Blau, Nenad

Heizmann, Claus W.

Kluge, Claudia

Leimbacher, Walter

Thöny, Beat

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 Kluge et aZ.: PTPS-cDNAs from human brain Pteridines Vol. 7, 1996, pp. 91- 93 Isolation of 6-Pyruvoyl-tetrahydropterin Synthase cDNAs from Human Brain -:audia Kluge, Walter Leimbacher, Claus W. Heizmann, Nenad Blau and Beat Thony§ ~)l\'ision of Clinical Chemistry, Department of Pediatrics, University of Zurich, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland Received July 15, 1996) Introduction The recent update summary of tetrahydrobio-pterin deficiency due to autosomal recessive mu-tations in 6-pyruvoyl-tetrahydropterin synthase (PTPS) compiled 186 cases in total, i.e. 156 cases with the more common severe form and 30 cases with the rare mild or peripheral form (1). Pa-tients with the mild type of deficiency, as opposed to the severe form, do not show any ab-normalities of monoamine neurotransmitter meta-bolites in CSF and thus do not require re-placement therapy with L-DOPA and 5-0H-typ-tophan. Genetic as well as biochemical properties of the normal human PTPS in comparison with mutant alleles might be a key to the understanding of these phenotypical differences. As an example, tissue-specific splicing was uncovered for the Dro-sophila purple gene that expresses the fly PTPS ac-tivity (2). The single purple gene was shown to ex-press two transcripts with the same putative cod-ing region from two different promoters; i.e. a weak constitutive body promoter and a strong transient head-specific promoter, expressing the alternatively spliced mRNAs with exon structures a-bl-c-d-e and b2-c-d-e, respectively. In-terestingly, the molecular characterization of the first patient with the mild form of PTPS de-ficiency revealed one allele, RI6C, with the cor-responding mutation located in exon 1 in the sin-gle human gene (3,4). In the case of a similar tis-§ Author to whom correspondence should be addressed. sue-specific splicing as observed for the Dro-sophila purple gene, a brain-specific, non-mutated 5 I exon might be present in the human neuronal PTPS-cDNA. In this work we addressed the question wheth-er the brain-specific form (s) of the human PTPS-cDNA is identical to the liver-specific form. We first isolated PTPS-cDNAs from human brain li-braries by using the liver-specific cDNA as a probe. As we did not find any clones with 5 '-ends comparable to the known liver cDNA, we applied RACE technology to examine 5 I -stretches from freshly prepared RNA of neuronal cells expressing the active PTPS enzyme. Analysis of these am-plified cDNAs revealed that the neuron-specific PTPS was identical to the liver-specific form. These findings argue against e.g. tissue-specific splicing as a molecular mechanism to develp the rare peripheral form of PTPS deficiency as op-posed to the more common central form. Materials and Methods Screening of Agtll-vector derived human brain cDNA libraries (hypothalamus 5 '-stretch, Clon-tech HL1l72b and Alzheimer patient, Clontech HLI 028) was carried out following conventional protocols. As a probe, the human liver PTPS-eDNA was hybridized at a temperature of 62°C. To isolate 5' ends of the PTPS eDNA, a stan-dard protocol describing the 'rapid amplification of cDNA ends' (5 ' -R..-\CE ) by Frohman et al. was ap-plied (5). The two PTPS-specific primers PTPS7B Pteridines / Vol. 7 / No. 3 92 (5'-GGCACATCCATATCCAG-3') and PTPS9 ( 5 ' - C G G GAT C C G G G G C T G CAT AA-TCGCCTCC-3 ') were used for the first and second round, respectively, of PCR amplification. As a template, we used reverse transcribed total RNA (2 j..Lg) prepared from the cultured human neuroblastoma cells SK-N-BE. Restriction frag-ments from positive A-phage clones or from 5'-RACE products were cloned into pUC18/ 19 plasmids and sequenced using the AutoRead sequencing kit and an Automated Laser Flu-orescent (A.L.F.) DNA sequencer from Pharmacia Biotech. Results and Discussion Previous Western blot analysis of PTPS-cross-reactive material in extracts from human brain tis-sues (hypothalamus, mesencephalon, hypophysis) and the neuroblastoma cell line SK-N-BE sug-gested the presence of the same PTPS in neu-ronal tissue as in human liver cells or fibroblasts. Similarly, Northern blot analysis of human SK-N-BE (neuroblastoma cells), Hep G2 (hepatoma cells) and fibroblasts showed no difference in PTPS transcript size between the different RNA extracts (unpublished observations). Nevertheless, we performed extensive screening of two comm-ercially available human brain cDNA libraries, Al-zheimer patient and hypothalamus, with the liver PTPS-cDNA as a probe and isolated three clones with different 5'-and 3'-ends (Fig. 1). Apart from the fact that none of these isolated had a poly-A tail, each exhibited a different length of its 3 '-end. While the longest clone had an extension of 415 bp downstream from the TAG stop codon, the two other clones matched either the previous cDNA isolates from liver (6 ) or leukemia cells (7) with an extension of 198 bp and 249 bp, respec-tively, of untranslated DNA. Comparison of the 3' untranslated sequence of the PTS gene revealed that the entire 415 bp are part of exon 6 . Moreov-er, polyadenylation signals, a classical (AATAAA) and a non-classical (ATTAAA), were present 15 bp and 16 bp upstream of the 3' end of the clones with the 415 bp and 249 bp non-coding DNA, respectively. These findings suggest that up to 3 different 3' -ends of the PTPS transcript may be present in neuronal and other human tissues. Regarding the 5'-ends of the cDNA library iso-lates, the two longer clones were deleted for exon 3 , and only one extended into exon 1. A deletion of the 23 bp in the PTPS-cDNA, comprising exon 3 on genomic DNA, leads to a frame shift Pteridines / Vol. 7 / No.3 Kluge et aZ. : PTPS-cDNAs from human brain I II I I , ptll y A li ver eDNA libr.uy tThuny el OIL, 1992) I II I I I I I I pul y A i\1n lt -4, IYl11phuhl., .!> ti c leukemia I I I I I I cells (A.!>hida et il l.. 1 'JlJ3) I I II I I I I -I I br,\ill e DNA IihriHy. A lzheim e r ~[ I I II I I I I I I I II I I I I I I II I -==:J I I brain eDNA libr.uy, h ypo-Ihal,HulI!. Z I II I '2 I I II I I brain eON A li br.uy. h y pu. I ~ th a l;tmllS I I II I I ~[ I I II I I S'-HA CE. SK-N-B E ,"' e ll s, I I I neu fu hl .ls lun, ,, I I II I I ~ I S'·It.A CE, SK -N-BE ce lls, I I I Ilcurol> laSlo m.l I I II I I I I -' I , ""1'5 · 1'1 rclrupseuJ.vsenc un chromo 9p12-p13 (Kluge e l .. I., 1996) Figure 1 . Organization of the human PTS gene and ex-tension of corresponding cDNA clones isolated from various sources. The PTS gene located on chromosome 11q 22.3-q23.3 is composed of 6 exons asincticated by the boxes. The coding regions are in black, whereas the 5' -and 3'-untranslated stretches are depicted as open bars. The position of the two putative polyadenylation signals (ATTAAA and AATAAA) are incticated by arrows . The different sources of inctividual cDNA isolates are given on the right. Polyadenin stretches found at the 3'-ends of cDNAs are incticated by 'polyA' . The hatched box is a spliced fragment which originates from intron 1 (see text for details) . For comparison, the extension of the 74%-identiy to the PTS-Pl retropseudogene is given at the bottom. and a subsequent stop codon. A similar mutant cDNA allele has been isolated previously from a PTPS patient (K54X allele; 8). The cause for ex-pressing such a mutant RNA was a genomic T to A transversion at position-7 in the 3 '-acceptor splice site of the patient 's intron 2, which leads to exon 3 skipping (4). Interestingly, a sequence alignment between the liver PTPS cDNA and the PTS-Pl retropseudogene revealed a significant homology gap of 23 bp extending over the exon 3 region . This implicates that exon 3 skipping does not occur exclusively due to 3 '-acceptor splice mu-tations, but might also be caused by a splicing po-lymorphism. In addition to a deletion of exon 3, we found in one clone from the hypothalamus cDNA li-brary a DNA fragment which was fused at the 5 ,-end of exon 2. This fragment turned out to be an internal 226 bp stretch from intron 1 (nucleotide  Kluge et al.: PTPS-cDNAs from human brain poslt:J.on 1451-1676 in the deposited GenBank se-quence L76259). This cDNA clone, which is a splice product consisting of the 226 bp 'exonic' fragment plus exons 2, 4, 5 and 6, did also not yield any open reading frame in its 5 I -portion of the cDNA. Although at this stage we could not exclude any functional role for the isolated brain cDNA clones, we assumed that the exon 3 deletion in two clones and the spliced fragment from intron 1 in one clone were due to some artifacts present in the neuronal cDNA libraries analyzed. Furthermore, as we detected PTPS enzyme ac-tivity (9) and cross-reactive material in human hypothalamus extract (not shown), we expected to find a cDNA that encodes a functional PTPS. To discriminate between erroneously spliced cDNAs present in the brain cDNA libraries and potentially alternative splicing in neuronal cells, we carried out 5 ' -RACE experiments with fresh-ly prepared RNA from cultured · neurobla-stoma cells that expressed the human PTPS en-dogenously (SK-N-BE cells, unpublished ob-servation) . The PTPS-specific downstream prim-ers were selected to amplifY the cDNA upstream from exon 5 (PTPS7B and PTPS9; for details see Materials and Methods). The 5 '-RACE pro-ducts were gel purified, subcloned into plasmid vectors, and sequenced. The two clones that we analyzed did neither show any alternatively splic-ed forms of PTPS-cDNA, nor a deletion of exon 3 (Fig. 1). Furthermore, one clone had the com-plete coding sequence of exon 1 plus 18 bp of the non-coding 5 ' -upstream sequence. Un-fortunately, we did not isolate any clones that were extended up to the previously described 68 bp upstream from the translational start site (Ashida et ai., 1993). In summary, our findings argue against alt-ernative 5 '-exons in the human PTS gene for ex-pression of a functional enzyme, since no such tis-sue- or cell-specific transcripts for the human PTPS were isolated . Whether the occurrence of a deletion of exon 3 in processed transcipts is a po-lymorphism or has some functional meaning ne-eds to be clarified. Furthermore, to unravel the molecular basis of clinical and biochemical diff-erences of the various forms of PTPS insufficiency, more data about the molecular defects in patients especially with the peripheral type of PTPS de-ficiency are required. 93 Acknowledgments We thank M. Killen for help with the pre-paration of the mansucript. This work was sup-ported by grants from the Swiss National Science Foundation (project No. 31-43380.95) and the Hartmann-Muller Stiftung. References 1. Blau N . Barnes 1. Dhondt, 1.L. (1996). International database of tetrahydrobiopterin deficiencies. 1. Inher. Metab . Dis., 1996; 19: 8 -14. 2. Kim N. Kim, J. Park D. Rosen C . Dorsett D. Yim 1. Structure and expression of wild-type and suppres-sible alleles of the Drosophila purple gene. Genetics, 1995; 142: 1157-1168. 3. Thony B. Leimbacher W. Blau N. Harvie A. Heizmann C.W. Hyperphenylalaninemia due to de -fects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahy-dropterin synthase. 1994: Am. 1. Hun. Genet, 54: 782-792. 4 . Kluge C. Brecevic L. Heizmann C.W. Blau N . Thony B. Chromosomal localization, genomic structure and characterization of the human gene and a retropseu-dogene for 6-pyruvoyltetrahydropterin synthase. 1996: Eur. 1. Biochem. 240,477-484. 5. Frohman M .A. Rapid amplification of eDNA ends (RACE): user-friendly eDNA cloning. Perkin Elmer Cetus amplifications, a forum for PCR users: 1990: 11-15 . 6. Thony B. Leimbacher W. Burgisser D . Heizmann C. W. Human 6 -pyruvoyl-tetrahydropterin synthase: eDNA cloning and heterologous expression of the re-combinant enzyme. Biochem. Biophys. Res. Comm. 1992; 189: 1437-1443. 7. Ashida A. Hatakeyama K Kagamiyama H. cDNA clon-ing, expression in Escherichia coli and purification of human 6-pyruvoyl-tetrahydropterin synthase. 1993: Biochem. Biophys. Res. Comm.: 195: 1386-1393. 8. Oppliger T. Thony B. Nar H. Burgisser D . Huber R . Heizmann C .W. Blau N. Structural and functional consequences of mutations in 6-pyruvoyl-tetrahy-dropterin synthase causing hyperphenylalaninemia in humans: phosphorylation is a requirement for in vivo activity. 1995; 1. BioI. Chern., 270: 29498-29506. 9. Heizmarm C .W. Leimbacher W. Kierat L. Blau N. Measurement of enzymes involved in the biosyn-thesis of tetrahydrobiopterin, pterins and nu-urotransmitter metabolites in various regions of the human brain . In: Blau N. Curtius H. C. Le"ine R.A. Cotton R .GH . eds. Pteridines and Biogenic Amines in Neurology, Pediatrics and Immunology. Lak-eshore Publishing 1991; 95 -99 . Pteridines / Vol. 7 / No. 3 

Isolation of 6-Pyruvoyl-tetrahydropterin Synthase cDNAs from Human Brain

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