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

Open AccessPublished:October 12, 2001DOI:https://doi.org/10.1074/jbc.M104818200
      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 ω-hydroxylation (Km = 0.68 μm) but has low activity for arachidonic acid (Km = 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 higherKm for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for ω-hydroxylation (Km = 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.
      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)
      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.
      Web address: drnelson.utmem.edu/CytochromeP450.html.
      2Web address: drnelson.utmem.edu/CytochromeP450.html.
      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 (
      • Fisslthaler B.
      • Popp R.
      • Kiss L.
      • Potente M.
      • Harder D.R.
      • Fleming I.
      • Busse R.
      ,
      • Node K.
      • Huo Y.
      • Ruan X.
      • Yang B.
      • Spiecker M.
      • Ley K.
      • Zeldin D.C.
      • Liao J.K.
      ). CYP4 enzymes catalyze ω-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 (
      • Nowicki S.
      • Chen S.L.
      • Aizman O.
      • Cheng X.J.
      • Li D.
      • Nowicki C.
      • Nairn A.
      • Greengard P.
      • Aperia A.
      ,
      • Ma Y.-H.
      • Gebremedhin D.
      • Schwartzman M.L.
      • Falck J.R.
      • Clark J.E.
      • Masters B.S.
      • Harder D.R.
      • Roman R.J.
      ,
      • Amlal H.
      • LeGoff C.
      • Vernimmen C.
      • Soleimani M.
      • Paillard M.
      • Bichara M.
      ,
      • Muthalif M.M.
      • Benter I.F.
      • Karzoun N.
      • Fatima S.
      • Harper J.
      • Uddin M.R.
      • Malik K.U.
      ,
      • McGiff J.C.
      • Quilley J.
      ). Enzymes in the CYP4A subfamily have high activity for arachidonic acid in animals (
      • Loughran P.A.
      • Roman L.J.
      • Aitken A.E.
      • Miller R.T.
      • Masters B.S.
      ). The identity and distribution of enzymes generating 20-HETE in humans is poorly understood. This determination is complicated by wide-ranging Kmvalues 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 (
      • Palmer C.N.
      • Richardson T.H.
      • Griffin K.J.
      • Hsu M.H.
      • Muerhoff A.S.
      • Clark J.E.
      • Johnson E.F.
      ), but it exhibits very low activity for arachidonic acid (
      • Hoch U.
      • Zhang Z.
      • Kroetz D.L.
      • Ortiz de Montellano P.R.
      ). Recently it was suggested that ω-hydroxylation of arachidonic acid in human liver and kidney is mediated primarily by CYP4F2 (
      • Powell P.K.
      • Wolf I.
      • Jin R.
      • Lasker J.M.
      ,
      • Lasker J.M.
      • Chen W.B.
      • Wolf I.
      • Bloswick B.P.
      • Wilson P.D.
      • Powell P.K.
      ).
      In addition to activating arachidonic acid, CYPs can inactivate LTB4 and prostaglandins by ω-hydroxylation. CYP4F3 was originally identified as the enzyme that catalyzes ω-oxidation of the 5-lipoxygenase pathway product LTB4 with a lowKm (0.5–1.0 μm) in human neutrophils (
      • Shak S.
      • Goldstein I.M.
      ,
      • Soberman R.J.
      • Harper T.W.
      • Murphy R.C.
      • Austen K.F.
      ,
      • Kikuta Y.
      • Kusunose E.
      • Endo K.
      • Yamamoto S.
      • Sogawa K.
      • Fujii-Kuriyama Y.
      • Kusunose M.
      ). LTB4 functions as a chemoattractant of neutrophils and monocytes (
      • Ford-Hutchinson A.W.
      • Bray M.A.
      • Doig M.V.
      • Shipley N.E.
      • Smith M.J.H.
      ,
      • Migliorisi G.
      • Folkes E.
      • Pawlowski N.
      • Cramer E.B.
      ), 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 (
      • Christmas P.
      • Ursino S.R.
      • Fox J.W.
      • Soberman R.J.
      ) 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 aKm for arachidonic acid of 22 μm and generates 20-HETE as the major product of ω-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.

      DISCUSSION

      CYP4F3 was originally identified as an LTB4ω-hydroxylase which provides the major pathway of inactivation of LTB4 in human neutrophils (
      • Shak S.
      • Goldstein I.M.
      ,
      • Soberman R.J.
      • Harper T.W.
      • Murphy R.C.
      • Austen K.F.
      ,
      • Kikuta Y.
      • Kusunose E.
      • Endo K.
      • Yamamoto S.
      • Sogawa K.
      • Fujii-Kuriyama Y.
      • Kusunose M.
      ). Subsequently, we detected the existence of a second CYP4F3 isoform in liver (
      • Christmas P.
      • Ursino S.R.
      • Fox J.W.
      • Soberman R.J.
      ). 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. TheKm 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 aKm for arachidonic acid of 22 μm and generates 20-HETE as the major product of ω-hydroxylation (Fig. 2). This is in contrast to CYP4F3A, which has low activity for arachidonic acid (Km = 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 Km for LTB4 of <1 μmis 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 ω-hydroxylation in human liver (
      • Powell P.K.
      • Wolf I.
      • Jin R.
      • Lasker J.M.
      ), and produce bioactive 20-HETE in human kidney (
      • Lasker J.M.
      • Chen W.B.
      • Wolf I.
      • Bloswick B.P.
      • Wilson P.D.
      • Powell P.K.
      ). 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 (Km = 24 μm,Vmax = 7.4 min−1) (
      • Powell P.K.
      • Wolf I.
      • Jin R.
      • Lasker J.M.
      ) appear similar to CYP4F3B. Other tissues that express CYP4F3B, including ileum and trachea, are important sites of 20-HETE activity (
      • Macica C.
      • Balazy M.
      • Falck J.R.
      • Mioskowski C.
      • Carroll M.A.
      ,
      • Jacobs E.R.
      • Effros R.M.
      • Falck J.R.
      • Reddy K.M.
      • Campbell W.B.
      • Zhu D.
      ).
      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 (
      • Bylund J.
      • Finnstrom N.
      • Oliw E.H.
      ) and catalyzes ω-1 hydroxylation of PGH1 and PGH2 to 19R-hydroxy-PGH1 and 19R-hydroxy-PGH2 (
      • Bylund J.
      • Hidestrand M.
      • Ingelman-Sundberg M.
      • Oliw E.H.
      ). Preliminary studies indicate that CYP4F12 has the capacity to catalyze ω-hydroxylation of various eicosanoids (
      • Bylund J.
      • Bylund M.
      • Oliw E.H.
      ,
      • Hashizume T.
      • Imaoka S.
      • Hiroi T.
      • Terauchi Y.
      • Fujii T.
      • Miyazaki H.
      • Kamataki T.
      • Funae Y.
      ). 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 (
      • Simpson E.R.
      • Michael M.D.
      • Agarwal V.R.
      • Hinshelwood M.M.
      • Bulun S.E.
      • Zhao Y.
      ,
      • Mullick J.
      • Addya S.
      • Sucharov C.
      • Avadhani N.G.
      ), neutral changes to the coding region (
      • Desrochers M.
      • Christou M.
      • Jefcoate C.
      • Belzil A.
      • Anderson A.
      ), or arise from mutations that generate non-functional transcripts (
      • Miles J.S.
      • McLaren A.W.
      • Wolf C.R.
      ,
      • Huang Z.
      • Fasco M.J.
      • Kaminsky L.S.
      ,
      • Chen W.
      • Kubota S.
      • Teramoto T.
      • Nishimura Y.
      • Yonemoto K.
      • Seyama Y.
      ,
      • Kuehl P.
      • Zhang J.
      • Lin Y.
      • Lamba J.
      • Assem M.
      • Schuetz J.
      • Watkins P.B.
      • Daly A.
      • Wrighton S.A.
      • Hall S.D.
      • Maurel P.
      • Relling M.
      • Brimer C.
      • Yasuda K.
      • Venkataramanan R.
      • Strom S.
      • Thummel K.
      • Boguski M.S.
      • Schuetz E.
      ). For example, alternative splicing of the 5′-untranslated region of CYP19 (aromatase) transcripts has been well characterized (
      • Simpson E.R.
      • Michael M.D.
      • Agarwal V.R.
      • Hinshelwood M.M.
      • Bulun S.E.
      • Zhao Y.
      ), but this does not alter the kinetic properties of the enzyme. Single nucleotide polymorphisms inCYP3A genes cause alternative splicing and protein truncation resulting in no expression of the enzymes in tissues (
      • Kuehl P.
      • Zhang J.
      • Lin Y.
      • Lamba J.
      • Assem M.
      • Schuetz J.
      • Watkins P.B.
      • Daly A.
      • Wrighton S.A.
      • Hall S.D.
      • Maurel P.
      • Relling M.
      • Brimer C.
      • Yasuda K.
      • Venkataramanan R.
      • Strom S.
      • Thummel K.
      • Boguski M.S.
      • Schuetz E.
      ). 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.

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