©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence and Expression of a Candidate for the Human Secretor Blood Group (1,2)Fucosyltransferase Gene (FUT2)
HOMOZYGOSITY FOR AN ENZYME-INACTIVATING NONSENSE MUTATION COMMONLY CORRELATES WITH THE NON-SECRETOR PHENOTYPE (*)

(Received for publication, November 8, 1994; and in revised form, December 2, 1994)

Robert J. Kelly (1) Sylvie Rouquier (3)(§) Dominique Giorgi (3)(§) Gregory G. Lennon (3) John B. Lowe (1) (2)(¶)

From the  (1)Howard Hughes Medical Institute and the (2)Department of Pathology, University of Michigan Medical Center, Medical Sciences Research Building I, Ann Arbor, Michigan 48109-0650 and the (3)Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Synthesis of soluble A, B, H, and Lewis b blood group antigens in humans is determined by the Secretor (Se) (FUT2) blood group locus. Genetic, biochemical, and molecular analyses indicate that this locus corresponds to an alpha(1,2)fucosyltransferase gene distinct from the genetically-linked H blood group alpha(1,2)fucosyltransferase locus. The accompanying paper (Rouquier, S., Lowe, J. B., Kelly, R. J., Fertitta, A. L., Lennon, G. G., and Giorgi, D.(1995) J. Biol. Chem. 270, 4632-4639) describes the molecular cloning and mapping of two human DNA segments that are physically linked to, and cross-hybridize with, the H locus. We present here an analysis of these two new DNA segments. One of these, termed Sec1, is a pseudogene, because translational frameshifts and termination codons interrupt potential open reading frames that would otherwise share primary sequence similarity with the H alpha(1,2)fucosyltransferase. The other DNA segment, termed Sec2, predicts a 332-amino acid-long polypeptide, and a longer isoform, that share 68% sequence identity with the COOH-terminal 292 residues of the human H blood group alpha(1,2)fucosyltransferase. Sec2 encodes an alpha(1,2)fucosyltransferase with catalytic properties that mirror those ascribed to the Secretor locus-encoded alpha(1,2)fucosyltransferase. Approximately 20% of randomly-selected individuals were found to be apparently homozygous for an enzyme-inactivating nonsense allele (Trp ter) at this locus, in correspondence to the frequency of the non-secretor phenotype in most human populations. Furthermore, each of six unrelated non-secretor individuals are also apparently homozygous for this null allele. These results indicate that Sec2 corresponds to the human Secretor blood group locus (FUT2) and indicate that homozygosity for a common nonsense allele is responsible for the non-secretor phenotype in many non-secretor individuals.


INTRODUCTION

The ABO blood group antigens consist of oligosaccharides synthesized by the sequential action of glycosyltransferases. While these molecules were classically defined as polymorphic red cell antigens, subsequent studies demonstrated that soluble forms of these molecules may be found in the saliva and in other secretions in some, although not all, humans (reviewed in (1, 2, 3) ). The ability to elaborate soluble A, B, and H antigens is determined by the Secretor blood group locus. Homozygosity for null alleles at this locus occurs in approximately 20% of most populations (4) and yields the non-secretory phenotype, characterized by absence of normal amounts of soluble A, B, and H substance in the saliva(1) . By contrast, most humans maintain the secretor phenotype and elaborate soluble blood group substance in amounts easily detectable by standard hemagglutination-inhibition assays(5, 6) . The penultimate step in the pathway leading to synthesis of soluble and membrane-associated A and B antigens is catalyzed by alpha(1,2)fucosyltransferases(^1)(1, 2, 3) . These enzymes form a precursor oligosaccharide substrate (Fuc alpha(1,2)Galbeta-) termed the H antigen, which is an essential substrate for the final step in the pathway, catalyzed by allelic glycosyltransferases encoded by the ABO locus(1) . Genetic (7, 8, 9, 10) and biochemical (10, 11, 12, 13) analyses indicate that the H blood group locus represents an alpha(1,2)fucosyltransferase gene expressed in the erythroid lineage and determines expression of the H antigen (along with A and/or B antigens) on red cells. By contrast, these studies are consistent with a hypothesis (7) that the Secretor locus corresponds to a closely linked but distinct alpha(1,2)fucosyltransferase gene whose expression is restricted to secretory epithelial cells in the salivary glands, gastrointestinal tract, and elsewhere, where it controls expression of soluble H antigen (and thus A or B antigen synthesis) in saliva and other secretions (reviewed in (3) ).

In the accompanying paper(14) , we report the isolation of two human DNA segments that represent candidates for the Secretor alpha(1,2)fucosyltransferase locus because they cross-hybridize with the H blood group alpha(1,2)fucosyltransferase gene and are in close physical proximity to this locus on chromosome 19. We report here the results of our structural and functional analysis of these two newly identified sequences. One of these, termed Sec1, represents a pseudogene. The other sequence, termed Sec2, encodes a polypeptide of 332 amino acids in length (and an isoform that is longer by 11 NH(2)-terminal amino acids) that in turn functions as an alpha(1,2)fucosyltransferase with catalytic properties corresponding to those assigned previously to the Secretor locus-encoded alpha(1,2)fucosyltransferase. We furthermore find that each of six unrelated non-secretor individuals maintains homozygosity for an allele at this locus which contains an enzyme-inactivating translational termination codon corresponding to amino acid residue 143. 10 of 52 (19%) randomly chosen individuals were determined to be homozygous for the mutant allele, in close correspondence to the frequency of the non-secretor phenotype in most populations. These results confirm that this newly described alpha(1,2)fucosyltransferase gene is the FUT2 gene corresponding to the human Secretor blood group locus and provide molecular confirmation of the hypothesis (7) that the H and Secretor loci correspond to two distinct alpha(1,2)fucosyltransferase genes.


MATERIALS AND METHODS

Molecular Cloning and Sequencing of the Sec1 and Sec2 DNA Segments

Cosmid 31553 (14) was mapped using a 1.3-kb probe made from the coding region of the human H blood group alpha(1,2) fucosyltransferase gene (8, 9) (FUT1). Two sequences related to FUT1, termed Sec1 and Sec2 (Secretor candidates 1 and 2), were identified in this cosmid. The Sec1 sequence is contained within an 8.2-kb EcoRI fragment that cross-hybridizes with the H alpha(1,2)fucosyltransferase coding region probe(14) . This 8.2-kb fragment was subcloned into the EcoRI site of the mammalian expression vector pcDNAI (Invitrogen) to create the vector pcDNAI-Sec1. Approximately 3.0 kb of this 8.2-kb fragment, representing the cross-hybridizing portion of this fragment, was subjected to DNA sequencing by the dideoxy chain termination method (15) using T7 DNA polymerase (Sequenase, Amersham). Both strands were sequenced using oligonucleotide primers corresponding to the insert sequence. The Sec2 sequence is contained within an 18.5-kb EcoRI restriction fragment of the cosmid that cross-hybridizes with the H alpha(1,2)fucosyltransferase coding region probe. Four cross-hybridizing and co-linear PstI fragments were subcloned into the PstI site of pTZ19R (Pharmacia Biotech, Inc.), and their sequence was determined. Direct sequencing of the cosmid was also performed to verify restriction fragment junctions.

Molecular Cloning and Sequencing of Two Alleles at the Sec2 Locus

Preliminary sequence analysis of the Sec2 allele within cosmid 31553 suggested that a nonsense codon at codon 143 might be an alternative to a tryptophan codon in a potential functional, wild type allele, by way of comparison to the H alpha(1,2)fucosyltransferase DNA and protein sequence at this position. This analysis predicted that a putative wild type allele at this position would maintain an intact BstNI cleavage site (CCTGG), whereas this restriction site would not exist at this position in the putative Trp ter null allele (CCTGA) within the cosmid sequence. Preliminary analysis of a secretor-positive individual indicated that he was heterozygous for the BstI restriction site at this position. We therefore cloned the coding regions of both alleles at the Sec2 locus from this individual, using the PCR (16) and primers that flank the codon corresponding to the Trp ter polymorphism. Each primer contains 30 bp derived from the Sec2 DNA sequence (below, underlined) and 10 bp that contain a restriction site (EcoRI, GAATTC; XbaI, TCTAGA) to facilitate subcloning. One primer pair amplifies the DNA sequence between base pairs -216 and -187 (GCGCGAATTCTATAAACACACTTGAGATACATGCCTGTGC; Sec2 sequence underlined), 5` to the ``short'' initiation codon, and between base pairs 535 and 564 (GCGCTCTAGAATGGACCCCTACAAAGGTGCCCGGCCGGCT; Sec2 sequence underlined) within the putative coding region of Sec2, and 3` to the polymorphic DNA sequence corresponding to codon 143. The second primer pair amplifies the DNA sequence between base pairs 369 and 398 (GCGCGAATTCGAGGAATACCGCCACATCCCGGGGGAGTAC; Sec2 sequence underlined) of the coding sequence, at a position 5` to the polymorphic site, and between base pairs 1049 and 1088 (GCGCTCTAGAGAACCATGTGCTTCTCATGCCCGGGCACTC; Sec2 sequence underlined), 50 base pairs 3` to the termination codon. Genomic DNA isolated (17) from this secretor-positive individual was subjected to 30 cycles of PCR amplification (denaturation at 94 °C for 1.5 min, annealing/extension for 2.5 min at 72 °C). Fragments generated with these primers were restricted with EcoRI and XbaI, gel-purified, and cloned into EcoRI/XbaI-cleaved pTZ19R. Clones corresponding to the putative wild type allele, and the putative Trp ter allele, were distinguished by restriction digestion with BstNI. The DNA sequences of a representative number of fragments from each allele were determined(15) . Both strands were sequenced using oligonucleotide primers corresponding to the insert sequence. Multiple clones corresponding to each allele were sequenced to distinguish PCR errors from actual sequence polymorphisms.

Expression Vector Construction and Analysis

For expression of Sec1, we used the vector pcDNAI-Sec1 (described above) and a related vector (pcDNAI-Sec1-rev) containing the identical 8.2-kb EcoRI fragment of Sec1 but cloned in the opposite orientation in pcDNAI. For the expression of the Sec2 allele in cosmid 31553(14) , the PCR and cosmid 31553 template DNA were used to construct an expression vector (pcDNAI-alpha(1,2)FTse) with an insert encompassing the coding sequence of Sec2. The pair of PCR primers used amplifies the Sec2 sequence from a position bounded by base pair -15 and base pair 15 (assigning the A of the initiation codon, highlighted in bold type, as position 1; GCGCGAATTCCCTTTCTCCTTTCCCATGGCCCACTTCATC; Sec2 sequence underlined), to a position bounded by base pairs 1000 and 1029, immediately 3` to the stop codon at base pairs 997-999 (GCGCTCTAGAGGAGAAAAGGTCTCAAAGGACGGGCCAGCA; Sec2 sequence underlined). PCR conditions were used that minimize PCR-mediated DNA sequence alterations(18) . The product from this amplification was restricted with EcoRI and XbaI, gel-purified, and ligated into EcoRI-XbaI-doubly digested pcDNAI. Clones with a single insert in the correct orientation were subjected to DNA sequence analysis to identify one without PCR-mediated DNA sequence alterations; one such clone was termed pcDNAI-alpha(1,2)FTse. Other expression vectors containing single or multiple DNA sequence polymorphisms were assembled with restriction fragment exchange procedures (17) using restriction sites within pcDNAI-alpha(1,2)FTse. The vector pcDNAI-alpha(1,2)FTSe-int, containing the wild type sequence corresponding to codon 143 (TGG; Trp) along with wild type protein sequence-neutral DNA sequence polymorphisms at DNA sequence positions 171 (wild type = A; cosmid-derived = G) and 216 (wild type = C; cosmid-derived = T), was constructed by replacing the 0.4-kb AgeI/PstI restriction fragment (base pairs 90 to 524) in pcDNAI-alpha(1,2)FTse, that encompasses these three polymorphisms, with the corresponding 0.4-kb AgeI/PstI restriction fragment from the wild type allele. This latter fragment was derived from a 599-bp fragment encompassing this 150-bp EcoRI-AgeI fragment, using the PCR, genomic DNA template from the heterozygous secretor-positive individual described above, a synthetic oligonucleotide PCR primer (GCGCGAATTCTATAAACACACTTGAGATACATGCCTGTGC; Sec2 sequence underlined) corresponding to a position approximately 100 bp upstream from the short initiation codon, and a primer corresponding to base pairs 535 and 564 of the Sec2 coding region (GCGCGAATTCATGGACCCCTACAAAGGTGCCCGGCCGGCT; Sec2 sequence underlined). A vector encompassing the short form of the entire wild type allele (pcDNAI-alpha(1,2)FTSe-short) was constructed by exchanging the 0.5-kb PstI/XbaI restriction fragment (base pairs 524 to 1029) of pcDNAI-alpha(1,2)FTSe-int (encodes Ser at codon 247) with the same fragment from the wild type allele (encodes Gly at codon 247) prepared from the heterozygous secretor-positive individual described above, using the PCR. To reiterate, this exchange replaces the serine codon found at protein sequence position 247 in the allele derived from cosmid 31553 with a glycine codon found at this position in the wild type allele. Finally, plasmid pcDNAI-alpha(1,2)FTSe-long was constructed by replacing a 130-bp EcoRI-AgeI in pcDNAI-alpha(1,2)FTSe-short (encompasses the short initiation codon) with a 150-bp EcoRI-AgeI fragment encompassing the ``long'' initiation codon. This latter fragment was generated using the PCR, wild type allele template DNA, and a primer pair that amplifies the DNA sequence between base pairs -35 and -6 of the Sec2 sequence (GCGCGAATTCCCATGCTGGTCGTTCAGATGCCTTTCTCCT; Sec2 sequence underlined), corresponding to the long initiation codon (indicated in bold in the PCR primer sequence), and between base pairs 535 and 564 of the Sec2 coding region (GCGCGAATTCATGGACCCCTACAAAGGTGCCCGGCCGGCT; Sec2 sequence underlined). Plasmid pcDNAI without an insert served as the negative control vector.

COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected with expression vectors using a DEAE-dextran procedure, all as described previously(8, 9) . A control plasmid (pCDM7-CAT; (9) ), encoding bacterial chloramphenicol acetyltransferase, was co-transfected to allow normalization for transfection efficiency. Cell extracts were prepared 72 h after transfection and were used in alpha(1,2)fucosyltransferase assays as described below. An aliquot of the cell culture medium was also subjected to assay for alpha(1,2)fucosyltransferase activity. Cell extracts were also subjected to chloramphenicol acetyltransferase activity assays(17) .

Fucosyltransferase Assays

Cell extracts containing 1% Triton X-100, 10% glycerol were prepared from transfected COS-7 cells using procedures previously described(9) . Cell extract protein concentrations were determined using the micro-BCA assay reagent (Pierce Chemical Co.). Fucosyltransferase assays were performed in a volume of 20 µl and contained 3 µM GDP-[^14C]fucose, various concentrations of unlabeled GDP-fucose, 5 mM ATP, 25 mM sodium phosphate at pH 6.0, 2-8 µl of cell extract (approximately 25-42 µg of protein), and various concentrations of different low molecular weight glycan acceptors, as described previously(9, 18, 19) . Preliminary assays were used to adjust the amount of cell extract added to each assay to ensure that reactions were linear (no more than 10% of GDP-fucose consumed prior to assay termination). All assays were performed in duplicate, along with parallel control reactions containing no added acceptor. Reactions were incubated at 37 °C for 2 h and then terminated by adding 20 µl of ethanol and either 1 ml of water (for phenyl-beta-D-galactoside acceptor assays) or 560 µl of water (for all other acceptor assays). The reactions were centrifuged for 5 min at 15,000 times g, and the supernatant was collected and used to determine the amount of fucosylated product formed. When phenyl-beta-D-galactoside was used as the acceptor, a hydrophobic interaction chromatography procedure was used, in which the product of the reaction is retained by, and then eluted from, a Sep-Pak column (Waters-Millipore)(19) . When neutral acceptors were used (lacto-N-biose and N-acetyllactosamine), a portion of the reaction was applied to a 500-µl column of Dowex 1X 2-400, formate form(18) . Unincorporated GDP-fucose is retained on the column, and radiolabeled product is recovered in the column flow-through fractions and quantitated by liquid scintillation counting. For all acceptors tested, background counts obtained in the absence of added acceptor was no more than 1% of the total added radiolabel. When total enzyme activity was measured in a plate of transfected COS-7 cells, and in the media prepared from those cells, units were defined as picomoles of product (phenyl-beta-D-galactoside acceptor) formed per h, per unit of 1-µl volume of cell extract or cell media assayed.

For pH optimum determination, reactions were buffered with either 25 mM sodium acetate (pH 4.5-6.0), 25 mM sodium phosphate (pH 5.5-7.5), or 25 mM Tris-HCl (pH 7.0-9.0), using concentrated solutions of these buffers previously titrated to the various pH values. The final pH of each reaction was directly determined with a micro pH electrode as described previously(19) . Phenyl-beta-D-galactoside was used as the acceptor in these assays. These experiments were completed using the enzyme derived from the pcDNAI-alpha(1,2)FTSe-short vector.

In assays to determine the apparent K(m) values, the concentration of the acceptors was varied as follows: phenyl-beta-D-galactoside, 0-170 mM; lacto-N-biose I, 0-10 mM; N-acetyllactosamine, 0-12.5 mM. These assays were performed in 25 mM sodium phosphate, pH 6.0, using 3 µM GDP-[^14C]fucose. To determine the apparent K(m) value for GDP-fucose, 3 µM GDP-[^14C]fucose was supplemented with different amounts of unlabeled GDP-fucose (19) to achieve final GDP-fucose concentrations that ranged from 3 µM to 400 µM. The GDP-fucose K(m) determination was completed in reactions containing 25 mM phenyl-beta-D-galactoside and buffered with 25 mM sodium phosphate, pH 6.0. Apparent Michaelis constants were derived from Lineweaver-Burk plots of substrate concentration-rate determinations. These experiments were completed using the enzyme derived from the pcDNAI-alpha(1,2)FTSe-short vector.

Pedigree Analysis

Human genomic DNA was prepared from peripheral blood samples(11) , or from freshly plucked hairs(20) , from 60 individuals. The secretor status of eight of these individuals was determined with standard blood group typing methods, using their red blood cells and saliva(21) . PCR analyses were performed using conditions described above. The primers used to sample the Trp ter position (GAGGAATACCGCCACATCCCGGGGGAGTAC and ATGGACCCCTACAAAGGTGCCCGGCCGGCT) correspond to positions 369 to 398 and 535 to 564, respectively, of the Sec2 coding sequence. PCR products were fixed to nylon hybridization membranes and probed with P-labeled allele-specific oligonucleotides(9, 22) (wild type probe TGCTCCTGGACCTTC; Trp ter specific probe, TGCTCCTAGACCTTC). Filters were hybridized at 37 °C in 5 times SSPE (1 times SSPE is 15 mM sodium citrate, 12 mM NaCl, 13 mM sodium phosphate, 1 mM EDTA, pH 7.2), 5 times Denhardt's solution, 0.5% SDS, and 0.1 mg of sheared salmon sperm DNA per ml, rinsed twice at room temperature in 2 times SSPE, 0.1% SDS, washed for 10 min at either 42 °C (Trp ter probe) or 46 °C (wild type probe) in 2 times SSPE, 0.1% SDS, and subjected to autoradiography.


RESULTS

Molecular cloning and mapping studies described in the accompanying paper (14) identify two cross-hybridizing human sequences, termed Sec1 and Sec2, that represent candidates for the human Secretor blood group locus. The cross-hybridizing portions of each of these sequences were subcloned, sequenced, and tested for function, in order to determine if either represent the Secretor blood group fucosyltransferase locus.

The Sec1 DNA Segment Is Most Probably a Pseudogene

Sequence analysis of the cross-hybridizing portion of Sec1 identified substantial primary sequence similarity to the human H blood group alpha(1,2)fucosyltransferase gene (Fig. 1). This sequence yields an open reading frame with primary protein sequence similarity to the H alpha(1,2)fucosyltransferase, beginning at a methionine codon at a position that roughly corresponds to the initiator codon of the H alpha(1,2)fucosyltransferase. This open reading frame predicts an amino-terminal hydrophobic segment typical of the membrane-spanning signal-anchor sequences found in mammalian glycosyltransferases(24) . Nonetheless, the DNA sequence of the Sec1 fragment does not yield a single long translational reading frame corresponding to the H locus, due to the presence of frameshift and nonsense mutations that disrupt this reading frame, and each of the other two, relative to the H alpha(1,2)fucosyltransferase reading frame. Maximal and sustained alignment of amino acid sequence residues predicted by the Sec1 DNA sequence with the H alpha(1,2)fucosyltransferase sequence may be accomplished, however, by conceptual suppression of a single frameshift mutation and a single nonsense mutation, as shown in Fig. 1. This conceptual sequence maintains several predicted asparagine-linked glycosylation sites(25) , including some that are at positions precisely corresponding to those in the H alpha(1,2)fucosyltransferase. Expression of this segment in alpha((1,2)fucosyltransferase-deficient COS-7 cells (via vectors pcDNAI-Sec1 or pcDNAI-Sec1-rev; see ``Materials and Methods'') generates no detectable alpha(1,2)fucosyltransferase activity (data not shown). Furthermore, transcripts corresponding to this gene are not detectable by Northern blot or cDNA selection approaches (data not shown), although these approaches would not detect unstable Sec1-derived transcripts. Thus, these observations, together with the absence of an uninterrupted open reading frame in the Sec1 segment and its inability to encode detectable alpha(1,2)fucosyltransferase activity, lead us to conclude that the Sec1 segment isolated from cosmid 31553 represents a pseudogene.


Figure 1: Comparison between the DNA and derived protein sequences of the H blood group locus and the Sec1 sequence. The DNA and derived protein sequences for the Sec1 DNA segment and the H gene (8, 9) are aligned to maximize DNA sequence identity using the GCG program Align(23) . Amino acid sequence residue numbers are indicated at the left and right of the sequence listings. Nucleotide sequences are numbered in a similar manner, assigning the A residue of the translation initiation codon as position number 1. Nucleotide sequence identity is denoted by a vertical line between the two sequences. Dots denote gaps introduced to maximize sequence identity between the DNA or protein sequences. The derived protein sequences are displayed above (for Sec1) or below (for H) the corresponding DNA sequence. The reading frame used to predict the maximally similar Sec1-derived amino acid sequence is indicated at left by a, b, or c. Amino acid residues within the Sec1-derived protein sequence that are identical with corresponding residues in the H alpha(1,2)fucosyltransferase sequence are indicated by a dash in the Sec1-derived amino acid sequence. Amino acid residues within putative membrane-spanning hydrophobic segments in each amino acid sequence are doubly underlined. A methionine codon that yields an open reading frame with amino acid sequence similarity to the H alpha(1,2)fucosyltransferase is singly underlined, as is the initiator methionine in the H alpha(1,2)fucosyltransferase. Potential asparagine-linked glycosylation sites are indicated by dotted underlining. The frameshift and nonsense mutations that must be conceptually suppressed to maintain amino acid sequence similarity between the protein sequence predicted by the Sec1 DNA sequence are indicated above that sequence.



An Open Reading Frame in the Sec2 DNA Segment Shares Primary Sequence Similarity with the Human H Blood Group alpha(1,2)Fucosyltransferase

Sequence analysis of the Sec2 segment reveals substantial primary DNA sequence similarity to the H blood group alpha(1,2)fucosyltransferase gene (Fig. 2). Translation of the Sec2 sequence, beginning at either of two closely-spaced, in-frame methionine codons, yields a long open reading frame with substantial primary amino acid sequence similarity to the human H blood group alpha(1,2)fucosyltransferase (Fig. 2). This similarity is most marked, and sustained, beginning at a position corresponding to an alanine residue at position 66 in the H alpha(1,2)fucosyltransferase. Each of the two closely-spaced in-frame methionine codons is an appropriate candidate to be an initiator codon; each falls within a consensus splice acceptor splice site(27) , as does the initiator codon at the beginning of the single coding exon in the human H alpha(1,2)fucosyltransferase gene (9) (Fig. 2). Furthermore, the sequence context of each putative initiation codon (distal to the predicted splice acceptor junction ; NCCATGC; NCCATGG) is substantially similar to the Kozak consensus translation initiation sequence(28) . This arrangement suggests the possibility that alternative splicing events might lead to the synthesis of two different polypeptides that differ by the presence, or absence, of an 11-amino acid NH(2)-terminal extension.


Figure 2: Sequence and predicted structure of the enzyme encoded by Sec2. A, comparison between the DNA and derived protein sequences of the H blood group locus and the Sec2 sequence. The DNA and derived protein sequences for the Sec2 and H genes are aligned to maximize DNA sequence identity, using the GCG program Align(23) . Amino acid sequence residue numbers are indicated at the left and right of the sequence listings. Nucleotide sequences are numbered in a similar manner, assigning the A residue of the translation initiation codon of the shortest predicted polypeptide (indicated by the downward pointing arrow) as position number 1. Nucleotide sequence corresponding to coding sequence is displayed by capital letters, whereas sequence predicted to correspond to nontranslated regions is denoted by lower case letters. Nucleotide sequence identity is denoted by a vertical line between the two sequences. Gaps introduced to maximize sequence identity between the DNA sequences are indicated by dotted lines. The derived protein sequences are displayed above (for Sec2) or below (for H) the corresponding DNA sequence. Amino acid residues within the Sec2-derived sequence that are identical with corresponding residues in the H alpha(1,2)fucosyltransferase sequence are indicated by the bullet symbol above the Sec2-encoded residue. Amino acid residues within putative membrane-spanning hydrophobic segments in each protein are doubly underlined. The methionine codon that may initiate the longest of two potential Sec2-encoded polypeptides is singly underlined, as is the initiator codon for the H gene. This predicted protein is 11 amino acid residues longer (indicated in lower case letters) than the shorter Sec2-encoded alpha(1,2)fucosyltransferase (see text for details). The (second) methionine codon tentatively assigned as an initiator codon for this latter protein is singly underlined, and the corresponding methionine residue is indicated by an arrow. Potential asparagine-linked glycosylation sites are indicated by dotted underlining. The polymorphic DNA sequence residues that either inactivate the enzyme (Trp ter), or that are functionally neutral (Gly Ser), are underlined (wild type nucleotide sequence is shown). The corresponding amino acid residue is indicated in bold type. The sequence of the alternative allele and its corresponding protein sequence change are indicated above these positions. Protein sequence-neutral DNA sequence polymorphisms are also indicated (wild type nucleotide sequence is shown). The sequence of the alternative allele is indicated above these positions. The singly underlined section of the Sec2 sequence 3` to the coding region corresponds exactly to the DNA sequence of the partial cDNA derived from this locus and reported in the companion paper(14) . B, schematic diagram of the alpha(1,2)fucosyltransferase encoded by the Sec2 sequence. Proposed domain structure, above, and hydropathy plot(26) , below, for the 332-amino acid polypeptide predicted by the Sec2 DNA sequence. The relative positions of the potential asparagine-linked glycosylation sites (), the inactivating nonsense mutation (Trp stop), and transmembrane segment (&cjs2108;) are indicated on the rectangular schematic representation of the fucosyltransferase. Hydrophobic regions within the predicted protein are indicated by the shading above the horizontal axis of the hydropathy plot. The predicted intracellular locations of the NH(2)-terminal (Cytosol), hydrophobic transmembrane (T.M.), and catalytic (Golgi Lumen) domains are indicated with their amino acid lengths. The potential 11-amino acid-long NH(2)-terminal extension is indicated by the dotted rectangle appended to the shorter Sec2-encoded alpha(1,2)fucosyltransferase. The positions of the AgeI and PstI restriction sites and synthetic (see ``Materials and Methods'') AgeI and EcoRI sites (in parentheses) used to create various expression vectors are also displayed.



Translation from these putative initiator codons predicts the synthesis of either a 332-amino acid-long polypeptide, or a 343-amino acid-long polypeptide, each of which shares 68% sequence identity with the human alpha(1,2)fucosyltransferase protein sequence, across 292 corresponding amino acid residues distal to the conserved alanine residue (Fig. 2). The NH(2) terminus of the protein(s) consists of 3 residues (or 14 residues) that precede a 14-residue hydrophobic segment. This hydrophobic segment is flanked by charged residues and is predicted to function as a signal-anchor sequence (Fig. 2), in a motif that corresponds to the type 2 transmembrane topology typical of mammalian glycosyltransferases(24) . By analogy to these enzymes, it is predicted that the 315 residues that comprise the COOH-terminal catalytic domain reside within the lumen of the Golgi apparatus. Three potential asparagine-linked glycosylation sites are present in this COOH-terminal domain; two of these sites are at positions corresponding to the two potential asparagine-linked glycosylation sites previously identified in the H alpha(1,2)fucosyltransferase (Fig. 2). Sequence analysis also identifies a region of sequence identity between a region 3` to the predicted termination codon in the Sec2 sequence and the hybrid-selected cDNA derived from human small intestinal tissue described in the accompanying manuscript (14) (Fig. 2). This confirms data reported in the accompanying manuscript indicating that the Sec2 sequence is transcribed in the small bowel and suggests that the Sec2 sequence encodes an alpha(1,2)fucosyltransferase gene expressed in some gastrointestinal epithelial cells.

The Open Reading Frame in the Sec2 DNA Segment Encodes an alpha(1,2)Fucosyltransferase Activity

To confirm that this segment encodes an alpha(1,2)fucosyltransferase, a segment encompassing the 332 amino acid residues initiated at the second putative initiator methionine codon was cloned into a mammalian expression vector (see ``Materials and Methods''), to create pcDNAI-alpha(1,2)FTSe-short. A similar vector, termed pcDNAI-alpha(1,2)FTSe-long, was assembled to direct synthesis of the 343 amino acid residues initiated at the first putative initiator methionine codon. These vectors, or a control vector, were then transfected into alpha(1,2)fucosyltransferase-deficient COS-7 cells (see ``Materials and Methods''). Extracts prepared from the transfected COS-7 cells, or control transfected COS-7 cells, were then subjected to assays for alpha(1,2)fucosyltransferase activity. Preliminary assays using an acceptor substrate specific for alpha(1,2)fucosyltransferase activity (phenyl-beta-D-galactoside, (29) ) indicated that cells transfected with pcDNAI-alpha(1,2)FTSe-short, or with pcDNAI-alpha(1,2)FTSe-long, contain substantial amounts of alpha(1,2)fucosyltransferase activity (data not shown). Since previous work indicates that the Secretor locus-encoded alpha(1,2)fucosyltransferase maintains characteristic pH-activity profiles and apparent Michaelis-Menten constants for acceptor substrates and GDP-fucose(10, 11, 12, 13, 19) , we sought to determine these parameters for the alpha(1,2)fucosyltransferase encoded by the Sec2 segment.

The Sec2-encoded alpha(1,2)Fucosyltransferase Is Catalytically Similar to the alpha(1,2)Fucosyltransferase Determined by the Human Secretor Blood Group Locus

The pH optimum of the alpha(1,2)fucosyltransferase activity in transfected cell extracts (see ``Materials and Methods'') is approximately 6.5 (Fig. 3A). This value is similar to that reported previously for the Secretor alpha(1,2)fucosyltransferase(19) . Kinetic analyses demonstrate that this alpha(1,2)fucosyltransferase activity maintains an apparent Michaelis-Menten constant of 11.5 mM (Fig. 3B) for the artificial acceptor phenyl-beta-D-galactoside. This K(m) is similar to the apparent K(m) of 15.1 mM obtained for a preparation of human Secretor alpha(1,2)fucosyltransferase partially purified from human milk(11, 19) and is essentially identical with the apparent K(m) of 11.4 mM reported for a preparation of the human Secretor alpha(1,2)fucosyltransferase purified from human serum(13) . The apparent K(m) values for the acceptor substrates lacto-N-biose I (3.6 mM, Fig. 3C) and N-acetyllactosamine (3.8 mM, Fig. 3D) are also generally quite similar to those determined by others for the Secretor locus-encoded alpha(1,2)fucosyltransferase (10, 11, 13) (Table 1). Likewise, this enzyme exhibits an apparent K(m) of 197 µM for the substrate GDP-fucose (Fig. 3E). This is similar to the relatively high apparent Michaelis constant for this substrate reported previously (13, 19) for an alpha(1,2)fucosyltransferase activity believed to be encoded by the Secretor locus (Table 1).


Figure 3: Catalytic properties of the Sec2-encoded alpha(1,2)fucosyltransferase activity. A, effect of pH on alpha(1,2)fucosyltransferase activity. Enzyme activity encoded by the Sec2 segment in transfected COS-7 cell extracts was measured using 25 mM phenyl-beta-D-galactoside and 3 µM GDP-[^14C]fucose, as described under ``Materials and Methods.'' Buffers used in assays at various pH values are indicated by the symbols in the boxed legend. The pH values displayed here were determined by measuring the pH of the assay solution, as described under ``Materials and Methods.'' B, apparent Michaelis constant for phenyl-beta-D-galactoside. The apparent K (K = 11.5 mM) for the Sec2-encoded alpha(1,2)fucosyltransferase was determined using 3 µM GDP-[^14C]fucose, as described under ``Materials and Methods.'' C, apparent Michaelis constant for lacto-N-biose I. The apparent K (K = 3.6 mM) for the Sec2-encoded alpha(1,2)fucosyltransferase was determined using 3 µM GDP-[^14C]fucose as described under ``Materials and Methods.'' D, apparent Michaelis constant for N-acetyllactosamine. The apparent K (K = 3.8 mM) for the Sec2-encoded alpha(1,2)fucosyltransferase was determined using 3 µM GDP-[^14C]fucose as described under ``Materials and Methods.'' E, apparent Michaelis constant for GDP-fucose. The apparent K(K = 197 µM) for the Sec2-encoded alpha(1,2)fucosyltransferase was determined using 25 mM phenyl-beta-D-galactoside as described under ``Materials and Methods.''





The Secretor locus-determined alpha(1,2)fucosyltransferase activity is detectable in soluble form in milk and in other body fluids (3, 29) . To determine if this biosynthetic process is recapitulated in COS-7 cells, we transfected COS-7 cells with pcDNAI-alpha(1,2)FTSe-short, or with pcDNAI-alpha(1,2)FTSe-long, and quantitated the cell-associated and soluble alpha(1,2)fucosyltransferase activity produced by each vector (see ``Materials and Methods''). We found that media isolated from a 100-mm dish of COS-7 cells transfected with pcDNAI-alpha(1,2)FTSe-short contained approximately 868 total units of activity. By contrast, the cell extract prepared from the same plate of transfected cells contained approximately 118 total units of alpha(1,2)fucosyltransferase activity, for a media/cell extract ratio of approximately 7.4. Similar results were obtained when pcDNAI-alpha(1,2)FTSe-long was used in these experiments (357 units in media versus 55 units in cell extract; media/cell extract ratio of approximately 6.5). These data indicate that both forms of this enzyme are released from the transfected COS-7 cells in a relatively efficient manner. This contrasts with the H blood group cDNA-encoded alpha(1,2)fucosyltransferase activity, which is largely cell-associated when expressed in COS-7 cells(8) .

In aggregate, these data confirm that the Sec2 segment encodes an alpha(1,2)fucosyltransferase. They are also consistent with the hypothesis that this sequence corresponds to an alpha(1,2)fucosyltransferase locus, presumed to be the Secretor blood group locus, that encodes an alpha(1,2)fucosyltransferase found in the serum and milk of secretor-positive individuals.

A Naturally-occurring Nonsense Mutation (Trp ter) Yields an Inactive Sec2 Allele

It is well established that approximately 20% of humans are homozygous for null alleles at the Secretor locus(4, 5) . Naturally-occurring coding sequence mutations yield null alleles at other blood group glycosyltransferase loci, including the human ABO(30) , H(9) , and Lewis(31) blood group loci. We therefore imagined that if the Sec2 alpha(1,2)fucosyltransferase locus does correspond to the Secretor locus, then one or more coding sequence mutations might be identified that would be responsible for the null alleles at the Secretor locus in non-secretor individuals. Indeed, during the course of our DNA sequence analysis of alleles corresponding to the Sec2 DNA fragment, we identified a DNA sequence polymorphism that yields a translation termination codon within the open reading frame in Sec2, as shown in Fig. 2. This DNA sequence polymorphism corresponds to the tryptophan residue at codon number 143 (numbered from the putative initiator methionine of the short protein, Fig. 2), yields a stop codon at this position, and is predicted to truncate a large part (189 amino acid residues) of the enzyme's COOH-terminal segment.

To determine if this polymorphism yields an inactive allele, the expression vector pcDNAI-alpha(1,2)FTse (see ``Materials and Methods''), containing the termination codon at position 143, was tested by transfection for its ability to encode a functional alpha(1,2)fucosyltransferase. No alpha(1,2)fucosyltransferase activity was detected in COS-7 cells transfected with vector pcDNAI-alpha(1,2)FTse (data not shown). One other DNA sequence polymorphism that yields a protein coding sequence alteration is also present in the Trp ter allele derived from cosmid 31553 (Gly Ser; Fig. 2). An expression vector (pcDNAI-alpha(1,2)FTSe-int, see ``Materials and Methods'') containing this other amino acid sequence polymorphism, on the wild type sequence background, was constructed and tested for its ability to express alpha(1,2)fucosyltransferase activity. This vector determined expression of wild type levels of alpha(1,2)fucosyltransferase activity when expressed in COS-7 cells. The results indicate that the DNA sequence polymorphism that creates the translation termination codon inactivates this allele, whereas the other polymorphism is functionally neutral in a qualitative alpha(1,2)fucosyltransferase activity assay.

The Frequency of the Trp ter Mutation Corresponds to the Frequency of the se Allele and Is Present in Double Dose in Non-secretors but Not in Secretor-positive Individuals

We next considered the possibility that this inactivating DNA sequence polymorphism might represent a common null allele of the Secretor locus. Allele-specific oligonucleotide analyses (Fig. 4A) indicated that 10 of 52 unselected, unrelated individuals were homozygous for the Trp ter null allele. This frequency (19%) is virtually identical with the frequency of the non-secretor phenotype in most populations (20%, (4) and (5) ). The remaining persons maintain at least one functional allele at this locus, an observation consistent with the possibility that these persons are secretor-positive by virtue of maintaining at least one functional copy of the Sec2 locus.


Figure 4: Allele-specific oligonucleotide (ASO) hybridization analysis of the Trp ter codon polymorphism. A, ASO analysis of 52 random individuals and 8 control samples. A DNA segment encompassing the polymorphic site was amplified from genomic DNA obtained from each of 52 randomly sampled individuals and probed with a radiolabeled oligonucleotide corresponding to the Trp allele (Wild-type probe) or with a radiolabeled oligonucleotide corresponding to the ter allele (Mutant probe), using reagents and conditions described under ``Materials and Methods.'' Results obtained with control samples derived from cloned versions of each allele, or from individuals with known phenotypes, are displayed in the boxed area at right. B, ASO analysis of six non-secretor individuals, two secretor individuals, and cloned control samples, using reagents and procedures identical with those used in A, above.



To further explore the possibility that homozygosity for the Trp ter null allele is commonly responsible for the non-secretor phenotype, we analyzed the Sec2 sequence at this position in a group of individuals whose secretor phenotypes had been previously determined (see ``Materials and Methods''). Each of six non-secretor individuals were found to be homozygous for the Trp ter null allele, whereas all secretor-positive persons were found to maintain at least one wild type allele at codon 143 (Fig. 4B). Taken together with the physical linkage analyses reported in the accompanying paper(14) , and the biochemical analyses reported here, these genetic results lead us to conclude that the Sec2 locus corresponds to a structural gene encoding the Secretor locus alpha(1,2)fucosyltransferase. They further indicate that the non-secretor phenotype is often, although perhaps not exclusively, due to homozygosity for a common Trp ter null allele at this locus.


DISCUSSION

The results reported here, and in the accompanying paper, indicate that the Sec2 DNA segment corresponds to the human Secretor blood group locus. They also confirm the hypothesis (3) that the H and Secretor loci represent two distinct but closely linked alpha(1,2)fucosyltransferase loci and demonstrate, with other data(9) , that homozygosity for null alleles at these two loci can explain virtually all known recessively inherited H-deficient phenotypes in humans. Nonetheless, it will be useful to obtain additional genetic support for this assignment, by completing genetic studies of this locus in families informative for the secretor and non-secretor phenotypes, analogous to previous studies performed with the H(9) and Lewis(31) blood group loci.

It will also be interesting to determine if this locus maintains structurally distinct alleles with idiosyncratic catalytic properties, analogous to the allelic transferases encoded by the ABO (A and B subgroups; refs. 32-34) and Lewis(31) blood group loci. In particular, the molecular cloning of this locus will facilitate a determination of the molecular basis for the postulated weak Secretor allele (Se(w)) proposed to account for the unusual Lewis blood group phenotype observed in selected individuals of Polynesian ancestry(35, 36) .

The results reported here raise a number of additional issues to be explored. For example, while the Sec1 sequence we analyzed most probably represents a pseudogene, it remains a formal possibility some individuals may maintain an Sec1 allele in which the inactivating frameshift and termination codon ``mutations'' we found are instead represented by DNA sequence (``suppressed'') that encodes a functional alpha(1,2)fucosyltransferase. It is also interesting to consider the possibility that a ``corrected'' Sec1 allele, present either as an endogenous sequence, or derived by ``correction'' events associated with mutation or RNA editing (37-39, reviewed in (40) ), may account for the observation that a cultured human carcinoma cell line expresses an alpha(1,2)fucosyltransferase activity that is catalytically distinct from the H and Secretor locus-encoded enzymes(41) . These issues may be resolved by the construction and testing of an expression vector in which the translational frameshift and termination codons in the Sec1 sequence have been corrected by site-directed in vitro mutagenesis procedures and by a thorough search for transcripts corresponding to the Sec1 locus in human tissues and cell lines.

The presence of two functional initiation codons in the Sec2 locus, in the context of two potential splice acceptor sites, suggests the possibility that alternative splicing events might yield two distinct polypeptides. Since these two polypeptides are predicted to maintain identical catalytic domains, it seems unlikely that the catalytic activities of the corresponding enzymes would differ. However, it is conceivable that splice acceptor site-dependent polymorphism in the length of the proteins' NH(2) termini might in turn influence sub-Golgi localization processes, post-translational proteolytic processing events that liberate catalytically active fucosyltransferase fragments, and functionally relevant consequences of these events (including differential access to different glycoconjugate acceptors). Alternatively, the hypothetical alternatively spliced transcripts might be translated with differential efficiencies. Resolution of these questions will require an exploration of the structures and functions of the transcript(s) and corresponding polypeptides derived from this locus.

Unfortunately, and perhaps not surprisingly, these studies do not address the biological relevance of the oligosaccharides whose expression is determined by the Secretor locus. While there is evidence for associations between secretor status and susceptibility to a variety of malignant and infectious diseases, virtually all of these associations are rather weak (reviewed in (42) ). However, one especially intriguing association concerns the observation that non-secretors maintain an increased relative risk (1.5-fold) for duodenal ulcers(43) . This observation is especially interesting in the context of recent work demonstrating that the bacterial organism Helicobactor pylori can attach to gastric epithelium via type I H and Lewis b oligosaccharide determinants(44) , whose expression in gastrointestinal tissues is determined by the Secretor locus, or by Secretor and Lewis blood group loci, respectively(3) . Since colonization of the gastrointestinal tract by this organism in humans has been associated with histologic gastritis(45) , gastric lymphoma(46) , gastric carcinoma(47, 48) , gastric ulcer(49) , and recurrence of duodenal ulcers(50) , it is possible to speculate that Secretor locus-dependent expression of soluble H-active and Lewis b-active glycoconjugates in the gastrointestinal tract prevents (or allows) H. pylori colonization and indirectly determines susceptibility to the gastrointestinal diseases noted above. The relationship between secretor phenotype and susceptibility to these gastrointestinal diseases remains weak, however, and there is evidence that H. pylori colonization and secretor phenotype may be independent risk factors for these disorders(51) .

In summary, the evolutionary basis for polymorphism at the Secretor locus remains a mystery. Given that there seems to be no obvious or strong selective disadvantage associated with the non-secretor phenotype, one might have initially presumed that the Trp ter null allele would be no more common than any other inactivated allele and would be found, therefore, in only a small fraction of the individuals we examined. Instead, we observed a high frequency of the Trp ter null allele in our studies, high enough to suggest the surprising possibility that most, if not all, non-secretors are homozygous for this allele. It is also surprising that this was the only inactivating DNA sequence alteration we found in the cosmid-derived Sec2 allele. These observations suggest the possibility that this particular inactivated allele once conferred, or still does confer, some specific selective advantage upon members of the human species. Alternatively, it is intriguing to speculate that this locus may indeed have an essential function and that this specific termination codon is suppressed in a tissue-specific manner to yield a functional enzyme in some tissues or at a particular developmental stage, although not in tissues where the secretor phenotype is usually assayed. In particular, it is formally possible that RNA editing mechanisms (40) might suppress this nonsense codon, by changing the A residue in the termination codon at position 143 in the se-derived transcript to a G residue, to yield a wild type tryptophan codon at this position. We note that there is precedence for an RNA editing activity in rodent and human neuronal cells that is capable of catalyzing an A to G change in glutamate receptor transcripts(37, 38, 39) . We also note that there is evidence for editing of a mammalian glycosyltransferase transcript(52) . A better understanding of these issues may be obtained by a systematic determination of the frequency of the Trp ter null allele in a large number of ethnically diverse non-secretor individuals and by determining if non-secretor alleles generally maintain an otherwise wild type coding sequence, in consideration of the possibility that other null alleles can also yield the non-secretor phenotype. Experiments designed to to determine if Se locus-derived transcripts are subject to RNA editing mechanisms might also be informative.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant 1R01HL48859 and under the auspices of the U.S. Department of Energy at Lawrence Livermore National Laboratory under Contract No. W-7405-ENG-48. GCG-related computer sequence alignment services were supported by the General Clinical Research Center at the University of Michigan, funded by a Grant M01RR00042 from the National Center for Research Resources, National Institutes of Health, United States Public Health Service. 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.

This paper is dedicated to the memory of Dr. Mark S. Roth.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17894 [GenBank]and 17895.

§
Present address: Centre National de la Recherche Scientifique, UPR 9008, BP5051, 34033 Montpellier Cedex, France.

Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Institute, MSRBI, Room 3510, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0650. Tel.: 313-747-4779; Fax: 313-936-1400.

(^1)
The abbreviations used are: alpha(1,2)-fucosyltransferase, GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Dr. Rafael Oriol, Dr. Rosella Mollicone, and Dr. David Ginsburg for their helpful comments about this work.


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