*Department of Biology, University of San Diego, 5998 Alcalá Park, San Diego, CA 92110; §Department of Biology, Lafayette College, Easton, PA 18042; Departments of ¶Pathology and ÝBiochemistry & Biophysics, University of California, San Francisco, CA 94143
Journal of Neurogenetics, 1999, 13(3): 157-180
*Corresponding author, Tel. (619) 260-4129. Fax: (619) 260-6804. E-mail: cloer@acusd.edu.
We have identified an aromatic amino acid hydroxylase gene from the nematode C. elegans that likely encodes the worm phenylalanine hydroxylase (PheH). The predicted amino acid sequence is most similar to that of other PheH and TrpH proteins. Reporter gene fusions and staining with an antibody to mammalian PheH indicate the gene is expressed in hypodermal cells. A fusion protein expressed in bacteria can convert phenylalanine to tyrosine, and, to a lesser extent, tryptophan to 5-hydroxytryptophan. We hypothesize that the protein is necessary to produce additional tyrosine for protein cross-linking in the nematode cuticle.
Keywords: aromatic amino acid hydroxylase; tryptophan hydroxylase; tyrosine hydroxylase; cuticle; pterin-dependent monooxygenase; tyrosine cross-linking
The family of aromatic amino acid hydroxylases (AAAHs) in the vertebrates comprises three enzymes: phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1, PheH), tryptophan hydroxylase (tryptophan 5- monooxygenase, EC 1.14.16.4, TrpH), and tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2, TH). TrpH and TH catalyze the rate-limiting steps for serotonin and catecholamine synthesis, respectively; PheH synthesizes tyrosine, but is probably more important for phenylalanine catabolism (see review by Hufton et al., 1995). It has been suggested that PheH in some invertebrates is important as the first step in melanin synthesis used in a host defense system (Wiens et al., 1998). Common epitopes, cofactor requirements, and ultimately, sequence analysis have revealed a common ancestry for these genes (Coker et al., 1990). Although Neckameyer and White (1992) have presented evidence that the fruit fly Drosophila possesses only a single enzyme that encodes both PheH and TrpH activities, genomic sequencing in C. elegans has revealed three AAAH genes, probably corresponding in function to the three familiar genes from the vertebrates (The C. elegans Sequencing Consortium, 1998; Bargmann, 1998). Here we describe the cloning and sequencing of an aromatic amino acid hydroxylase gene from the nematode C. elegans, its expression pattern in situ, and the substrate specificity of the encoded protein expressed in vitro. We present evidence that this gene encodes a protein in the Phe/TrpH class of AAAH proteins, that it is expressed in the hypodermis of the worm, and that the protein effectively hydroxylates Phe in vitro. We propose that this gene encodes the phenylalanine hydroxylase of C. elegans, and hypothesize that the enzyme synthesizes tyrosine in the hypodermis to be used for extensive tyrosine cross-linking in the nematode cuticle.
Routine culturing of Caenorhabditis elegans was performed as described by Brenner (1974). Nomenclature used here for C. elegans genetics conforms to the conventions set forth by Horvitz et al. (1979). Strains used include N2 (wild type); CB1490: him-5(e1490)V; DP19: unc-119(e2498). The him-5(e1490) strain generates approximately 30% males by increased X chromosome non-disjunction (Hodgkin et al., 1979), but is otherwise essentially wild-type. We used it to examine expression patterns in males because the standard wild type generates only about 0.2% males.
Cloning of a C. elegans Aromatic Amino Acid Hydroxylase Gene
We aligned amino acid sequences of several aromatic amino acid hydroxylase (AAAH) genes, identified conserved regions and designed degenerate oligonucleotide primers for polymerase chain reaction (PCR). The 20 ml PCR reaction mixture was as previously described (Kamb et al., 1989); template DNA was ~1 µg genomic DNA from N2 (wild type) worms. Conditions for PCR were 94deg C (1 min), 55deg C (1 min), 72deg C (2 min) for 40 cycles in a MJ Research Thermocycler. Primer sequences were as follows: TH-1: AAGAATTCCAYGARYTNYTNGGNCA, which encodes the amino acid sequence HELLGH and contains an EcoRI site. TH-2: AAAAGCTTNCCRAAYTCNACNGTRAA, which encodes the reverse complement of the amino acid sequence FTVEFG and contains a HindIII site. TH-3: AAAAGCTTNCCNGCNCCRTANGCYTT, which encodes the reverse complement of the amino acid sequence KAYGAG and contains a HindIII site. The degeneracy of primers TH-1 and TH-3 was 1024 and that of TH-2 was 512. Among a number of variable bands, a reliable band of ~250 base pairs (bp) was seen when amplification was performed with primers TH-1 and TH-3. This reaction mixture was diluted 1:100 with water and 1 ml used as template in PCR using TH-1 and TH-2 as primers. From this reamplification reaction, a single, strong band of ~210 bp resulted. This was the size predicted for a genuine AAAH gene (i.e., the distance between TH-1 and TH-2 primer sites is 42 bp less than the distance between TH-1 and TH-3 primer sites.) The reamplified band was excised from the gel, purified with GeneClean (Bio 101), cut with EcoRI and HindIII and ligated into EcoRI/HindIII-cut vector pIBI30 in agarose (Struhl, 1985). Several white colonies were selected and purified, and DNA from these were sequenced using a Sequenase kit (US Biochemical). Two such clones yielded identical sequences encoding an AAAH gene.
We used one of these clones (pCL-62) as a probe to screen 200,000 lambda phage plaques representing a C. elegans genomic library (courtesy of A. Kamb). From this library we recovered 2 identical hybridizing clones. A large (~12.5 kb) SalI fragment was subcloned (pCL-S12) into pIBI30. This clone was restriction-mapped and restriction fragments hybridizing to pCL-62 were identified. We subcloned a strongly hybridizing ~2.4 kb HindIII fragment (pCL-H2.4) and subsequently used this to screen a cDNA library (in lambda-ueber2, courtesy of Chris Martin). 500,000 plaques were screened and 38 phage clones were isolated and purified. Six clones were converted to plasmids and analyzed. All genuine AAAH clones yielded similar restriction digests, suggesting they were identical. Based on PCR analysis of inserts, we selected the two longest clones for further analysis. The longest cDNA clone (pCL-41) was sequenced in its entirety; identical sequence was derived from a slightly shorter clone (pCL-92). We obtained sufficient sequence from genomic clone pCL-H2.4 and other subclones of pCL-S12 to identify the locations of all introns.
Construction of lacZ Reporter Fusions
Sequence of cDNA and restriction analysis of genomic DNA was used to select a fragment of genomic AAAH gene and upstream region that could be cloned into the vector pPD21.28 to make an in-frame protein coding sequence fusion with the reporter gene lacZ (Fire et al., 1990). Two different fusions were constructed. 1) pCL-S12BN-lacZ, with ~6.7 kb upstream sequence: A 6.9 kb NcoI fragment of pCL-S12 was isolated, blunted with T4 DNA polymerase, cut with BglII and inserted into BamHI/MscI-cut pPD21.28. 2) pCL-S12XN-lacZ, with ~4.2 kb upstream sequence: The same 6.9 kb blunted NcoI fragment was cut with XbaI and inserted into pPD21.28 cut with XbaI and MscI. In both cases, junctions were sequenced to confirm the predicted in-frame fusion. The NcoI site is located near the beginning of the 2nd exon, 141 bp downstream of the predicted translation start site.
LacZ reporter construct DNAs, purified with Qiagen, were microinjected either with the dominant marker plasmid pRF4 containing the mutant gene rol-6(su1006) (Mello et al., 1991) into him-5(e1490) worms or with an unc-119 rescuing plasmid (pDP#M011B, courtesy Morris Maduro) into unc-119(e2498) mutant worms (Maduro and Pilgrim, 1995). Transgenic Roller or non-Unc progeny were isolated and propagated. Eventually, ten independent heritable pRF4 lines containing the 6.7 kb insert and two pRF4 lines containing the 4.2 kb insert were analyzed for their expression pattern in him-5 worms. Four heritable unc-119-rescued lines with the 6.7 kb insert were examined. Animals were stained with X-gal by a modification of a procedure described by Fire (1989).
Staining with the PH8 Monoclonal Antibody
Worms were fixed and permeabilized by two different methods, both derived from methods originally described by Ruvkun and colleagues (Ruvkun and Giusto, 1989; Finney and Ruvkun, 1990). Similar results were obtained with respect to hypodermal cell staining; however, staining of serotonergic neurons was seen only with the second method. Method 1: Worms were fixed in 1% paraformaldehyde in 1X MRWB for 1 hr at 4degC, frozen in liquid nitrogen, thawed and rinsed 2 times with 1% Triton X-100/0.1M Tris (pH 7.4). Worms were then incubated for 1 hr at 37deg C in 1% beta-mercaptoethanol/1% Triton X-100/0.1M Tris, then rinsed in 25 mM borate (pH 9.2), then 15 min in 10 mM DTT/borate buffer, rinsed in borate buffer, then 15 min 0.3% H2O2/borate buffer, and rinsed 2 times with borate buffer. Worms were 'blocked' by a 1 hr incubation in 1% BSA/0.5% Triton X-100/PBS (Soln A), then incubated with 1:100 PH8 in Soln A for 1 hr at 37deg C, rinsed 3 times with 0.1% BSA/0.5% Triton X-100/PBS (Soln B) and incubated 30 min, then incubated for 1 hr at 37deg C with 1:100 TRITC-conjugated Goat anti-Mouse IgG in Soln A, rinsed 3 times with Soln B, and viewed with epifluorescence. Method 2: Worms were fixed in 1% paraformaldehyde in 1X MRWB, shaken for 20 min at RT, frozen in liquid nitrogen, thawed and shaken for another 20 min. They were then rinsed in 100% methanol, 100% ethanol, 50% ethanol/50% xylene, then incubated for 1.5 hr in 100% xylene. This was followed by 50% ethanol/50% xylene, 100% ethanol incubated overnight, 50% ethanol/50% PBTS (PBS / 0.1% Tween 20/0.01% SDS), then 2 rinses with PBTS. Worms were digested in Proteinase K (100 µg/ml)/PBTS for 30 min, rinsed 2 times with 2 mg/ml glycine in PBTS, 2 times with PBTS, and post-fixed with 4% paraformaldehyde for 20 min at RT. Worms were then rinsed 3 times with 0.5% Triton X-100/PBS. From this point, the protocol was essentially the same as method 1, beginning with the 'blocking' step.
Expression of a Hydroxylase Fusion Protein in Bacteria
An in-frame protein fusion of the complete worm hydroxylase protein with maltose binding protein was created by directionally cloning the 4.1 cDNA into the pMAL-c2 vector (IBI). The pCL4.1 cDNA was PCR-amplified with primers that added a 5' BamHI site and a 3' XbaI site. This PCR fragment was gel purified with GeneClean (Bio 101), cut and ligated into appropriately cut vector in agarose. The construct was transformed into the DH5alpha strain of E. coli. Cells in log phase growth were induced with 0.4 µg/ml IPTG for 1-2 hr. PAGE of induced cells showed high levels of a ~93 kD protein, which is the size expected for a maltose binding protein-aromatic amino acid hydroxylase fusion protein (MBP-AH).
Enzyme Assays
The ability of the K08F8.4 protein (expressed as a fusion protein in bacteria) to make tyrosine from phenylalanine (phenylalanine hydroxylase activity) and 5-hydroxytryptophan from tryptophan (tryptophan hydroxylase activity) was tested using assays after those of Neckameyer and White (1992), which are based on the phenylalanine and tryptophan hydroxylase assays of Geltosky and Mitchell (1980) and Kuhn et al. (1980), respectively. Cells were induced with IPTG for 1 hr, then harvested and lysed by sonication (four 30 sec pulses at 50% power using a Virsonic 50 cell disrupter) into 1/10th volume of assay reaction buffer (50 mM Tris pH 7.0, 2 mM DTT), and centrifuged at 16,000 g for 5 min. PAGE analysis of supernatant and pellets showed that a large percentage of the induced fusion protein was in the supernatant following sonication. Therefore, supernatant (soluble protein) from cell lysates was used for the reactions. One hundred microliter reactions containing 50 mM Tris (pH 7.0), 2 mM DTT, 0.1 mg/ml catalase, with or without 1 mM DMPH4 synthetic biopterin cofactor (6,7-dimethyl-5,6,7,8-tetrahydropterine, Sigma) were incubated for 1 - 2 hr in open air at room temperature. Reactions were also prepared with or without substrate: in PheH assays, substrate was 4 mM phenylalanine; in TrpH assays, substrate was 2 mM tryptophan. In most experiments, a positive control using rat phenylalanine hydroxylase (Sigma) was performed in parallel with the C. elegans fusion protein. Heat denaturation of protein was accomplished by boiling the supernatant for 2 min. Reactions were stopped and protein precipitated by the addition of cold 15% TCA or perchloric acid. The reaction was centrifuged and supernatant taken. In the PheH assay, tyrosine was converted to a highly fluorescent product by a nitrosonaphthol reaction, then detected in a Perkin-Elmer spectrofluorometer at 470 excitation, 565 emission. For the TrpH assay, 5-HTP was detected directly after addition of ethanol to the supernatant taken following protein precipitation. For both assays, standards containing known amounts of product were prepared in reaction buffer and measured in the same way.
To find a C. elegans aromatic amino acid hydroxylase (AAAH) gene, we first aligned amino acid sequences encoded by several known AAAH genes, identified conserved regions and designed degenerate oligonucleotide primers for polymerase chain reaction (PCR). Using C. elegans genomic DNA as template, we amplified, cloned and then sequenced a fragment that encoded a highly conserved region of AAAH genes and which contained one small intron at a site conserved among many AAAH genes. A C. elegans genomic Southern blot probed with the PCR-derived fragment at high stringency indicated a single copy gene (not shown). We used this fragment to probe a C. elegans genomic lambda library from which we isolated a single ~12 kb genomic clone. The genomic clone was fingerprinted and mapped to chromosome II just to the right of rol-6 (A. Coulson, personal communication). The complete genomic sequence of this region has been determined by the C. elegans Genome Sequencing Consortium (GSC). The AAAH gene we identified corresponds to a predicted gene on cosmid K08F8 designated K08F8.4; we use this designation through the rest of this paper. (The designation bas-2 for biogenic amine synthesis-related has also been used for this gene.) Two other predicted AAAH genes have been identified by the GSC: a TH homolog (B0432.5) and a TrpH homolog (ZK1290.2). The TH homologous sequence is located at the extreme right end of chromosome II, the genetic location of cat-2, mutants of which are dopamine-deficient (Sulston et al., 1975).
We used a 2.4 kb genomic subclone to which the original PCR fragment hybridized to probe a cDNA library. We isolated several cDNA clones and completely sequenced the longest. This 1531 bp cDNA contained a 1371 base open reading frame encoding an aromatic amino acid hydroxylase protein of 457 amino acids (Fig. 1). The cDNA contained a consensus translation start site (GAAAATG), polyadenylation signal (AATAAA), and poly-A tail. The sequence of the cDNA is identical* with that predicted from the sequence of cosmid K08F8.
Comparisons with other AAAH proteins showed that the K08F8.4 protein was most similar to a Drosophila Phe/TrpH protein and chordate PheH proteins (Table I, Figure 2). Although only 334 amino acids could be easily aligned in all three types of AAAH proteins, more than 430 amino acids could be aligned between K08F8.4 (457 amino acids) and several vertebrate PheH and TrpH proteins. For example, in a pairwise comparison of K08F8.4 with Rat PheH, 436 amino acids could be aligned with 57% identity, with the introduction of a single gap. Among AAAH proteins, K08F8.4 was less similar to mammalian TH proteins (54-55% identity/334 residues) and least similar to C. elegans putative TrpH (ZK1290.2) and TH (B0432.5) proteins (Table I).
We also compared the location of introns in K08F8.4 with those of other AAAH genes for which genomic structure is known, and the predicted introns of C. elegans putative TrpH and TH genes (Figure 3). K08F8.4 shares all of its 7 introns with the human PheH gene, which contains 12 introns, 11 of which are conserved among various AAAH genes. Coker and others (1990) proposed that an ancestral hydroxylase gene contained up to 14 introns, although genomic structures from only a few mammalian PheH and TH genes were available at that time. The putative C. elegans TrpH gene shares 8 intron positions with human PheH, and contains 3 non-conserved introns, although these are only predicted splice sites. The putative C. elegans TH gene contains 3 conserved introns, and 2 predicted non-conserved introns (None of the non-conserved introns of the C. elegans TH gene are the same as those in the C. elegans TrpH).
Expression Pattern
To learn where the K08F8.4 gene might be expressed, we made reporter fusion constructs with the lacZ gene, injected the DNA into worms and isolated transgenic lines that heritably transmitted the constructs. Two different constructs were tested, both with in-frame protein coding fusions in the 2nd exon of the gene, but with different amounts of upstream sequence (6.7 or 4.2 kb). Both constructs showed essentially the same pattern of expression in all transgenic lines. The transgene was predominantly expressed in hypodermal cells of the worm, especially in the posterior (Fig. 4A, C). Frequently, staining was seen only in a few cells in the tail; we saw this especially in larvae. In many animals we noted a strong anterior-posterior gradient in staining, with the tail being strongly stained, and the anterior being weakly stained or unstained. We observed no staining in neurons of the ventral nerve cord, head or tail in either hermaphrodites or males. Staining was apparent, however, in ventral hypodermal cells (derived from Pn.p cells) associated with the ventral nerve cord (Fig. 4B). Staining was occasionally seen also in body wall muscle nuclei in adults. Essentially the same expression pattern of the transgene was seen with two different co-injection markers in different genetic backgrounds: rol-6 dominant plasmid in wild type worms and unc-119 (+) rescuing plasmid in unc-119 mutant worms. One small difference was seen in the frequency of non-hypodermal cells staining. In a minority of transgenic worms containing the unc-119 marker, a few random nerve ring, ventral nerve cord, body wall, and pharyngeal nuclei were stained. There was no consistent pattern to which additional cells stained, although most seemed to be neurons. We believe this expression may be from the influence of unc-119 enhancers in the transgene array; the unc-119 gene is expressed in most neurons (Maduro and Pilgrim, 1995).
We used a monoclonal antibody (PH8) that recognizes mammalian PheH (and, under appropriate conditions, also mammalian TrpH and TH; Haan et al, 1987) to confirm the expression pattern suggested by the reporter gene fusions. The amino acid sequence recognized by PH8 (Cotton et al., 1988) is found virtually unchanged in the protein encoded by K08F8.4 (Fig. 5A). Staining with the PH8 monoclonal frequently showed staining in the hypodermis and especially in the tail (Fig. 5B, C). No staining was seen in animals prepared with secondary antibody alone. In contrast with the lacZ fusion expression, in which a nuclear localization signal was attached to the beta-galactosidase protein, PH8 staining was clearly cytoplasmic. Nuclei were apparent as unstained spots within cells. Staining was often clearly apparent in the seam cells (lateral hypodermis) or ventral hypodermis (Fig. 5C), apparently stronger than staining in the large body hypodermal syncitium (hyp7). Like the pattern of expression seen with the reporter fusions, staining was often very strong in a small number of cells in the tail, especially in larvae (Fig 5B). Some of these cells are associated with the anus of the worm and appear to be interfacial hypodermal cells of the rectum and anus. Tail cell staining also included hypodermal cells forming the tail spike. Staining with PH8 in male C. elegans was similar to that of hermaphrodites but more complex in the tail, consistent with greater number of cells and complexity of the male copulatory apparatus in the tail.
Based on the predicted amino acid sequence of the putative C. elegans TrpH protein (ZK1290.2, Fig. 5A), we expected that PH8 would also recognize this protein. When worms were prepared by a different fixation and permeabilization technique ("method 2" - see Methods), hypodermal staining was somewhat decreased and we then saw staining in serotonergic neurons. We saw in many worms staining of cell bodies and processes of NSM's, identified serotonergic neurons in the pharynx (data not shown). We occasionally saw one or two small cells in the head in the location of other known serotonergic neurons, although strong hypodermal staining made such observations difficult. In a few hermaphrodites, we identified a cell near the vulva located sublaterally and with a process extending ventrally as the egg-laying neuron HSN. In many adult and some late L4 males, we observed 6 neurons with processes in the ventral nerve cord (Fig. 5D); these are the serotonergic CP neurons found only in the male (Loer and Kenyon, 1993). The CP neurons were the most reliably staining neurons with the antibody. We did not observe staining of dopaminergic neurons with the PH8 monoclonal.
Enzyme Assays
We expressed the K08F4.8 cDNA in E. coli as a fusion protein with maltose binding protein (MBP) with a Factor X protease cleavage site between the two proteins, using the pMAL-c2 vector system. Although we were able to purify the MBP-AH fusion protein using an amylose column and were able to cleave the protein with Factor X, we were unable to recover enzymatic activity from either affinity-purified MBP-AH or cleaved protein. Therefore, we tested the enzymatic activity of the MBP-AH fusion protein in crude lysates of induced E. coli. Induced bacteria containing the recombinant plasmid expressed high levels of a ~93 kD protein; sonication released much of this protein into the supernatant (data not shown). We compared supernatants of lysates from these cells with those of induced cells containing the parental plasmid (pMAL-c2), which produce MBP upon induction. Crude lysates containing MBP-AH, but not MBP, produced tyrosine in a time-dependent fashion (Fig. 6A). Tyrosine synthesis did not occur in the absence of a synthetic biopterin cofactor, phenylalanine, or following heat-inactivation of the lysate. During a typical 1 hr incubation, reactions containing all necessary reagents converted up to 5% of available phenylalanine (initial concentration, 4 mM) to tyrosine (~0.2 mM at end of 60 min).
We also tested whether the MBP-AH fusion protein could hydroxylate tryptophan. Crude lysates containing MBP-AH produced detectable 5-hydroxytryptophan (5-HTP) in a time-dependent fashion in the presence of substrate (tryptophan) and biopterin cofactor (Fig. 6B). As with tyrosine synthesis, no 5-HTP synthesis occurred in the absence of cofactor, substrate, or following heat inactivation of the lysate. The MBP-AH protein was much less efficient at synthesizing 5-HTP than tyrosine. During a typical 1 hr incubation, reactions containing all necessary reagents converted about 0.25% of available tryptophan (at 2 mM) to 5-hydroxytryptophan (~5 micromolar at end of 60 min).
Preincubation of PheH protein with phenylalanine can increase enzymatic activity (Bel et al., 1992; Doskeland et al., 1996). Reduced iron (Fe++) is found in all AAAH's and is essential to their function (Hufton et al., 1995); addition of iron to reactions can also improve enzymatic activity in cases where it may be limiting (Bel et al., 1992). We lysed induced bacteria directly into reaction buffer containing substrate (4 mM Phe in PheH reactions, 2 mM Trp in TrpH reactions) and/or iron (1 mM FeSO4) and incubated for 1 hr prior to using this lysate in complete reactions. Preincubation with substrate, iron or both had little or no effect on product formation (data not shown).
Three AAAH genes have now been identified in C. elegans including the K08F8.4 gene we describe here; since the C. elegans genomic sequence is essentially complete, there are unlikely to be more members of this family. We propose that this gene encodes the phenylalanine hydroxylase of C. elegans. The cDNA we isolated and sequenced is likely to contain the entire coding sequence because of the presence of a good translation initiation consensus sequence, although we do not know whether this is a full-length cDNA. It is noteworthy also that the size of the predicted protein is similar to that of Phe/TrpH proteins from other organisms (ranging 437 to 453 amino acids in length). Conservation of phosphorylation sites suggests that this protein may be regulated by phosphorylation like other AAAH proteins.
The predicted amino acid sequence is about equally similar to other AAAH's in the Phe/TrpH class. Interestingly, the AAAH sequences in C. elegans have diverged considerably, such that K08F8.4 is least similar to the putative C. elegans TH and TrpH among the AAAH's. This may reflect a more rapid molecular evolution within the nematodes than in other metazoan lineages (Fitch & Thomas, 1997). The sequence of K08F8.4 alone does not allow us to decide whether the protein is likely to act as a PheH, TrpH or both (as seems to be the case in Drosophila), although it is clearly not in the TH class. Assuming that C. elegans has three AAAH's with functions like those of the vertebrates, K08F8.4 could be assigned the role of PheH by exclusion. The AAAH gene found on cosmid ZK1290 is a likely to be a C. elegans Trp hydroxylase gene based on its sequence homology and expression in identified serotonergic neurons (Loer and Anderton, 1997; Sze & Ruvkun, personal communication). The AAAH encoded by B0432.5 is likely the C. elegans TH: it is most homologous to other TH's, it is expressed in known dopaminergic neurons and the gene rescues the dopamine-deficient mutant cat-2 (R. Lints and S. Emmons, personal communication).
As seen with reporter gene fusions, the K08F8.4 gene is expressed in the hypodermis and not in identified serotonergic or dopaminergic neurons, making it a poor candidate for a neurotransmitter synthetic enzyme gene. We believe staining seen with the PH8 monoclonal comes from both the K08F8.4 (PheH) and ZK1290.2 (TrpH) gene products. It is noteworthy that serotonergic neurons were stained only using a protocol that decreased hypodermal staining. Although PH8 recognizes TH in mammals, we have not seen staining of dopaminergic neurons in C. elegans; the predicted sequence of C. elegans TH is sufficiently different from that of the defined epitope that this is not surprising.
We hypothesize that PheH is expressed in the hypodermis to make tyrosine for cuticle synthesis and maintenance. Beside reducible disulfide bond cross-links in the cuticle, non-reducible covalent bonds that cross-link collagens and cuticlin are also present. First discovered in the parasitic nematode Ascaris, these cross-links are in the form of dityrosine, trityrosine and isotrityrosine (Fujimoto, 1975; Fujimoto et al., 1981; Kramer, 1997). Cuticle collagens typically contain a conserved tyrosine residue that may be used for isotrityrosine cross-links within triple-helical collagen proteins. Other tyrosine cross-linking is likely to occur between collagens and non-collagenous cuticle proteins as well (Kramer, 1997). Expression of K08F8.4 was particularly strong in the tail spike and anal region. Perhaps this reflects some special structural requirements in these locations and thus a greater need for tyrosine cross-linking.
Additional evidence in support of this hypothesis comes from the phenotype of cat-4 mutants. These mutants are serotonin- and dopamine-deficient (Sulston et al., 1975; Desai et al., 1988) and seem to have a defective cuticle (Loer, 1995; Loer, unpublished). These mutant worms are hypersensitive to a variety of agents, consistent with a leaky, poorly cross-linked cuticle. The cat-4 gene maps very close to a GTP cyclohydrolase I-homologous sequence and therefore may encode this enzyme, which is required for synthesis of biopterin cofactor. The phenotype of the cat-4 mutant could therefore be explained by absence of function of all three AAAH proteins caused by lack of cofactor. Loss or reduction in serotonin and dopamine alone does not appear to affect the cuticle, as serotonin- and dopamine-deficient bas-1 and cat-1 mutants do not show hypersensitivity like cat-4 mutants (Loer, unpublished).
We found that a K08F8.4 fusion protein expressed in bacteria is able to hydroxylate phenylalanine, and to a lesser extent, tryptophan. Although the addition of MBP to the N-terminal end could alter the function of the protein, studies with mammalian AAAH proteins suggest that substrate specificity will not be altered. Mammalian AAAH proteins have an N-terminal regulatory domain and a C-terminal catalytic domain. Although some treatments can broaden specificity of PheH (Kaufman and Mason, 1982), substrate specificity seems largely to be determined by the catalytic domain of the proteins. Deletion of the N-terminal third of the protein typically changes the activation of the C-terminal catalytic core, but does not alter substrate specificity of TrpH or PheH; expression of either full-length or truncated TrpH as a fusion protein also did not alter substrate specificity (D'Sa et al., 1996). A chimeric protein with the N-terminal regulatory domain of TH and the catalytic domain of TrpH retained a substrate specificity for tryptophan (Mockus et al., 1997); chimeras with regulatory and catalytic domains of TH and PheH always retained the substrate specificity of catalytic domain (Daubner et al., 1997). Expression of human PheH as a MBP fusion protein with a Factor X cleavage site, virtually an identical situation to our experiments, resulted in a protein with a Km for L-Phe essentially the same as or higher than a cleaved fusion protein or purified PheH (Martinez et al., 1995). Therefore, it seems likely based on our results that this protein in C. elegans is able to act as a phenylalanine hydroxylase.
We wish to thank the following persons: Cynthia Kenyon, Dept. of Biochemistry & Biophysics, University of California, San Francisco, in whose laboratory much of this work was performed, for generous support and guidance; Alexander (Sasha) Kamb and Steve Salser, for initiating CL into the mysteries of molecular biology and PCR; David Husic, Dept. of Chemistry, Lafayette College, for help with the spectrofluorometer and kinetics discussions; Deepali Deka, John McCartney, Willy Christian and Patrick Merritt for technical assistance; Phil Auerbach (Lafayette) and Don Gennero (USD) for help with digital imaging; Morris Maduro and David Pilgrim for the unc-119 mutant and rescuing plasmid; Andy Fire and lab for reporter gene fusion vectors; Ian Jennings and R.G.H. Cotton for a generous supply of the PH8 monoclonal antibody; Jamie Ahner (USD) for her work with PH8; members of the Kenyon, McKerrow and Loer labs for advice and discussions. Some of the strains used were obtained from the Caenorhabditis Genetics Center, which is funded by the NIH National Centers for Research Resources. C.L. was supported by an NIH Postdoctoral Fellowship and Traineeship, startup funds from Lafayette College, a National Science Foundation RUI grant (Developmental Neuroscience), and the Fletcher Jones Foundation; B.D. by a Merck/AAAS Summer Research Fellowship; and J.McK. by the Edna McConnell Clark Foundation.
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