 4Gal-T functions as both P^k and P₁ synthases. This is based on the findings that p individuals do not produce P₁ antigens, and it is supported by the finding that erythrocytes of P₁ individuals contain relative less LacCer and more Gb₃ than P₂ individuals (40). Generally, glycosyltransferases with similar functions are encoded by homologous glycosyltransferase gene families (24); however, recently two non-homologous 3GlcNAc-transferases both functioning as poly-N-acetyllactosamine synthases have been identified (41, 42). The third possibility is unlikely, as the coding region of 4Gal-T1 was found in a single exon. However, there are precedents for multiple protein forms originating from such genes by differential usage of splice donor sites of introns placed in 3'-UTR (43). Although the Northern analysis with mRNA from human organs only revealed one transcript of 2.3 kilobases (Fig. 5), the Northern analysis with total RNA from human cell lines may suggest the presence of additional larger transcripts (Fig. 6). The nature of these transcripts required further analysis to explore the possibility that they may encode a novel form of the 4Gal-T1 protein with P₁ synthase activity. A possible mechanism for the P₁ blood group polymorphism would include sequence polymorphisms in the 3'-UTR directing different donor splice sites in the 3' coding region of the characterized 4Gal-T1 coding sequence. It is important to note, however, that a total of three polymorphisms were identified in P₁+/ individuals (Table II), and none of these correlated with the phenotype. This finding argues against the existence of another polymorphism in this gene (3'-UTR) responsible for the P₁+/ polymorphism, because it would be expected that at least some of these would have arisen after the P₁+/ polymorphism and hence co-segregate with this as found e.g. for the polymorphisms in the ABO gene locus (30).

4Gal-T1 is homologous to an 4GlcNAc-T located at 3p14.3 (17). The 4GlcNAc-T forms the linkage GlcNAc1-4Gal1-3/4R, where R can be GalNAc, GlcNAc, or less effectively, glucose. Preference for mucin oligosaccharides of the core 2 structure was found, and the gene was shown to control expression of Con-A-binding class-III mucins in stomach and pancreas. Genetic polymorphisms in expression of the 4GlcNAc structures have not been reported. The sequence similarity with 4Gal-T1 (35% overall amino acid sequence similarity) is similar to that found among other homologous glycosyltransferases with similar functions, and the characteristic feature of conserved spacings of cysteine residues (five cysteine residues align, Fig. 2) is also found. Both enzymes transfer to galactose, but, whereas the acceptor disaccharide specificity of the 4GlcNAc-T appears to be broad, 4Gal-T1 is apparently highly specific for the glycolipid, lactosylceramide. Lopez et al. (44) recently characterized an 4Gal-T activity in insect cells and found it had preferred acceptor substrate specificity for Gal1-3GalNAc1-R rather than lacto-series structures. Thus, the acceptor substrate specificity is similar to that of the 4GlcNAc-T and different from 4Gal-T1.

The N. gonorrhoeae lgtC 4Gal-T (12) exhibits no significant sequence similarity to the 4Gal-T1 reported here. However, two human genes homologous to lgtC were identified with significant sequence similarities and conserved DXD motif (data not shown). We have been unable to demonstrate glycosyltransferase activity with these two human genes using full coding and secreted recombinant constructs expressed in insect cells. Nevertheless, they may represent glycosyltransferase genes and encode 4Gal-Ts, but they are unlikely to be involved in the P₁ polymorphism as they are not located on chromosome 22. One of the genes is located at 8q24 distal to the c-myc oncogene, and hence part of region of chromosome 8, which is translocated in Burkitt's lymphoma. Since Burkitt's lymphoma is associated with high level expression of P^k (45), this represented a candidate for the P^k gene. However, Northern analysis of various B cell lines (Fig. 6) shows that 4Gal-T1 expression clearly correlates with P^k synthase activity and antigen expression in Burkitt's lymphoma cell lines (22, 23).

The P blood group system is implicated in important biological phenomena. Blood group p individuals have strong anti-P₁PP^k IgG antibodies and these are implicated in high incidence of spontaneous abortions (46). The globo-series glycolipid antigens constitute major receptors for microbial pathogens with the Gal1-4Gal linkage being an essential part of the receptor site (for a review, see Ref. 47). The P^k glycolipid is the CD77 antigen, a B cell differentiation antigen, which is able to transduce a signal leading to apoptosis of the cells (48). Furthermore, the association of this glycolipid with the type I interferon receptor or with the human immunodeficiency virus type 1 co-receptor, CXCR4, seems to be crucial for the functions of these receptors (49, 50). Cloning of the P^k synthase is an important step toward understanding the biological roles of the globo-series class of glycolipids, and a first step in elucidating the molecular genetics of the P blood group system.

	ACKNOWLEDGEMENTS

We are indebted to Dr. Bo Samuelsson for support and encouragement during these studies. We are grateful to Dr. Alan Chester for helpful suggestions and critical reading of the manuscript. We thank Cécile Tétaud and Yann Lécluse for technical assistance.

	FOOTNOTES

^* This work was supported by the PhD Foundation Aalborg Sygehus, Aalborg Frivillige Bloddonorers Foundation, Nordjyllands Amts Foundation, Det Obelse Familiefond, Peder Kristen Tøfting, and Dagmar Tøftings Foundation, Ebba and Aksel Schølins Foundation, the Danish Cancer Society, the Velux Foundation, the Danish Research Council, Grant 5 P41 RR05351 from the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates, and Grant ARC9588 from the Association pour la Recherche sur le Cancer.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ245581.

^§§ To whom correspondence should be addressed. Tel.: 45-35326835; Fax: 45-35326505; E-mail: henrik.clausen@odont.ku.dk.

Published, JBC Papers in Press, March 21, 2000, DOI 10.1074/jbc.M000728200

² R. Steffensen, J. Wiels, E. P. Bennett, and H. Clausen, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: 4Gal-T, UDP-galactose:-D-galactose-R 4--D-galactosyltransferase; UTR, untranslated region; EST, expressed sequence tag; PCR, polymerase chain reaction; bp, base pair(s); nt, nucleotide(s); EBV, Epstein-Barr virus; CDH, ceramide dihexoside; CTH, ceramide trihexoside; Gb₃, globotriaosylceramide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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