- Email: [email protected]
Asparagine repeats are rare in mammalian proteins
LETTER
TIBS 25 – JUNE 2000
translucid vacuoles. These are only a few examples of the experimental developments that could originate from the model. Moreover, once their mechanism of action is firmly established, the presynaptic PLA2 neurotoxins could be employed as tools to investigate specific aspects of SSV fusion and recycling.
Acknowledgements
presynaptically. Annu. Rev. Pharmacol. Toxicol. 20, 307–336 Kelly, R.B. et al. (1976) Beta-bungarotoxin, a phospholipase that stimulates transmitter release. Cold Spring Harbor Symp. Quant. Biol. 40, 117–125 Lee, C.Y. et al. (1984) Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom. Arch. Int. Pharmacodyn. Ther. 268, 313–324 Chen, I.L. and Lee, C.Y. (1970) Ultrastructural changes in the motor nerve terminals caused by betabungarotoxin. Virchows Arch. B. Cell Pathol. 6, 318–325 Cull-Candy, S.G. et al. (1976) The effect of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1, 175–180 Gopalakrishnakone, P. and Hawgood, B.J. (1984) Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon 22, 791–804 Dixon, R.W. and Harris, J.B. (1999) Nerve terminal damage by β-Bungarotoxin: its clinical significance. Am. J. Pathol. 154, 447–455 Schmid, S.L. et al. (1998) Dynamin and its partners: a progress report. Curr. Opin. Cell Biol. 10, 504–512 Kini, R.M. and Evans, H.J. (1989) A model to explain the pharmacological effects of snake venom phospholipase A2. Toxicon 27, 613–635 Rufini, S. et al. (1990) β-Bungarotoxin-mediated liposome fusion: spectroscopic characterization by fluorescence and ESR. Biochemistry 29, 9644–9651 Karli, U.O. et al. (1990) Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system. Proc. Natl. Acad. Sci. U. S. A. 87, 5912–5915 Nishio, H. et al. (1996) Ca21-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro. Biochem. J. 318, 981–987 Meers, P. et al. (1988) Free fatty acid enhancement of cation-induced fusion of liposomes: synergism with synexin and other promoters of vesicle aggregation. Biochemistry 27, 6784–6794 Wilschut, J. et al. (1992) Ca21-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients. Biochemistry 31, 2629–2636 Zellmer, S. et al. (1994) Temperature- and pH-controlled fusion between complex lipid membranes. Examples with the diacylphosphatidylcholine/fatty acid mixed liposomes. Biochim. Biophys. Acta 1196, 101–113 Langner, M. et al. (1995) Interaction of free fatty acids with phospholipid bilayers. Biochim. Biophys. Acta 1236, 73–80 Simpson, L.L. et al. (1993) Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. Toxicon 31, 13–26 Montecucco, C. and Schiavo, G. (1995) Structure and function of tetanus and botulinum neurotoxins. Q. Rev. Biophys. 28, 423–472 Dennis, E.A. (1994) Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269, 13057–13060 Nagy, A. et al. (1976) The preparation and characterization of synaptic vesicles of high purity. Brain Res. 109, 285–309
31 Deutsch, J.W. and Kelly, R.B. (1981) Lipids of synaptic vesicles: relevance to the mechanism of membrane fusion. Biochemistry 20, 378–385 32 Michaelson, D.M. et al. (1983) Asymmetry of lipid organization in cholinergic synaptic vesicle membranes. Biochem. J. 211, 155–162 33 Ledeen, R.W. et al. (1993) Ganglioside composition of subcellular fractions, including pre- and postsynaptic membranes, from Torpedo electric organ. Neurochem. Res. 18, 1151–1155 34 Carlson, S.S. and Kelly, R.B. (1983) A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles. J. Biol. Chem. 258, 11082–11091 35 Scranton, T.W. et al. (1993) The SV2 protein of synaptic vesicles is a keratan sulfate proteoglycan. J. Neurochem. 61, 29–44 36 Stadler, H. and Dowe, G.H.C. (1982) Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata. EMBO J. 1, 1381–1384 37 Schmidt, R. et al. (1980) Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: effect of stimulation-induced transmitter release. Neuroscience 5, 625–638 38 Schuldiner, S. et al. (1995) Vesicular neurotransmitter transporters: from bacteria to humans. Physiol. Rev. 75, 369–392 39 Kamp, F. and Hamilton, J.A. (1993) Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry 32, 11074–11086 40 Cullis, P.R. and De Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559, 399–420 41 Chernomordik, L.V. et al. (1995) Lipids in biological membrane fusion. J. Membr. Biol. 146, 1–14 42 Chernomordik, L.V. et al. (1997) An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J. Cell Biol. 136, 81–93 43 Schiavo, G. et al. (1998) Synaptotagmins: more isoforms than functions? Biochem. Biophys. Res. Commun. 248, 1–8 44 Schmidt, A. et al. (1999) Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133–141 45 Weigert, R. et al. (1999) CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature 402, 429–433 46 Sudhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature 375, 645–653 47 Nicholls, D.G. et al. (1985) Bioenergetic actions of β-bungarotoxin, dendrotoxin and bee-venom phospholipase A2 on guinea-pig synaptosomes. Biochem. J. 229, 653–662 48 Rugolo, M. et al. (1986) The mechanism of action of β-bungarotoxin at the presynaptic plasma membrane. Biochem. J. 233, 519–523 49 Esquerda, J.E. et al. (1982) Binding of β-bungarotoxin to Torpedo electric organ synaptosomes. A highresolution autoradiographic study. Neuroscience 7, 751–758
prevalent of these disorders is Huntington’s disease. The protein encoded by the Huntington gene functions normally with repeats of up to ~38 Q residues, whereas longer stretches form aggregates. We have compared the number of Q repeats with poly-asparagines (N repeats) and analysed sequences in both SWISSPROT (a manually curated protein database) and, to ensure that our results are not affected by a bias in the selection of proteins in this database, SPTREMBL. Eight different genes have been described with disease-causing expansions of CAG triplets1,2. In addition,
Q repeats ranging from 30 to 38 residues are present in four proteins deposited in SWISS-PROT. One is from the slime mold Dictyostelium discoideum (accession no. P54683) and two are from yeast (P18480, P14922). The fourth is a human TATAsequence-binding protein (P20226), which contains a stretch of 38 glutamines. In SPTREMBL, 15 entries were found with Q repeats ranging from 30 to 41 residues. These are from diverse organisms (human, mouse, quail, Drosophila, Dictyostelium). It is noteworthy that, as observed from the pathology of human diseases, Q repeats containing .41 residues have not been found in other
12
13
14
15
We are grateful to R.W. Dixon, J.B. Harris and A. Menez for discussions on snake neurotoxins and to M.G. Cifone, J. Meldolesi, T. Pozzan and G. Schiavo for critical reading of the manuscript. Work in the authors’ laboratory is supported by Biomed2 BMH4-97 2410, by Telethon grant no. 1068 and by the Armenise-Harvard Medical School Foundation. We apologize to the many colleagues, whose relevant papers could not be quoted owing to space restrictions.
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References 1 Fontana, F. (1782) Sui veleni della vipera e sui veleni americani, Firenze 2 Rappuoli, R. and Montecucco, C. (1997) Protein Toxins and Their Use in Cell Biology, Oxford University Press 3 Kini, R.M., ed. (1997) Venom Phospholipase A2 Enzymes. Structure, Function and Mechanism, John Wiley & Sons, Chichester 4 Dennis, E.A. (1997) The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22, 1–2 5 Lambeau, G. and Lazdunski, M. (1999) Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol. Sci. 20, 162–170 6 Carredano, E.B. et al. (1998) The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2. Toxicon 36, 75–92 7 Scott, D.L. (1997) In Venom Phospholipase A2 Enzymes. Structure, Function and Mechanism. (Kini, R.M., ed.), pp. 97–128, John Wiley & Sons, Chichester 8 Harris, J.B. (1985) Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol. Ther. 31, 79–102 9 Chang, C.C. et al. (1973) Studies of the presynaptic effect of β-bungarotoxin on neuromuscular transmission. J. Pharmacol. Exp. Ther. 184, 339–345 10 Kamenskaya, M.A. and Thesleff, S. (1974) The neuromuscular blocking action of an isolated toxin from the elapid (Oxyuranus scutellactus). Acta Physiol. Scand. 90, 716–724 11 Howard, B.D. and Gundersen, C.B. (1980) Effects and mechanisms of polypeptide neurotoxins that act
Asparagine repeats are rare in mammalian proteins It has been shown in recent years that the expansion of CAG triplets within the coding regions of several human genes yields proteins with glutamine repeats (Q repeats), which, beyond a certain length, cause neurodegenerative disorders1,2. This was first observed for a muscular atrophy caused by CAG expansions in the androgen receptor gene3. The most
270
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25
26
27
28
29
30
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
PII: S0968-0004(00)01594-2
LETTER
TIBS 25 – JUNE 2000
Table 1. Frequency of Q and N repeats in proteinsa SPTREMBL
SWISS-PROT Mammals
>41 30–41 20–29 10–19 6–9 5 4 3
Mammals
Q
N
Q
N
Q
N
Q
N
0 15 56 322 886 841 1746 15 574
7 10 35 184 610 480 1511 20 190
0 7 27 90 120 78 206 1149
0 0 0 0 0 4 58 570
0 6 26 183 448 417 879 4343
2 2 8 49 174 165 537 3833
0 1 11 31 50 63 165 878
0 0 0 0 1 2 34 413
Total number 199 794
23 465
80 000
16 813
aData are from SWISS-PROT release 38 and SPTREMBL release 11 (Ref. 7). Data collection employed the SRS software version 5.1 (Ref. 8). Results obtained for the whole data collection and the subset of mammalian proteins are shown.
eukaryotes. Alternatively, repeats of the corresponding triplets might somehow impair transcription or translation. In our search we found that, in all reading frames, repeats of AAY triplets (Y being T or C) are not present in coding sequences of mammalian genes. The longest repeat is an (AAC)8 sequence in the gene coding for protein P81122 mentioned earlier4. This is also surprising given that (AAT)n is the most frequent trinucleotide repeat in non-coding regions of, for example, the human genome6. Recombination of gene constructs containing AAT or AAC repeats into the genome of various cell types to study the stability of transcripts and their translation could possibly discern between the alternatives mentioned above.
References organisms, including unicellular ones like yeast and Dictyostelium where late-onset defects might not exist. In the case of asparagine, repeats of 30, 32, 42 and 50 residues, respectively, are present in proteins P18160, P54675, P54674 and P54637 from D. discoideum. SPTREMBL contains 17 further examples of N repeats of >30, including seven with 42–46 residues. It is noteworthy that N repeats composed of up to 50 residues appear to be innocuous. In view of the importance of Q repeats for some human diseases, we compared the frequency of these and N repeats in mammals. This yielded an unexpected result (Table 1). Although Q repeats of all lengths are present in mammalian proteins, no long N repeats have been found. In SWISS-PROT, there is a single entry, insulin receptor substrate-2 (Ref. 4), with eight consecutive asparagines (P81122), and two with (Asn)5 sequences (P38432, O60238). A search for N repeats in all vertebrate proteins yielded only one additional entry [P11885; (Asn)5]. Also in SPTREMBL, only four entries with N repeats of five have been found. A comparison of tetra- and tri-peptides demonstrated that those containing glutamine were approximately five and two times more frequent, respectively, than those composed of asparagines (Table 1). These amino acids are, however, present in approximately equal amounts in all mammalian proteins listed in the databases (~4.5 mol percent Q and 3.8 mol percent N). We have also surveyed the repeat lengths for the other amino acids. Only in the case of asparagine does this large difference between proteins from all organisms and the subset from mammals exist. For those residues that occur in longer homopeptides, we also find many examples in mammalian proteins. The actual numbers of repeats composed of more than five residues in mammalian
proteins are as follows: 181 for glycine, 237 for alanine, 131 for serine, 17 for threonine, 51 for aspartic acid, 247 for glutamic acid, 93 for glutamine, 205 for proline, 57 for histidine, 76 for arginine, 38 for lysine, ten for valine and 270 for leucine. This is in marked contrast with asparagine, where only one such sequence has been found. For the remaining amino acids, repeats are rare or do not exist in proteins from all organisms. In SWISS-PROT, the numbers of repeats with more than five residues for all organisms versus mammals are: 5/2 for cysteine, 5/3 for methionine, 8/0 for isoleucine, 3/0 for phenylalanine, 2/0 for tyrosine and 0/0 for tryptophane. In an earlier analysis, proteins from humans and from Drosophila were listed that contain multiple amino acid repeats. Even though this was not addressed by the authors, in the tables presented in Ref. 5, there is not a single N repeat in the human proteins, although 16 with 5–20 asparagines were listed for Drosophila. By now, four years later, this number has risen to 53 SWISS-PROT entries. This result suggests that N repeats have properties that, for unknown reasons, are harmful to mammalian and probably vertebrate cells in general, but not for those of invertebrates and lower
1 Perutz, M.F. (1999) Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem. Sci. 24, 58–63 2 Reddy, P.S. and Housman, D.E. (1997) The complex pathology of trinucleotide repeats. Curr. Opin. Cell Biol. 9, 364–372 3 La Spada, A.R. et al. (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352, 77–79 4 Sun, X.J. et al. (1995) Role of IRS-2 in insulin and cytokine signalling. Nature 377, 173–177 5 Karlin, S. and Burge, C. (1996) Trinucleotide repeats and long homopeptides in genes and proteins associated with nervous system disease and development. Proc. Natl. Acad. Sci. U. S. A. 93, 1560–1566 6 Gastier, J.M. et al. (1995) Survey of trinucleotide repeats in the human genome: assessment of their utility as genetic markers. Hum. Mol. Genet. 4, 1829–1836 7 Bairoch, A. and Apweiler, R. (1999) The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Res. 27, 49–54 8 Kreil, D.P. and Etzold, T. SRS – access to molecular biological databanks and integrated data analysis tools. In Bioinformatics: a Practical Approach, Oxford University Press (in press)
DAVID P. KREIL European Bioinformatics Institute, Cambridge, UK CB10 1SD. Email: [email protected]
¨ NTHER KREIL GU Institute of Molecular Biology, 5020 Salzburg, Austria. Email: [email protected]
Letters to TiBS TiBS welcomes letters on any topic of interest. Please note, however, that previously unpublished data and criticisms of work published elsewhere cannot be accepted by this journal. Letters should be sent to: Emma Wilson, Editor Trends in Biochemical Sciences Current Trends, Elsevier Science London 84 Theobald’s Road, London, UK WC1 8RR
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translucid vacuoles. These are only a few examples of the experimental developments that could originate from the model. Moreover, once their mechanism of action is firmly established, the presynaptic PLA2 neurotoxins could be employed as tools to investigate specific aspects of SSV fusion and recycling.
Acknowledgements
presynaptically. Annu. Rev. Pharmacol. Toxicol. 20, 307–336 Kelly, R.B. et al. (1976) Beta-bungarotoxin, a phospholipase that stimulates transmitter release. Cold Spring Harbor Symp. Quant. Biol. 40, 117–125 Lee, C.Y. et al. (1984) Mode of neuromuscular blocking action of toxic phospholipases A2 from Vipera ammodytes venom. Arch. Int. Pharmacodyn. Ther. 268, 313–324 Chen, I.L. and Lee, C.Y. (1970) Ultrastructural changes in the motor nerve terminals caused by betabungarotoxin. Virchows Arch. B. Cell Pathol. 6, 318–325 Cull-Candy, S.G. et al. (1976) The effect of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience 1, 175–180 Gopalakrishnakone, P. and Hawgood, B.J. (1984) Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon 22, 791–804 Dixon, R.W. and Harris, J.B. (1999) Nerve terminal damage by β-Bungarotoxin: its clinical significance. Am. J. Pathol. 154, 447–455 Schmid, S.L. et al. (1998) Dynamin and its partners: a progress report. Curr. Opin. Cell Biol. 10, 504–512 Kini, R.M. and Evans, H.J. (1989) A model to explain the pharmacological effects of snake venom phospholipase A2. Toxicon 27, 613–635 Rufini, S. et al. (1990) β-Bungarotoxin-mediated liposome fusion: spectroscopic characterization by fluorescence and ESR. Biochemistry 29, 9644–9651 Karli, U.O. et al. (1990) Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system. Proc. Natl. Acad. Sci. U. S. A. 87, 5912–5915 Nishio, H. et al. (1996) Ca21-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro. Biochem. J. 318, 981–987 Meers, P. et al. (1988) Free fatty acid enhancement of cation-induced fusion of liposomes: synergism with synexin and other promoters of vesicle aggregation. Biochemistry 27, 6784–6794 Wilschut, J. et al. (1992) Ca21-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients. Biochemistry 31, 2629–2636 Zellmer, S. et al. (1994) Temperature- and pH-controlled fusion between complex lipid membranes. Examples with the diacylphosphatidylcholine/fatty acid mixed liposomes. Biochim. Biophys. Acta 1196, 101–113 Langner, M. et al. (1995) Interaction of free fatty acids with phospholipid bilayers. Biochim. Biophys. Acta 1236, 73–80 Simpson, L.L. et al. (1993) Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. Toxicon 31, 13–26 Montecucco, C. and Schiavo, G. (1995) Structure and function of tetanus and botulinum neurotoxins. Q. Rev. Biophys. 28, 423–472 Dennis, E.A. (1994) Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269, 13057–13060 Nagy, A. et al. (1976) The preparation and characterization of synaptic vesicles of high purity. Brain Res. 109, 285–309
31 Deutsch, J.W. and Kelly, R.B. (1981) Lipids of synaptic vesicles: relevance to the mechanism of membrane fusion. Biochemistry 20, 378–385 32 Michaelson, D.M. et al. (1983) Asymmetry of lipid organization in cholinergic synaptic vesicle membranes. Biochem. J. 211, 155–162 33 Ledeen, R.W. et al. (1993) Ganglioside composition of subcellular fractions, including pre- and postsynaptic membranes, from Torpedo electric organ. Neurochem. Res. 18, 1151–1155 34 Carlson, S.S. and Kelly, R.B. (1983) A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles. J. Biol. Chem. 258, 11082–11091 35 Scranton, T.W. et al. (1993) The SV2 protein of synaptic vesicles is a keratan sulfate proteoglycan. J. Neurochem. 61, 29–44 36 Stadler, H. and Dowe, G.H.C. (1982) Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata. EMBO J. 1, 1381–1384 37 Schmidt, R. et al. (1980) Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: effect of stimulation-induced transmitter release. Neuroscience 5, 625–638 38 Schuldiner, S. et al. (1995) Vesicular neurotransmitter transporters: from bacteria to humans. Physiol. Rev. 75, 369–392 39 Kamp, F. and Hamilton, J.A. (1993) Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry 32, 11074–11086 40 Cullis, P.R. and De Kruijff, B. (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559, 399–420 41 Chernomordik, L.V. et al. (1995) Lipids in biological membrane fusion. J. Membr. Biol. 146, 1–14 42 Chernomordik, L.V. et al. (1997) An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J. Cell Biol. 136, 81–93 43 Schiavo, G. et al. (1998) Synaptotagmins: more isoforms than functions? Biochem. Biophys. Res. Commun. 248, 1–8 44 Schmidt, A. et al. (1999) Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133–141 45 Weigert, R. et al. (1999) CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature 402, 429–433 46 Sudhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature 375, 645–653 47 Nicholls, D.G. et al. (1985) Bioenergetic actions of β-bungarotoxin, dendrotoxin and bee-venom phospholipase A2 on guinea-pig synaptosomes. Biochem. J. 229, 653–662 48 Rugolo, M. et al. (1986) The mechanism of action of β-bungarotoxin at the presynaptic plasma membrane. Biochem. J. 233, 519–523 49 Esquerda, J.E. et al. (1982) Binding of β-bungarotoxin to Torpedo electric organ synaptosomes. A highresolution autoradiographic study. Neuroscience 7, 751–758
prevalent of these disorders is Huntington’s disease. The protein encoded by the Huntington gene functions normally with repeats of up to ~38 Q residues, whereas longer stretches form aggregates. We have compared the number of Q repeats with poly-asparagines (N repeats) and analysed sequences in both SWISSPROT (a manually curated protein database) and, to ensure that our results are not affected by a bias in the selection of proteins in this database, SPTREMBL. Eight different genes have been described with disease-causing expansions of CAG triplets1,2. In addition,
Q repeats ranging from 30 to 38 residues are present in four proteins deposited in SWISS-PROT. One is from the slime mold Dictyostelium discoideum (accession no. P54683) and two are from yeast (P18480, P14922). The fourth is a human TATAsequence-binding protein (P20226), which contains a stretch of 38 glutamines. In SPTREMBL, 15 entries were found with Q repeats ranging from 30 to 41 residues. These are from diverse organisms (human, mouse, quail, Drosophila, Dictyostelium). It is noteworthy that, as observed from the pathology of human diseases, Q repeats containing .41 residues have not been found in other
12
13
14
15
We are grateful to R.W. Dixon, J.B. Harris and A. Menez for discussions on snake neurotoxins and to M.G. Cifone, J. Meldolesi, T. Pozzan and G. Schiavo for critical reading of the manuscript. Work in the authors’ laboratory is supported by Biomed2 BMH4-97 2410, by Telethon grant no. 1068 and by the Armenise-Harvard Medical School Foundation. We apologize to the many colleagues, whose relevant papers could not be quoted owing to space restrictions.
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References 1 Fontana, F. (1782) Sui veleni della vipera e sui veleni americani, Firenze 2 Rappuoli, R. and Montecucco, C. (1997) Protein Toxins and Their Use in Cell Biology, Oxford University Press 3 Kini, R.M., ed. (1997) Venom Phospholipase A2 Enzymes. Structure, Function and Mechanism, John Wiley & Sons, Chichester 4 Dennis, E.A. (1997) The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22, 1–2 5 Lambeau, G. and Lazdunski, M. (1999) Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol. Sci. 20, 162–170 6 Carredano, E.B. et al. (1998) The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2. Toxicon 36, 75–92 7 Scott, D.L. (1997) In Venom Phospholipase A2 Enzymes. Structure, Function and Mechanism. (Kini, R.M., ed.), pp. 97–128, John Wiley & Sons, Chichester 8 Harris, J.B. (1985) Phospholipases in snake venoms and their effects on nerve and muscle. Pharmacol. Ther. 31, 79–102 9 Chang, C.C. et al. (1973) Studies of the presynaptic effect of β-bungarotoxin on neuromuscular transmission. J. Pharmacol. Exp. Ther. 184, 339–345 10 Kamenskaya, M.A. and Thesleff, S. (1974) The neuromuscular blocking action of an isolated toxin from the elapid (Oxyuranus scutellactus). Acta Physiol. Scand. 90, 716–724 11 Howard, B.D. and Gundersen, C.B. (1980) Effects and mechanisms of polypeptide neurotoxins that act
Asparagine repeats are rare in mammalian proteins It has been shown in recent years that the expansion of CAG triplets within the coding regions of several human genes yields proteins with glutamine repeats (Q repeats), which, beyond a certain length, cause neurodegenerative disorders1,2. This was first observed for a muscular atrophy caused by CAG expansions in the androgen receptor gene3. The most
270
22
23
24
25
26
27
28
29
30
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
PII: S0968-0004(00)01594-2
LETTER
TIBS 25 – JUNE 2000
Table 1. Frequency of Q and N repeats in proteinsa SPTREMBL
SWISS-PROT Mammals
>41 30–41 20–29 10–19 6–9 5 4 3
Mammals
Q
N
Q
N
Q
N
Q
N
0 15 56 322 886 841 1746 15 574
7 10 35 184 610 480 1511 20 190
0 7 27 90 120 78 206 1149
0 0 0 0 0 4 58 570
0 6 26 183 448 417 879 4343
2 2 8 49 174 165 537 3833
0 1 11 31 50 63 165 878
0 0 0 0 1 2 34 413
Total number 199 794
23 465
80 000
16 813
aData are from SWISS-PROT release 38 and SPTREMBL release 11 (Ref. 7). Data collection employed the SRS software version 5.1 (Ref. 8). Results obtained for the whole data collection and the subset of mammalian proteins are shown.
eukaryotes. Alternatively, repeats of the corresponding triplets might somehow impair transcription or translation. In our search we found that, in all reading frames, repeats of AAY triplets (Y being T or C) are not present in coding sequences of mammalian genes. The longest repeat is an (AAC)8 sequence in the gene coding for protein P81122 mentioned earlier4. This is also surprising given that (AAT)n is the most frequent trinucleotide repeat in non-coding regions of, for example, the human genome6. Recombination of gene constructs containing AAT or AAC repeats into the genome of various cell types to study the stability of transcripts and their translation could possibly discern between the alternatives mentioned above.
References organisms, including unicellular ones like yeast and Dictyostelium where late-onset defects might not exist. In the case of asparagine, repeats of 30, 32, 42 and 50 residues, respectively, are present in proteins P18160, P54675, P54674 and P54637 from D. discoideum. SPTREMBL contains 17 further examples of N repeats of >30, including seven with 42–46 residues. It is noteworthy that N repeats composed of up to 50 residues appear to be innocuous. In view of the importance of Q repeats for some human diseases, we compared the frequency of these and N repeats in mammals. This yielded an unexpected result (Table 1). Although Q repeats of all lengths are present in mammalian proteins, no long N repeats have been found. In SWISS-PROT, there is a single entry, insulin receptor substrate-2 (Ref. 4), with eight consecutive asparagines (P81122), and two with (Asn)5 sequences (P38432, O60238). A search for N repeats in all vertebrate proteins yielded only one additional entry [P11885; (Asn)5]. Also in SPTREMBL, only four entries with N repeats of five have been found. A comparison of tetra- and tri-peptides demonstrated that those containing glutamine were approximately five and two times more frequent, respectively, than those composed of asparagines (Table 1). These amino acids are, however, present in approximately equal amounts in all mammalian proteins listed in the databases (~4.5 mol percent Q and 3.8 mol percent N). We have also surveyed the repeat lengths for the other amino acids. Only in the case of asparagine does this large difference between proteins from all organisms and the subset from mammals exist. For those residues that occur in longer homopeptides, we also find many examples in mammalian proteins. The actual numbers of repeats composed of more than five residues in mammalian
proteins are as follows: 181 for glycine, 237 for alanine, 131 for serine, 17 for threonine, 51 for aspartic acid, 247 for glutamic acid, 93 for glutamine, 205 for proline, 57 for histidine, 76 for arginine, 38 for lysine, ten for valine and 270 for leucine. This is in marked contrast with asparagine, where only one such sequence has been found. For the remaining amino acids, repeats are rare or do not exist in proteins from all organisms. In SWISS-PROT, the numbers of repeats with more than five residues for all organisms versus mammals are: 5/2 for cysteine, 5/3 for methionine, 8/0 for isoleucine, 3/0 for phenylalanine, 2/0 for tyrosine and 0/0 for tryptophane. In an earlier analysis, proteins from humans and from Drosophila were listed that contain multiple amino acid repeats. Even though this was not addressed by the authors, in the tables presented in Ref. 5, there is not a single N repeat in the human proteins, although 16 with 5–20 asparagines were listed for Drosophila. By now, four years later, this number has risen to 53 SWISS-PROT entries. This result suggests that N repeats have properties that, for unknown reasons, are harmful to mammalian and probably vertebrate cells in general, but not for those of invertebrates and lower
1 Perutz, M.F. (1999) Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem. Sci. 24, 58–63 2 Reddy, P.S. and Housman, D.E. (1997) The complex pathology of trinucleotide repeats. Curr. Opin. Cell Biol. 9, 364–372 3 La Spada, A.R. et al. (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 352, 77–79 4 Sun, X.J. et al. (1995) Role of IRS-2 in insulin and cytokine signalling. Nature 377, 173–177 5 Karlin, S. and Burge, C. (1996) Trinucleotide repeats and long homopeptides in genes and proteins associated with nervous system disease and development. Proc. Natl. Acad. Sci. U. S. A. 93, 1560–1566 6 Gastier, J.M. et al. (1995) Survey of trinucleotide repeats in the human genome: assessment of their utility as genetic markers. Hum. Mol. Genet. 4, 1829–1836 7 Bairoch, A. and Apweiler, R. (1999) The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Res. 27, 49–54 8 Kreil, D.P. and Etzold, T. SRS – access to molecular biological databanks and integrated data analysis tools. In Bioinformatics: a Practical Approach, Oxford University Press (in press)
DAVID P. KREIL European Bioinformatics Institute, Cambridge, UK CB10 1SD. Email: [email protected]
¨ NTHER KREIL GU Institute of Molecular Biology, 5020 Salzburg, Austria. Email: [email protected]
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