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J. Biol. Chem., Vol. 276, Issue 41, 38166-38172, October 12, 2001

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Alternative Splicing Determines the Function of CYP4F3 by Switching Substrate Specificity^*

Peter Christmas^§, Jeffrey P. Jones^¶, Christopher J. Patten, Dan A. Rock^¶, Yimin Zheng^**, Shing-Ming Cheng, Brittany M. Weber, Nadia Carlesso, David T. Scadden, Allan E. Rettie^**, and Roy J. Soberman

From the  Center for Immunology and Inflammatory Diseases and the  AIDS Research Center and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, the ^¶ Department of Chemistry, Washington State University, Pullman, Washington 99164, the ^** Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, and  Gentest Corporation, Woburn, Massachusetts 01801

Received for publication, May 25, 2001, and in revised form, July 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB₄ by -hydroxylation (K_m = 0.68 µM) but has low activity for arachidonic acid (K_m = 185 µM); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher K_m for LTB₄ compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for -hydroxylation (K_m = 22 µM) and generates 20-HETE, an activator of protein kinase C and Ca²⁺/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytochrome P450 (CYP)¹ monooxygenases catalyze the oxidation of a broad spectrum of lipophilic substrates that include endogenous products such as cholesterol, steroids, and fatty acids, or xenobiotics such as drugs. Fifty-five human CYP genes have been classified into 17 families and 40 subfamilies.² Phylogenetic studies indicate that all CYPs derive from duplication and divergence of an ancestral gene, and this radiation of CYP genes accounts for the diverse substrate utilization by the superfamily. To date, alternative splicing of CYP pre-mRNAs is not considered a mechanism that contributes to this functional diversity.

CYP-dependent oxidation of arachidonic acid generates biologically active eicosanoids that function as intracellular mediators in normal physiology and disease. Members of the CYP2C and CYP2J subfamilies act as arachidonic acid epoxygenases and generate eicosatrienoic acids (EETs), which regulate ion channels and are candidates for endothelium-derived hyperpolarizing factor (1-2). CYP4 enzymes catalyze -hydroxylation of fatty acids including arachidonic acid and are a potential source of 20-HETE. 20-HETE is a potent activator of protein kinase C and Ca²⁺/calmodulin-dependent kinase II and has roles in regulating vascular tone, natriuresis, and cell proliferation (3-7). Enzymes in the CYP4A subfamily have high activity for arachidonic acid in animals (8). The identity and distribution of enzymes generating 20-HETE in humans is poorly understood. This determination is complicated by wide-ranging K_m values of different enzymes for arachidonic acid, distinct patterns of tissue expression of the relevant enzymes, close homology between subfamily members, and variation between species. The human enzyme CYP4A11 has been shown to generate 20-HETE (9), but it exhibits very low activity for arachidonic acid (10). Recently it was suggested that -hydroxylation of arachidonic acid in human liver and kidney is mediated primarily by CYP4F2 (11, 12).

In addition to activating arachidonic acid, CYPs can inactivate LTB₄ and prostaglandins by -hydroxylation. CYP4F3 was originally identified as the enzyme that catalyzes -oxidation of the 5-lipoxygenase pathway product LTB₄ with a low K_m (0.5-1.0 µM) in human neutrophils (13-15). LTB₄ functions as a chemoattractant of neutrophils and monocytes (16, 17), and CYP4F3 has been projected to play a role in the termination of LTB₄-mediated inflammation. We identified an alternative splice form of CYP4F3 in liver (18) and designated the two isoforms CYP4F3A (the original isoform detected in neutrophils) and CYP4F3B (the isoform detected in liver). Both isoforms have 520 amino acids but are distinguished by the alternate use of exons that code for amino acids 67-114. CYP4F3B contains exon 3, whereas CYP4F3A contains exon 4. These exons are identical in size but code for sequences that share only 27% amino acid identity. CYP4F3B has lower activity for LTB₄ and is not expressed in myeloid cells, whereas CYP4F3A is not expressed in liver.

We analyzed the tissue distribution and kinetic properties of CYP4F3B to determine its functional significance. CYP4F3B has an expression pattern that is distinct from CYP4F3A and is the predominant CYP4F enzyme in liver and other non-hematopoietic tissues. It has a K_m for arachidonic acid of 22 µM and generates 20-HETE as the major product of -hydroxylation. In contrast, arachidonic acid is a very poor substrate for CYP4F3A. We used molecular modeling to predict the position of amino acids 67-114 within the molecule. These studies suggest that exons 3 and 4 code for a region that contributes to the active site and substrate access channel. Selection between these exons determines the ability of the enzyme to either inactivate LTB₄ (CYP4F3A) or activate arachidonic acid (CYP4F3B). The results demonstrate that tissue-specific alternative splicing of pre-mRNA must now be considered a mechanism for generating functional diversity of cytochrome P450s.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LTB₄ and Arachidonic Acid Hydroxylation Assays-- Microsomes containing human CYPs (specific content of 640 pmol of 4F3B/mg of protein or 36 pmol of 4F3A/mg of protein), P450 reductase, and cytochrome b₅ (Supersomes^TM) were prepared from baculovirus-infected BTI-TN-5B1-4 cells by Gentest Corporation. To assay the conversion of LTB₄ to 20-hydroxy-LTB₄, reaction mixtures containing Supersomes (10 pmol of CYP enzyme), LTB₄, 1 mM NADPH, and 100 mM KPO₄ buffer, pH 7.4 were incubated in a final volume of 0.1 ml for 20 min at 37 °C. The reaction was terminated with 0.1 ml of 94% acetonitrile, 6% glacial acetic acid and centrifuged at 10,000 × g for 3 min. The supernatant (60-85 µl) was injected into a Zorbax C18-SB reverse phase HPLC column (4.6 × 250 mm, 5 µm) coupled to a 5-µm Waters Sentry guard column and separated with a linear gradient of 30% acetonitrile, 2 mM perchloric acid to 70% methanol over 24 min at a flow rate of 1.0 ml/min. The product was detected by absorbance at 270 nm using a Waters 2487 UV detector and its identity confirmed by comparing the retention time of a 20-hydroxy-LTB₄ standard (Cayman). Kinetic results were analyzed by non-linear regression (Sigma Plot Software, SPSS inc., Chicago, IL) using eight substrate concentrations in a range of 0.2-20 µM (4F3A) or 2-80 µM (4F3B). To assay the conversion of arachidonic acid to 20-HETE, reaction mixtures containing Supersomes (10 pmol of CYP enzyme), arachidonic acid, 0.15 µCi of [¹⁴C]arachidonic acid (810 Ci/mmol), 1 mM NADPH, and 100 mM KPO₄ buffer, pH 7.4 were incubated in a final volume of 0.1 ml for 10 min at room temperature. The reactions were terminated and injected into a C₁₈ reverse phase HPLC column as before and separated with a linear gradient of 0.1% trifluoroacetic acid in H₂0 to 0.1% trifluoroacetic acid in acetonitrile over 24 min at a flow rate of 1.0 ml/min. The product was detected by on-line monitoring with a RadioFlow detector (Flo-One Beta 150TR, Packard BioScience). The retention time of a 20-HETE standard (Cayman) was determined by absorbance at 205 nm. Kinetic results were analyzed by linear regression using 8 substrate concentrations in a range of 1.0-150 µM (4F3A) or 0.5-60 µM (4F3B). The kinetic experiments were performed under conditions where less than 10% of the substrate was converted to product.

Hybridization of cDNA Probes-- CYP4F3 cDNA probes were labeled with [-³²P]dCTP by random priming. The full-length coding region of CYP4F3A and a partial cDNA between NsiI and EagI sites (nucleotides 561-1268) were used to generate probes of 1560 bp and 708 bp, respectively. Multiple Tissue Northern (MTN) Blots from CLONTECH were hybridized with the 1560-bp probe for the full-length coding region (5 ng/ml, ~10⁶ cpm/ng) in 10 ml of ExpressHyb (CLONTECH) for 1 h at 68 °C. The blots were washed in 2× SSC, 0.05% SDS at room temperature for 4 × 10 min and then 0.1× SSC, 0.1% SDS at 50 °C for 2× 20 min. A Human Multiple Tissue Expression (MTE) Array from CLONTECH was hybridized with the 708-bp probe (2 ng/ml, ~2 × 10⁶ cpm/ng) for 6 h at 65 °C in 10 ml of ExpressHyb supplemented with 300 µg of sheared salmon testes DNA (Sigma), 60 µg C₀t-1 DNA (Roche Molecular Biochemicals), and 100 µl of 20× SSC. The partial cDNA probe was used because it gave lower background than the full-length probe, and nucleotides 561-1268 were selected for this probe to ensure that both isoforms of CYP4F3 would be recognized with equal efficiency. The array was washed in 2× SSC, 1% SDS at 65 °C for 5× 20 min and then 0.1× SSC, 0.5% SDS at 55 °C for 2× 20 min. The MTN Blots and MTE Array were exposed to Kodak XAR film overnight at 70 °C with an intensifying screen. Hybridization signals on the MTE Array were quantified with a phosphorimager (Cyclone, Packard BioScience) and expressed relative to peripheral blood leukocytes.

RNA Isolation and RT-PCR-- RNA was extracted from freshly isolated human bone marrow cells (1-2 × 10⁶ cells) using the RNeasy Kit (QIAGEN) with QIAshredder columns for cell homogenization. Total RNA was purified from peripheral blood neutrophils as previously described (18). Total and poly(A)⁺ RNA from liver, fetal liver, kidney, prostate, ileum, and trachea were from CLONTECH. First-strand cDNA synthesis for PCR was performed using the cDNA Cycle Kit (Invitrogen) with AMV reverse transcriptase and random primers. The cDNA was purified by phenol/chloroform extraction and ethanol precipitation. Primers B, C, G, and H (Table I) were used to detect CYP4F3A (primer pair B-C), CYP4F3B (B-G), and CYP4F2 (H-G) by isoform-specific PCR as previously described (18). The PCR conditions were 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min; 30 cycles were followed by 1 cycle with a 10 min extension time. Primers specific for CYP4F8 (K-L) and CYP4F11 (M-N) were used under the same PCR conditions except for annealing temperatures of 65 °C and 62 °C, respectively.

Quantitative PCR Analysis of CYP4F Isoforms-- cDNA samples generated as described above were used to estimate the relative levels of expression of different CYP4F gene products. A variety of primers specific for CYP4F gene products (CYP4F3A, CYP4F3B, CYP4F2) and internal standards (GAPDH, cyclophilin A, 18 S rRNA) were tested in semiquantitative PCR experiments. Primer pair E-F (CYP4F3B-specific, 962-bp product), or I-J (CYP4F2-specific, 962-bp product), worked well in combination with the internal standard primers GDHF and GDHR (GAPDH-specific, 458-bp product). The PCR conditions were 94 °C for 1 min, 68 °C for 1 min, 72 °C for 1 min, 20 cycles. This cycle number was selected after comparing the linear range of ³²P incorporation for the different cDNAs and primer pairs used, and it represents a point within the linear range for all samples. Each reaction contained 5 µCi of [-³²P]dCTP, unlabeled dNTPs at a final concentration of 0.2 mM each, and 20 pmol of each primer. PCR products were separated by electrophoresis on a 6% sequencing gel (SequaGel System, National Diagnostics). The gel was dried onto filter paper, and the products were visualized by exposure to Kodak XAR film and quantified with a phosphorimager. The relative CYP4F isoform expression level in each sample was determined by comparing the intensity of the CYP4F and GAPDH signals. The results of four experiments using different RNA batches were used to determine the mean ± S.D.

A limited number of primers can be designed that are specific for CYP4F3A, and these failed to give PCR products when combined with primers for the internal standards required in quantitative PCR experiments. We attempted to use a variety of primer sequences, primer lengths, and annealing temperatures for CYP4F3A and internal standards that included GAPDH, cyclophilin A, and 18 S rRNA + competimers (Ambion) to eliminate this problem. None of these was successful, though the reasons for the inhibition were not clear.

An alternative method for quantifying the relative levels of CYP4F3A in tissues was developed using a generic CYP4F primer pair (A-D), which generates a 449-bp PCR product from CYP4F3A, CYP4F3B, CYP4F2, and CYP4F11. PCR was performed: 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min; 20 cycles were followed by 1 cycle with a 10 min extension time. The PCR products obtained from each tissue were then ligated into pCR2.1-TOPO vector (Invitrogen) and transformed into TOP10 cells. Individual colonies were picked for isolation of plasmid DNA and sequence analysis. A minimum of 50 colonies from each tissue were analyzed. Two methods of analysis of plasmid DNA were performed, which gave identical results: plasmid DNA from 50% of colonies were sequenced; alternatively, restriction digests were used to predict the outcome of sequence analysis. The 449-bp PCR products generated by primer pair A-D contain sites for both MscI and HincII (CYP4F2), HincII only (CYP4F11), MscI only (CYP4F3B), or neither enzyme (CYP4F3A). A comparison of the restriction pattern generated with MscI versus HincII predicts the identity of the PCR product. CYP4F8 and CYP4F12 are excluded from this analysis.

Homology Modeling of CYP4F3A and CYP4F3B-- Initial alignments of P450BM3 and CYP4A1 from rat were taken from the work of Hasemann et al. (19). CYP4A1 was aligned with the CYP4F enzymes with the multiple sequence alignment program ClustalW and the PAM250 similarity matrix. A second alignment was performed on amino acids 50-150 using ClustalW and the sequences for 4A7, 4A11, 4B1, 4F2, 4F3A, 4F3B, 4F12, and BM3. A three-dimensional homology model was constructed from these alignments using the program Modeler (20). This program uses a combination of molecular dynamics and restraints based on the known structures of homologous enzymes. Thus, this program will give a three-dimensional representation of an alignment that has a predicted structure similar to the known structure. The degree of similarity between target and model is dependent on the degree of implied homology in the alignments. Thus, structure in areas with inserts will be determined solely by the molecular dynamics force field, and areas without inserts will give a weighted average of the restraints and the force field.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Relationship of Human CYP4F Genes and cDNAs-- Five members of the human CYP4F subfamily have been described: 4F3 (15), 4F2 (21), and 4F8 (22) have 520 amino acids, and 4F11 (23) and 4F12 (24, 25) have 524. The proteins share ~ 80% amino acid identity. The genes exhibit a highly similar organization and were originally assigned 13 exons, with the ATG initiation codon at the beginning of exon 2 (22, 23, 26, 27). Our analysis of the 4F3 gene identified an additional exon which can participate in alternative splicing reactions (18). The 4F3 and 4F2 genes are now considered to have 14 exons as shown in Fig. 1. Exons 3 and 4 in the 4F3 gene are identical in size (145 bp) and are mutually exclusive, each coding for amino acids 67-114. The cDNA of 4F3 originally isolated from human neutrophils (now designated 4F3A) contains exon 4, which lacks a homologous counterpart in other 4F cDNAs. 4F3B contains exon 3, which shows high identity with the exon coding for amino acids 67-114 in all other 4F members. Exon 3 in 4F3B has 83, 75, 66, and 60% amino acid identity with the corresponding exon in 4F2, 4F8, 4F11, and 4F12, respectively. This identity is highest for 4F2, and in all cases is much greater than the 27% seen between exons 3 and 4 within the 4F3 gene. Analysis of genomic sequences determined that a homolog of 4F3 exon 4 is present in the 4F2 gene (18), but not the 4F8, 4F11, or 4F12 genes. A reassignment of 14 exons to the 4F2 gene, analogous to 4F3, is shown in Fig. 1. To determine the significance of alternative splicing we investigated whether 4F3B has a distinct substrate preference and tissue distribution compared with 4F3A, and whether a novel isoform of 4F2 containing exon 4 is expressed.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Organization of the human CYP4F subfamily. The homologous exons from the human CYP4F family of genes are aligned vertically, and known splicing pathways are schematically represented. Selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B) in CYP4F3 expression is mutually exclusive. Both exons are 145 bp and code for amino acids 67-114, but there is only 27% amino acid identity. Amino acids 67-114 in CYP4F2, CYP4F8, and CYP4F11 comprise an exon that has 83, 75, and 66% amino acid sequence identity, respectively, with CYP4F3 exon 3. Genomic sequence analysis predicts that the CYP4F2 gene but not the CYP4F8 or CYP4F11 genes, contains a homolog of CYP4F3 exon 4. Arrows indicate the positions of primers referred to in Table I. CYP4F12 is not shown.

Substrate Specificity of CYP4F3 Isoforms-- The 4F3B enzyme converts LTB₄ to 20-hydroxy-LTB₄ (Fig. 2A) with a K_m of 20.6 µM using a substrate concentration range of 1-80 µM. Substrate inhibition was observed at higher concentrations of LTB₄ (onset > 80 µM) for 4F3B, but not 4F3A. The K_m of 4F3A for LTB₄ was 0.68 µM, consistent with previous reports (14, 15). 4F3B utilizes arachidonic acid as a substrate and generates 20-HETE as its major product (Fig. 2B). Minor products with retention times of 16.8 min and 13.7 min likely correspond with the oxidation products 20-aldehyde-AA and 20-carboxy-AA, respectively, analogous to the conversion of 20-hydroxy-LTB₄ to 20-aldehyde-LTB₄ and 20-carboxy-LTB₄ (13, 28). These products were not observed with higher concentrations of arachidonic acid or at shorter times of incubation and appear to result from the sequential oxidation of 20-HETE. 4F3B has a K_m for arachidonic acid of 22 µM and a V_max of 13.3 pmol of product/min/pmol of P450. As a comparison, the K_m of 4F3A for arachidonic acid was determined to be 185.6 µM with a V_max of 11.5 pmol of product/min/pmol of P450. A summary of the kinetic data obtained for 4F3 isoforms using the Supersome system is shown in Table II. 4F3B has a V/K value that is 44-fold lower than 4F3A for LTB₄, and 10-fold higher than 4F3A for arachidonic acid.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Enzymatic conversion of substrates by CYP4F3B. A, HPLC chromatogram showing conversion of 1 µM LTB4 to 20-hydroxy-LTB4 by CYP4F3B. Retention time of 20-hydroxy-LTB4 is 10.8 min. B, conversion of 10 µM [14C] arachidonic acid to [14C]20-HETE by CYP4F3B. Retention time of 20-HETE is 14.9 min.

View this table: [in this window] [in a new window]   Table II LTB4 and arachidonic acid -hydroxylation by CYP4F3 isoforms

CYP4F Gene Products Show a Restricted Tissue Distribution-- To analyze the tissue distribution of mRNA products of the CYP4F subfamily, a human MTE array was hybridized with a ³²P-labeled cDNA probe (nucleotides 561-1268) for 4F3A (Fig. 3A). There is 100% identity with 4F3B within the 708-bp region of the probe, 95% identity with 4F2, and ~90% identity with 4F8, 4F11, and 4F12. Strong hybridization signals were detected in peripheral blood leukocytes, bone marrow, liver, kidney, fetal liver, and prostate. The signals were quantified and expressed relative to the measurement for peripheral blood leukocytes (Table III). Weaker hybridization signals were detected in trachea, ileum, and other gastrointestinal tract tissues including duodenum, jejunum, and colon. No hybridization was observed in the negative control grids in column 12 containing yeast total RNA, yeast tRNA, Escherichia coli rRNA, and E. coli DNA. A positive signal was obtained from human genomic DNA (100 ng of DNA, relative signal strength = 0.2; 500 ng of DNA, relative signal strength = 0.8) detecting the CYP4F gene family on chromosome 19. 

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Tissue distribution of CYP4F mRNA. A, MTE array analysis of CYP4F gene expression. A human MTE array was hybridized with the 708 bp 32P-labeled probe for CYP4F3A. Hybridization signals were quantified by phosphorimaging and expressed relative to the signal for peripheral blood leukocytes (Table III). B, Northern blot analysis of CYP4F gene expression. A human MTN blot (2 µg of poly(A)+ RNA per lane) was hybridized with the 1560-bp 32P-labeled probe corresponding to the coding region of CYP4F3A. The blot was analyzed by autoradiography.

Northern blotting confirmed the restricted tissue distribution of CYP4F mRNA, and identified transcripts of ~5 and ~2.4 kb in both liver and kidney, but not heart, brain, placenta, lung, skeletal muscle, or pancreas (Fig. 3B). 4F3 mRNA transcripts of 5.034 and 2.339 kb correspond to alternative polyadenylation signals in the 4F3 gene (18, 26) and are consistent with the pattern observed.

Quantification of CYP4F Gene Products in Different Tissues-- The identities of the CYP4F transcripts expressed in each tissue were determined by isoform-specific RT-PCR (Fig. 4A). Distinct patterns of expression were observed in different tissues. 4F3A was the only transcript that could be detected in RNA from peripheral blood neutrophils and bone marrow after 30 cycles of PCR. Fetal liver, kidney, prostate, and ileum contain 4F3B and 4F2 in addition to 4F3A. Trachea contains 4F3A and 4F3B, but not 4F2. Liver contains 4F3B and 4F2 but not 4F3A. The expression of exon 4 from the 4F2 gene was not detected in any of the tissues examined. Primers specific for 4F8 (K-L) or 4F11 (M-N) were included in RT-PCR experiments (data not shown). 4F8 could only be detected in prostate and required more than 30 cycles of PCR to give a visible band; 4F11 was detected in liver, kidney, prostate, ileum, and trachea.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Identification and quantification of CYP4F gene products. A, tissue-specific expression of CYP4F3 and CYP4F2 isoforms. Total RNA from each tissue was used as a substrate for reverse transcription using random primers. The cDNA was analyzed by PCR using primer pairs specific for CYP4F3A (B-C), CYP4F3B (B-G), a hypothetical form of CYP4F2 containing exon 4 (H-C), or the known form of CYP4F2 containing exon 3 (H-G). PCR products of 158 bp were resolved on a 2% agarose gel and stained with ethidium bromide. B, quantification of CYP4F3B and CYP4F2 in human tissues. The cDNA from each tissue shown was analyzed by 20 cycles of PCR using primer pairs specific for CYP4F3B (E-F, 962-bp product) or CYP4F2 (I-J, 962-bp product), in combination with primers for GAPDH (GDHF and GDHR, 458-bp product). The PCR products were labeled with 32P and visualized by autoradiography after separation on a 6% sequencing gel. The radiolabeled bands were quantified by phosphorimaging, and results are summarized in Table III.

The relative levels of 4F3B and 4F2 in tissues were determined by quantitative RT-PCR using GAPDH primers as an internal standard (Fig. 4B). The results are summarized in Table III. In addition, a sample of PCR products generated by the isoform-generic primer pair A-D (recognizes 4F3A, 4F3B, 4F2, and 4F11) was cloned and sequenced. Sequence analysis of cloned PCR products provides confirmation of isoform identity, and analysis of 50 PCR products (50 colonies from each tissue) predicted a similar ratio of 4F3B to 4F2 to that obtained by quantitative PCR (Table III). When determined by quantitative PCR, the levels of 4F3B and 4F2 are similar in kidney, but 4F3B exceeds 4F2 in liver, prostate, fetal liver, and ileum by 4-, 4-, 2.5-, and 2-fold, respectively. When determined by direct sequencing of subcloned PCR products, these ratios were 7-, 4-, 3.5-, and 2-fold, respectively, confirming the validity of both methods of analysis. Analysis of cloned PCR products enabled an estimation of the relative levels of 4F3A for which a specific quantitative assay could not be developed. Using this approach, it was determined that 4F3A is the most abundant 4F gene product in prostate: it exceeds 4F3B by 3-fold and 4F2 by 14-fold. In contrast, no colonies containing 4F3A PCR products were isolated from kidney. 4F3A could not be detected in kidney following 20 cycles of isoform-specific PCR (rather than 30 cycles shown in Fig. 4A) and must represent a small fraction of 4F gene products, possibly derived from endogenous myeloid cells or blood contamination. 4F11 colonies were only detected in trachea (24% of total, not included in Table III).

Homology Modeling-- We performed molecular modeling to make predictions about the location of the alternative exon (amino acids 67-114 encoded by exon 3 or exon 4) within the CYP4F3 molecule. A model was constructed based on the crystal structure of P450BM3 (Fig. 5). Amino acids 67-114 overlap and include the substrate recognition site 1 (SRS-1) defined by Gotoh (29). The model suggests that the alternative exon has a position that would enable it to contribute to interactions with substrate bound at the active site. It also has the potential to contribute to interactions in the substrate access channel, a region that is a determinant of substrate specificity in CYP4A enzymes (30-32).

View larger version (72K): [in this window] [in a new window]   Fig. 5.   Location of alternative exon within the CYP4F3 molecule. Homology model of CYP4F3A with LTB4 (magenta) bound at the active site. The heme group is shown in green. Amino acids 67-114 (yellow) comprise exon 4, and represent the region of the molecule that is switched for exon 3 in CYP4F3B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CYP4F3 was originally identified as an LTB₄ -hydroxylase which provides the major pathway of inactivation of LTB₄ in human neutrophils (13-15). Subsequently, we detected the existence of a second CYP4F3 isoform in liver (18). The two isoforms are generated by alternative splicing of mutually exclusive exons numbered 3 and 4 in the CYP4F3 gene (Fig. 1). Selection of exon 4 generates CYP4F3A in neutrophils, whereas selection of exon 3 generates CYP4F3B in liver. The K_m of CYP4F3B for LTB₄ (20.6 µM) is ~30-fold higher than CYP4F3A (0.68 µM), but may be sufficiently low for the enzyme to represent a significant source of LTB₄ metabolism in tissues such as the liver which lack CYP4F3A. CYP4F3B has a K_m for arachidonic acid of 22 µM and generates 20-HETE as the major product of -hydroxylation (Fig. 2). This is in contrast to CYP4F3A, which has low activity for arachidonic acid (K_m = 185.6 µM). The V/K values indicate that CYP4F3B is 10-fold more efficient than CYP4F3A at utilizing arachidonic acid, but 44-fold less efficient at utilizing LTB₄ (Table II). Therefore, each CYP4F3 isoform has a distinct profile of kinetic parameters.

The tissue distribution of CYP4F gene products was mapped using a human multiple tissue expression array (Fig. 3). Based on these results, RT-PCR analysis of individual tissue RNAs was performed to identify which enzymes were represented in each tissue (Fig. 4A). CYP4F gene products were then quantified by a combination of two independent PCR-based methods (Fig. 4B, Table III). CYP4F3A is the only isoform expressed in bone marrow and peripheral blood leukocytes, whereas CYP4F3B is selectively expressed in liver and kidney. The prostate was identified as a major source of CYP4F enzymes where CYP4F3A is the dominant isoform (it exceeds CYP4F3B by ~3-fold). Weaker hybridization signals for CYP4F gene products were detected in trachea and gastrointestinal tract, and these tissues contain transcripts for both CYP4F3A and CYP4F3B. In general, the tissue distribution of CYP4F3B shows greater correlation with CYP4F2 than with CYP4F3A. CYP4F3B was the most abundant CYP4F transcript detected in liver, fetal liver, ileum, and trachea, and in each case it exceeded the level of CYP4F2 by 2-5-fold.

The distinct patterns of expression of CYP4F3A and CYP4F3B, combined with their distinct substrate specificities, point to different functions of the two enzymes. Localization of CYP4F3A in myeloid cells of the blood and bone marrow enable it to participate in the fine control of LTB₄-mediated inflammation. Its very low K_m for LTB₄ of <1 µM is optimal for this function. The presence of CYP4F3A in trachea and gastrointestinal tract might enable it to play a role in the control of mucosal inflammation. CYP4F3B can promote clearance of both LTB₄ and arachidonic acid but is also a potential source of bioactive 20-HETE. Immunoinhibition studies provide evidence that CYP4F enzymes are responsible for most arachidonic acid -hydroxylation in human liver (11), and produce bioactive 20-HETE in human kidney (12). A polyclonal antibody to CYP4F2 was used in these studies, but this would not distinguish CYP4F3B, which shares 93% amino acid sequence identity and a similar level of expression. The reported kinetics of CYP4F2 for arachidonic acid (K_m = 24 µM, V_max = 7.4 min¹) (11) appear similar to CYP4F3B. Other tissues that express CYP4F3B, including ileum and trachea, are important sites of 20-HETE activity (33, 34).

The differences in substrate specificity exhibited by CYP4F3A and CYP4F3B must be determined by the alternative exon, which codes for amino acids 67-114, as this represents the only difference in sequence between the two proteins. The substrate specificity of cytochrome P450s may be determined by residues that mediate substrate binding to the surface of the molecule, substrate access to the active site via a hydrophobic channel, or substrate orientation within the active site. Molecular modeling with homology models based on CYP102 (P450BM3) suggests that amino acids 67-114 code for a region that contributes to both the active site and substrate access channel of CYP4F3 (Fig. 5). This would be consistent with the conclusion that exon switching has the capacity to modulate substrate specificity. We are currently performing site-directed mutagenesis and higher resolution molecular modeling studies to determine the precise amino acids that define the distinct kinetic properties of CYP4F3A and CYP4F3B.

Based on the data obtained with CYP4F3, it can be extrapolated that amino acids 67-114 will play a critical role in determining the substrate specificity of other CYP4F enzymes. This region is encoded by an exon of identical size in CYP4F2, CYP4F8, CYP4F11, and CYP4F12, which has 83, 75, 66, and 60% amino acid identity, respectively, with exon 3 in CYP4F3B. The overall identity of these enzymes with CYP4F3B is 93, 81, 86, and 82%, respectively. Amino acids 67-114 are seen to have a higher than average level of variation within the molecule, and this may contribute to differences in substrate preference and function. CYP4F8 has a restricted distribution to seminal vesicles (22) and catalyzes -1 hydroxylation of PGH₁ and PGH₂ to 19R-hydroxy-PGH₁ and 19R-hydroxy-PGH₂ (35). Preliminary studies indicate that CYP4F12 has the capacity to catalyze -hydroxylation of various eicosanoids (24, 25). The substrate specificity of CYP4F11 has not been determined. In general, the CYP4F subfamily is emerging as an important group for regulating eicosanoid activity through oxidation of arachidonic acid or its derivatives.

The amino acid identity between exons 3 and 4 of CYP4F3 is only 27%. However, their identical size and positioning in the molecule make it plausible that they are derived from duplication and divergence of a single exon. Exon 4 lacks a homologous counterpart in other CYP4F cDNAs. Analysis of genomic sequences reveals that a homolog of exon 4 is present in the CYP4F2 gene: it has 90% nucleotide identity, similar splice junction sequences, and is identical in size (145 bp). However, there is currently no evidence that this exon is used as a substrate for splicing in CYP4F2. Isoform-specific PCR reactions and direct sequencing of PCR products failed to detect a novel splice form of CYP4F2 containing exon 4 in any of the tissues tested. Tissues such as prostate, ileum, and trachea express both splice forms of CYP4F3 containing either exon 4 (CYP4F3A) or exon 3 (CYP4F3B), but these tissues only express a single form of CYP4F2 expressing exon 3. This has implications for the regulation of alternative splicing specific to CYP4F3, and for predictions based on genomic data in general.

Splicing variations in cytochrome P450s have been reported that either preserve the kinetic properties of the enzyme or abolish function: these involve changes in 5'-untranslated regions (36, 37), neutral changes to the coding region (38), or arise from mutations that generate non-functional transcripts (39-42). For example, alternative splicing of the 5'-untranslated region of CYP19 (aromatase) transcripts has been well characterized (36), but this does not alter the kinetic properties of the enzyme. Single nucleotide polymorphisms in CYP3A genes cause alternative splicing and protein truncation resulting in no expression of the enzymes in tissues (42). In contrast, alternate exons in the CYP4F3 gene enable expression of functionally distinct isoforms with different substrate specificities. There is potential to modulate the opposing capacities of CYP4F3 to generate a bioactive mediator (20-HETE) or inactivate one (LTB₄) by regulating splicing in response to microenvironmental stimuli. CYP4F3B is the major CYP4F enzyme in liver and other tissues, but it was difficult to identify because of close homology with other subfamily members. By analogy, it is likely that alternate exons are more widespread in the CYP superfamily but remain undetected. This would provide another level of diversity for a group of enzymes that already exhibit remarkable variation in substrate and product specificities. Alternative splicing of closely related CYP enzymes would also have broad implications for the functional assignment of CYP genes and for the design of large scale pharmacogenomic studies.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants 1K01DK59991-01 (to P. C.), 5R01GM-61823 (to R. J. S.), NIEHS ES09122 (to J. P. J.), 5R01GM9054-09 (to A. E. R.), 5R01DK52234 (to D. T. S.), and 5R01HL55718 (to D. T. S.), the Richard Saltonstall Charitable Foundation (to D. T. S.), and a grant by the Jewish Communal Fund (to R. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital East, 149 The Navy Yard, 13th St., Charlestown, MA 02129. Tel.: 617-724-4336; Fax: 617-726-5651; E-mail: christma@helix.mgh.harvard.edu.

Published, JBC Papers in Press, July 18, 2001, DOI 10.1074/jbc.M104818200

² Web address: drnelson.utmem.edu/CytochromeP450.html.

	ABBREVIATIONS

The abbreviations used are: CYP, cytochrome P450; LTB₄, leukotriene B₄; PGH₂, prostaglandin H₂; HETE, hydroxyeicosatetraenoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; PCR, polymerase chain reaction; AA, arachidonic acid; HPLC, high performance liquid chromatography; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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