Human Molecular Genetics, 2000, Vol. 9, No. 19 2837-2844
© 2000 Oxford University Press
Glutamate carboxypeptidase II: a polymorphism associated with lower levels of serum folate and hyperhomocysteinemia
1Department of Internal Medicine and 2Department of Nutrition, University of California, Davis, CA 95616, USA, 3Oxford Project to Investigate Memory and Ageing, Department of Pharmacology and 4Clinical Trial Service Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX1 3QT, UK
Received 15 May 2000; Revised and Accepted 25 September 2000.
DDBJ/EMBL/GenBank accession no. AF176574.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Low blood folate levels result in hyperhomocysteinemia, which has been associated with increased risk for cardiovascular disease, neural tube defects and cognitive deficits. Intake of dietary folates is the chief determinant of blood folate levels. Molecular defects in the intestinal absorption of dietary folates that precipitate low blood folate levels and hyperhomocysteinemia have not been investigated previously. Dietary folates are a mixture of polyglutamylated folates which are digested to monoglutamyl folates by the action of folylpoly-
-glutamate carboxypeptidase (FGCP), an enzyme that is anchored to the intestinal brush border membrane and is expressed by the glutamate carboxypepidase II (GCPII) gene. We cloned GCPII cDNA from human intestine and identified both a full-length transcript and a 93 bp shorter transcript lacking exon 18, consistent with the presence of a splice variant. In addition, we identified an H475Y polymorphism in GCPII in DNA samples from a healthy Caucasian population (n = 75). We found that membranes of transfected COS-7 cells expressing the H475Y variant GCPII cDNA had 53% less FGCP activity than did cells expressing wild-type GCPII. The presence of the H475Y GCPII allele was significantly associated with lower folate and higher homocysteine levels in this population. These data suggest that the presence of the H475Y GCPII allele impairs the intestinal absorption of dietary folates, resulting in relatively low blood folate levels and consequent hyperhomocysteinemia. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Folates are a family of vitamins that are required for DNA synthesis and the transfer of methyl groups in the methionine cycle (1). Low folate status induces alterations in methionine metabolism that result in hyperhomocysteinemia (2,3) which has been associated with increased risk of cardiovascular disease (4), cognitive deficit in the elderly (5–9) and neural tube defects (10). Blood folate levels are regulated in part by the intestinal absorption of dietary folylpoly-
-glutamates, the predominant form of naturally occurring dietary folates. The absorption of dietary folates involves the initial hydrolysis of terminal glutamate residues of folylpoly--glutamates by folylpoly--glutamate carboxypeptidase (FGCP), an exopeptidase which is anchored to the intestinal apical brush border membrane. The hydrolytic step is followed by membrane transport of monoglutamyl folate derivatives by a separate membrane protein, the reduced folate carrier (11). FGCP may regulate the availability of dietary folates, since folylpoly--glutamates are taken up by the perfused human jejunum at two-thirds of the efficiency of similar concentrations of monoglutamyl folic acid (11,12).
Purified FGCP from pig and human jejunal brush border membranes is a zinc-activated exopeptidase that hydrolyzes the terminal glutamate residues of folylpoly--glutamates (12–14). The nucleotide and predicted amino acid sequences of pig jejunal FGCP cDNA (15) are 88 and 92% identical to those of human prostate-specific membrane antigen (PSM), a marker for prostate cancer (16) and 83% identical to rat N-acetylated 
-linked acidic dipeptidase (NAALADase) (17), a brain enzyme which regulates glutamate neurotransmission by hydrolyzing the glutamate residue of N-acetyl-aspartyl-glutamate. The recent molecular characterization of human brain NAALADase cDNA confirmed its sequence identity to PSM (18). Purified pig jejunal brush border membranes and membranes of human prostate cancer PC3 cells transfected with the cDNA of pig FGCP exhibited both FGCP and NAALADase activity (15), consistent with previous findings that PSM is capable of hydrolysis of glutamate residues of folylpoly--glutamates and N-acetyl-aspartyl-glutamate (19,20). This cumulative evidence supports the concept that FGCP, NAALADase and PSM are functionally distinct expressions of a single gene that encodes 750 amino acids and is collectively known as glutamate carboxypeptidase II (GCPII). The genomic sequence of GCPII (characterized as PSM) is comprised of 19 exons (GenBank accession no. AF007544) and has been localized to chromosome 11p11.2 (21,22). Structural analysis of the predicted amino acid sequence of GCPII showed similarities with the M28 peptidase family and identified a single N-terminal membrane-spanning region and a catalytic region of 313 amino acids, which contains two co-catalytic zinc atoms (23). This model was substantiated by the demonstration that NAALADase activity is reduced by site-directed mutagenesis of the predicted zinc-binding sites of GCPII (24).
We propose that low levels of folate and higher levels of homocysteine in the blood may result from a molecular defect in GCPII that results in decreased absorption of dietary folates. Low levels of circulating folate are associated with decreased folate-dependent conversion of homocysteine to methionine and would predictably result in elevated levels of serum homocysteine. To address this hypothesis, our first objective was to isolate GCPII from human intestine and characterize its molecular structure including identification of potential functional polymorphisms that affect the activity of FGCP in cell culture. Our second objective was to correlate the incidence of a functional polymorphism of GCPII with relatively lower folate levels and hyperhomocysteinemia in a healthy Caucasian population (7).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of human intestinal GCPII
To characterize GCPII from human intestine and to confirm its identity with cDNAs of human PSM and NAALADase, we screened two commercial human intestinal cDNA libraries with an 852 bp PCR fragment of the open reading frame (ORF) of pig jejunal FGCP cDNA and identified two overlapping clones. Sequence analysis showed that these two clones were 100, 88 and 83% homologous to corresponding regions in human PSM cDNA (16), pig FGCP cDNA (15) and rat NAALADase cDNA (17), respectively, with the exception of a 93 bp deletion in the 3' end. Further sequence analysis using BLASTN (25) identified the 93 bp deletion as exon 18. Both the splice variant and the wild-type transcripts of GCPII were identified by RT–PCR of normal human jejunal mucosa obtained at elective gastric bypass surgery in an obese patient. Subsequently, we confirmed the presence of both wild-type and splice variant forms of GCPII by RT–PCR consistently in 40 intestinal biopsy samples from human subjects undergoing diagnostic endoscopic procedures (26). Analysis using rapid amplification of cDNA ends (RACE) recovered an additional 83 bp of the 5' untranslated region (UTR) of intestinal GCPII that placed the transcriptional start site at –138 relative to the translation initiation codon.
A commercial multiple human tissue northern blot (Clontech) was probed with human intestinal GCPII cDNA to determine the tissue distribution of GCPII mRNA. A predominant 2.8 kb band was identified in prostate, small intestine, brain, kidney, liver, spleen and colon, listed in order of decreasing intensity (Fig. 1). A less prominent 1.5 kb band was also identified in liver and kidney.
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Polymorphism analysis of GCPII
To identify a potentially significant polymorphism in GCPII associated with low folate levels and hyperhomocysteinemia, we screened for GCPII polymorphisms in a subset of 75 stored DNA samples obtained retrospectively from healthy Caucasian subjects (age >50 years) residing in Oxford, UK, who participated as a control group in a study of folate and homocysteine levels in Alzheimer’s disease (7). PCR primers were designed on the basis of the genomic sequence of human GCPII (21) in order to amplify the exon–intron borders and 19 exons of human GCPII (Fig. 2). Initially we amplified all exons in 20 DNA samples followed by polymorphism screening using temperature modulated heteroduplex chromatography (27) and identified a polymorphism in exon 13. Subsequent sequence analysis of the PCR product of exon 13 identified a polymorphism, characterized by a C
T single base substitution at nucleotide 1561 corresponding to codon 475, predicted to replace a histidine with tyrosine (Fig. 2). This polymorphism is located in the putative catalytic region of GCPII (23). The structural organization of human GCPII based on its genomic sequence and prior analysis (21,23) is shown in Figure 2.
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We next determined in vitro the functional significance of the H475Y variant in relation to wild-type GCPII. Mammalian COS-7 cells, predetermined to lack endogenous FGCP activity, were transfected with plasmids containing wild-type GCPII cDNA or H475Y GCPII cDNA, prepared as described in Materials and Methods. Compared with FGCP activity in wild-type GCPII COS-7 transfectants, the H475Y GCPII variant resulted in a significant 53% reduction (P < 0.01) in FGCP activity (Fig. 3a). Western blot analysis using the monoclonal antibody J591 against the extracellular domain of GCPII (28) (a gift from Dr N. Bander, Cornell University) identified a 110 kDa band in isolated human jejunal brush border membranes and in membranes from COS-7 transfectants expressing either wild-type GCPII or the H475Y variant of GCPII (Fig. 3b).
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We designed a PCR restriction enzyme method to genotype all the DNA samples. The C
T substitution in codon 475 creates an AccI site (Fig. 4). Amplification of exon 13 by PCR yields a 244 bp fragment, which is cut by AccI to produce 141 and 103 bp bands if the polymorphism is present (Fig. 4). We used PCR followed by digestion with AccI to screen the 75 stored DNA samples for the H475Y GCPII polymorphism, followed by sequence confirmation of positive samples. This process yielded six heterozygous variants and no homozygous variants. Of interest, all six subjects who had the H475Y GCPII allele were women. However, we cannot assume gender specificity since the sample was skewed towards women (44 of 75) and combined analysis of this group with a group of Alzheimer’s disease patients (7) showed equal distribution of the allele among both genders (29). The overall allele frequency of the H475Y GCPII polymorphism in the 75 healthy subjects was 0.04, with a genotype frequency of 8% heterozygote and 92% homozygote wild-type. According to the Hardy–Weinberg Law, the predicted frequency of homozygous genotype for the H475Y GCPII polymorphism in this population is 0.0016 or 1 in 625 subjects.
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Relationship of the H475Y GCPII polymorphism to folate status and hyperhomocysteinemia
Among the 75 healthy individuals with available DNA from the previous study (7), H475Y-positive subjects had significantly lower serum folate levels (P < 0.05) and higher serum homocysteine levels (P < 0.05) than wild-type subjects (Table 1). There was no relationship between the presence of the predetermined 677C
T methylenetetrahydrofolate reductase (MTHFR) allele (7) and H475Y GCPII. There was no significant relationship of the H475Y GCPII allele with red blood cell (RBC) folate or vitamin B12 levels, whereas RBC folate levels were significantly associated (P < 0.05) with vitamin B12 levels. Homocysteine varies by gender, age and by differences in blood levels of vitamin B12, folate and creatinine (28–30). After controlling for gender, age and blood levels of vitamin B12 and creatinine, the relationship of the H475Y GCPII allele remained positively associated with elevated homocysteine levels (P < 0.02). Serum folate was the only variable that influenced the relationship between GCPII genotype and homocysteine levels (P < 0.0001). These findings suggest that the significant association of higher levels of homocysteine with the H475Y allele is a consequence of the effect of serum folate on homocysteine levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We characterized the cDNA sequence of human intestinal GCPII and found near complete identity to human PSM (16) and NAALADase (18). This study confirms prior evidence that human GCPII is expressed as three functionally distinct proteins, namely: intestinal FGCP, brain NAALADase (17,18) and PSM (16). The transcriptional start site for GCPII in human jejunum was determined by RACE at nucleotide –138 relative to the translation initiation codon. This finding contrasts the multiple transcriptional start sites determined for PSM in prostate cancer cells at nucleotides –262, –235 and –195 relative to the translation initiation codon (16,21). The differences in start sites between intestine and prostate suggest that transcriptional regulation of GCPII is governed in a tissue-specific fashion. The present results show also that the 5' UTR of intestinal GCPII mRNA is shorter than that determined for PSM mRNA (16,21). A PSM splice variant, denoted as PSM', was identified in human prostate cancer LNCaP cells and characterized as lacking 266 bp of the 5' region of the PSM cDNA, corresponding to 146 bp of the 5' UTR and 120 bp of the ORF (30,31). However, it is unlikely that PSM' is present in the intestine since the 5' UTR region present in PSM' is not found in intestinal GCPII mRNA. On the other hand, an intracellular enzyme capable of hydrolyzing both internal and terminal
-glutamate residues of folylpoly--glutamates was previously purified from human intestinal mucosa (32) and is encoded by a separate gene (33). A previous study from our laboratory demonstrated that intestinal brush border FGCP and not the intracellular enzyme functions in the digestion of folylpoly--glutamates in the lumen of pig intestine (14). The identification of a GCPII splice variant lacking exon 18 in human jejunum is consistent with previous reports describing a similar 93 bp shorter NAALADase transcript in rat (34) and human brain (35). Regulation of the translation and the potential effect of varied amounts of the splice variant on FGCP and NAALADase activities remain to be determined.
Further studies of human GCPII expression using northern analysis showed its expression as a predominant 2.8 kb band in prostate, small intestine, brain, liver, kidney and colon (Fig. 1), consistent with previously reported findings of PSM expression in prostate cancer cells (16) and NAALADase expression in brain (17,18). We also identified a less predominant GCPII band at 1.5 kb in the kidney and liver, which has not been reported previously for PSM or NAALADase. The significance of this smaller transcript remains to be determined but may represent tissue-specific splice variants or products of GCPII mRNA degradation. These findings confirm the multiple tissue expression of GCPII and its expression in prostate, small intestine and brain (15–18). Further studies are required to determine the horizontal (proximal to distal) and vertical (crypt to villus) distribution of GCPII expression in human small intestinal epithelial cells.
Using DNA samples from a previous study of Caucasian subjects (7), we identified a CT polymorphism predicted to replace a histidine with a tyrosine at codon 475 in exon 13 of the catalytic region of GCPII (Fig. 2). The functional significance of the polymorphism was shown by findings in vitro that the H475Y GCPII cDNA expressed 47% of the activity of wild-type FGCP in membranes of transfected COS-7 cells (Fig. 3). Western blot analysis revealed that cell membranes expressing the H475Y variant exhibited GCPII protein with a band intensity that was somewhat less than that observed in membranes of cells expressing wild-type GCPII. Since the J591 antibody binds to the extracellular region of GCPII (28) that also contains the H475Y polymorphism, the lower band intensity observed for cell membranes expressing the H475Y variant of GCPII may simply reflect decreased efficiency of the binding of the antibody. Alternatively, the H475Y polymorphism may affect FGCP activity through altered post-translational processing and/or activity due to altered configuration of the catalytic region of the enzyme.
Intestinal FGCP cleaves glutamate residues from folylpoly--glutamates and thus plays an important regulatory role in the intestinal absorption of dietary folylpoly--glutamates (11). Therefore, polymorphisms in GCPII affecting the activity of FGCP would predictably decrease the intestinal absorption of dietary folylpoly--glutamates and consequently decrease folate levels and increase homocysteine levels in the body. To test this hypothesis, we analyzed DNA samples from a subset of healthy control subjects from a previous study of English subjects (7). Our hypothesis was corroborated by the present findings of significantly lower serum folate levels and higher homocysteine levels in the subjects with the H475Y GCPII allele than in subjects with wild-type GCPII alleles (Table 1). These findings are consistent with the concept that the H475Y GCPII allele affects folate absorption, resulting in lower serum folate levels in a healthy population.
Serum folate levels may be influenced by variations in dietary intakes of folates, which were not assessed in the original study of the present cohort (7). During the time when the majority of samples were collected the average daily folate intake for the population in the UK was 233 ± 90 µg/day according to a national survey carried out in 1994/1995 (36) which is less then the current recommended dietary intake of folate at 400 µg/day in the USA (37). Further work is required to determine whether the effect of the H475Y GCPII polymorphism on blood folate and homocysteine levels is influenced by the amount of dietary and/or supplemental folates in a similar way to the effect of the C677T MTHFR polymorphism (38).
That we observed no differences in RBC folate levels according to GCPII genotype may be explained by the confounding effect of vitamin B12 levels, which were unrelated to GCPII genotype but which affect levels of tissue folates. Methylated monoglutamyl folates must be demethylated and then polyglutamylated by folylpoly--glutamate synthase for intracellular storage in tissues as polyglutamyl folates (1). The demethylation reaction is catalyzed by vitamin B12 and, therefore, the ability of RBCs to store polyglutamylated folates is dependent on vitamin B12.
Serum homocysteine levels are influenced by nutritional deficiencies of folate, vitamin B12 or vitamin B6, by genetic defects in one or more of the proteins involved in the metabolism of methionine and by renal function, age and gender (39–42). The thermolabile 677CT polymorphism in MTHFR, an enzyme required for the synthesis of methyl tetrahydrofolate, the substrate for the conversion of homocysteine to methionine, has been implicated in the pathogenesis of hyperhomocysteinemia (42). In the present study, the association of the H475Y allele of GCPII with higher homocysteine levels was independent of gender, age, renal status and MTHFR genotype. Within the entire group, serum folate also influenced the relationship between GCPII genotype and homocysteine. This finding implies that the relationship of the H475Y GCPII allele with homocysteine levels is mediated by the effect of the variant allele on serum folate levels. Further studies with larger population groups are needed to establish the significance of the H475Y polymorphism in the expression of GCPII in clinically relevant conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cloning of human intestinal FGCP cDNA
Two separate human jejunal cDNA libraries in pcDNA2 (Invitrogen, Carlsbad, CA; a gift of B. Lonnerdal, University of California, Davis, CA) and
gt10 (Clontech, Palo Alto, CA; a gift of H. Said, University of California, Irvine, CA) were screened with an [-32P]dCTP-labeled 852 bp PCR fragment of the ORF of the pig jejunal FGCP cDNA (15). Standard protocols were followed for library screening (43). Two positive clones were identified and sequenced using the LI-COR 4200 automated sequencer (LI-COR, Lincoln, NE). The transcriptional start site was determined by 5' RACE using the Marathon Ready human small intestine cDNA and the Marathon RACE kit (Clontech). The first PCR reaction used the gene-specific primer 5'-GTGCTAGCTCAACAGAATCCAGGC-3' followed by a second PCR reaction with the nested gene-specific primer 5'-CCACAGCCGAGTCGGTTTCGTGAA-3'. Comparative analysis with the GenBank nucleic acid database using BLASTN (25) revealed that clones pFGCP52 and pFGCP72 were completely homologous to the human PSM cDNA sequence (16) except for a 93 bp deletion in the 3' end of clone pFGCP52. Further sequence analysis with PSM genomic sequence (21) revealed that the 93 bp deletion represented exon 18. The presence of the splice variant in human intestine was confirmed by RT–PCR of human jejunal mucosal RNA. A specimen of normal adult human jejunum was obtained at surgery from an obese patient undergoing elective gastric bypass with construction of gastro-jejunal anastomosis. The jejunal mucosa was scraped with a glass slide then immediately placed in TRIzol reagent (Life Technologies, Grand Island, NY), frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA was extracted using TRIzol reagent followed by reverse transcription using Superscript reverse transcriptase (Life Technologies) and PCR of the first strand cDNA using primers flanking exon 18, forward in exon 16 (5'-GAAACAAACAAATTCAGCGG-3') and reverse in exon 19 (5'-AGAGCATCATAAATTCCTGG-3'). BLASTP (25) was used to compare the predicted amino acid sequence of human intestinal GCPII cDNA with the GenBank protein database.
Northern analysis
Selected human multiple tissue northern blots of mRNA were obtained from a commercial source, each standardized to actin (Clontech) were hybridized at 42°C for 18 h with clone pFGCP52 labeled with [-32P]dCTP using the RadPrime DNA labeling system (Life Technologies). Following hybridization and washing, the blots were exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY) for 48 h with intensifying screens at –80°C.
Human GCPII polymorphism analysis
Genomic DNA samples were obtained retrospectively from 75 Caucasian subjects, aged 50 years and older, who participated as control subjects in a previously published study of folate metabolism in Alzheimer’s disease (7). Exons and exon–intron borders of GCPII were amplified in each DNA sample using the following primers designed from the reported genomic PSM sequence (21):
exon 2, 5'-GTATCTCATGCCTATTTTTGG-3'
and 5'-GTCCATATAAACTTTCGAGGA-3';
exon 3, 5'-CCACTTTCAATTTTTGTTCACC-3',
5'-ACATCATAATGAGCTAGCTCAA-3',
5'-CCTGTTGTCCTACCCAAA-3'
and 5'-AAATGGGGGGAATGTTTC-3';
exon 4, 5'-GTAATCAAACTTTAACTG-3'
and 5'-GTCATTTTTATTTGTTGC-3';
exon 5, 5'-AAGTCATTCCCGATCTCAA-3'
and 5'-AACTTTTTACATTGGGGGA-3';
exon 6, 5'-AAAGACACTGTGTTTTCTAG-3'
and 5'-AAGTTTCCTATGATATTCAA-3';
exon 7, 5'-AACTGAGTTTTTACTGGAAA-3'
and 5'-ACTGTCAATAAGAAATTAC-3';
exon 8, 5'-AACATTTTAATGCGTTACTTCAA-3'
and 5'-CCCCTTAAAAGAGTTAAAATTAA-3';
exon 9, 5'-AATTCCCTTAAACCTTATAG-3'
and 5'-ACTTGATCAATATTTGCTAA-3';
exon 10, 5'-AATCTTGTATCTATGCTTTCTAG-3'
and 5'-AGAAGACTAAACTGAGACTCAG-3';
exon 11, 5'-GGGCTTGGTAGTGTCCTGG-3'
and 5'-GCTTGGCAAACAAGTCCTGG-3';
exon 12, 5'-AATCTACATACATTAAATGTAG-3'
and 5'-AGTGCATAATTTATTAACTAGA-3';
exon 13, 5'-CATTCTGGTAGGAATTTAGCA-3'
and 5'-AAACACCACCTATGTTTAACA-3';
exon 14, 5'-AATGGACATCTAGTTTTTAA-3'
and 5'-ACTTAATGATTGAAAGAAAA-3';
exon 15, 5'-AAGAATGGGGTTTAGTTTAA-3'
and 5'-AGTCAGAATTATTTGTTTAG-3';
exon 16, 5'-AATAGTAAATATGG TGCTAA-3',
5'-ACAGTGAGGTGATATTTAAA-3',
5'-AAATATCACCTCACTGTGG-3'
and 5'-AATAGAACCATACAGATGAG-3';
exon 17, 5'-AAGCATTTGATGATC CCAA-3'
and 5'-AAAGCTAGTTCAGCAACAG-3';
exon 18, 5'-GGTAGAAACATATTTTCATGAA-3'
and 5'-AAAAAGTGATATTACAGAAAGG-3';
exon 19, 5'-GGGC ACCTAATAAACAGCAAA-3'
and 5'-GGCTACTTCACTCAAAGTCTC-3'.
Each PCR product was screened for polymorphisms by temperature-modulated heteroduplex chromatography (27) using the WAVE DNA Fragment Analysis System (Transgenomic, San Jose, CA). PCR samples found to contain polymorphisms and representative wild-type samples were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and both strands were sequenced using the PCR primers and an ABI Prism 377 DNA automated sequencer.
Biochemical analyses
Non-fasting blood samples were obtained in the period 1988–1995 as described previously (7). Serum and RBC folate levels were determined by microbiological assays with Lactobacillus casei in the Clinical Chemistry Labaoratory of the Radcliffe Infirmary, Oxford University, Oxford, UK. Homocysteine levels were determined by high-performance liquid chromatography with fluorescence detection (44). Vitamin B12 levels were measured by radioimmunoassay.
Transfection studies
COS-7 cells (ATCC, Rockville, MD) were transfected with DNA constructs of the wild-type GCPII cDNA or GCPII cDNA containing the H475Y polymorphism. The cells were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum, 50 U/ml penicillin G and 50 µg/ml streptomycin (Life Technologies). The cells were grown in at 37°C in a 5% CO2 incubator. The wild-type GCPII cDNA was synthesized by PCR of reverse transcribed human jejunal mucosal RNA using Pfu polymerase (Stratagene, La Jolla, CA) and primers at the 5' end based on the RACE sequence and the 3' end based on the clone pFGCP52 sequence. The wild-type GCPII cDNA was cloned into the mammalian expression vector, pTRACER-CMV2 (Invitrogen). The H475Y polymorphism was introduced into GCPII cDNA in pTRACER-CMV2 using the QuickChange Site-Directed Mutagenesis kit (Stratagene). The primers used in the PCR reaction were 5'-CCGCTGATGTACAGCTTGGTATACAACCTAACAAAAG-3' and 5'-CTTTTGTTAGGTGTATACCAAGCTGTACATCAGCGG-3'. The resulting product was transformed into XL1-Blue cells. Both the full-length wild-type GCPII cDNA and mutated GCPII cDNA constructs were sequenced using an ABI Prism 377 DNA automated sequencer (PE Biosystems, Foster City, CA). Transfections of COS-7 cells with wild-type and the H475Y variant forms of GCPII were accomplished using LipofectAMINE and OPTI-MEM I medium (Life Technologies) according to the manufacturer’s protocol. One day prior to transfection, the cells were seeded onto 6-well plates and grown in serum-containing DMEM so that on the day of transfection the cells were 60–80% confluent. Cell membranes were harvested at 48 h post-transfection by scraping into 50 mM Tris–HCl (pH 7.4) followed by homogenization and removal of supernatant after centrifugation, 35 000 g for 30 min. The complete membrane pellets were resuspended in 50 mM Tris–HCl and the protein concentration of the membranes was determined using the enhanced protocol BCA assay (BioRad, Hercules, CA). Expression of green fluorescence protein was used to monitor transfection efficiency and uniformity. Similar transfection efficiencies were observed for cells transfected with either wild-type GCPII or H475Y variant GCPII. Mock-transfected cells served as controls.
FGCP enzyme activity
FGCP activity was determined in COS-7 transfectant membranes using 12 µM folyl--glutamyl--[14C]glutamate substrate and a modification (14) of the method of Krumdieck and Baugh (45). As described previously, this method uses a saturable concentration of substrate and exhibits identical kinetics in purified human and pig intestinal FGCP (13,14). The method was validated previously for measurement of FGCP activity in membranes of PC-3 cells transfected with pig intestinal GCPII, in which we found similar Km values for transfectant membranes and purified pig jejunal brush border membranes (15).
Western blot analysis
Human jejunal brush border membranes were isolated as described previously (14). Membranes from cell transfectants (25 µg protein) were resolved on a 7.5% Tris–HCl SDS–polyacrylamide gel (BioRad) and transferred to PDVF membranes (Millipore, Bedford, MA). Immunodetection was performed using the J591 monoclonal antibody against the extracellular domain of GCPII (28) (gift of Dr N. Bander, Cornell University) followed by a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). Bands were detected using the SuperSignal West Dura Extended Duration Substrate (Pierce) and a CCD camera (NucleoTech, Hayward, CA) that detects chemiluminescence.
Statistical analyses
Two-sample Student’s t-test was used to determine whether the presence of the H475Y GCPII allele had an effect on serum folate, RBC folate, vitamin B12 and homocysteine. One-way analysis of covariance was use to further analyze the association of the H475Y GCPII allele with homocysteine levels controlling for the potentially confounding variables of gender, age, vitamin B12 and creatinine.
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ACKNOWLEDGEMENTS |
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We thank Dr Neil Bander, New York Presbyterian Hospital-Weill Medical College of Cornell University and BZL-Biologics for providing the J591 monoclonal antibody. We thank Lin Barnetson, Donald Warden and all other members of OPTIMA; Bristol-Myers Squibb for a grant to support OPTIMA; and P. Ueland and H. Refsum (Bergen, Norway) for the homocysteine assays. This work was supported by grants DK-45301 and DK-35747 from the United States Public Health Service.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 530 752 6778; Fax: +1 530 752 3470; Email: chhalsted@ucdavis.edu
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REFERENCES |
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