Cloning and Molecular Analysis of the Pea Seedling Copper Amine Oxidase
1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês
10.1074/jbc.270.28.16939
ISSN1083-351X
AutoresAlex J. Tipping, Michael J. McPherson,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoA pea seedling amine oxidase cDNA has been isolated and sequenced. A single long open reading frame has amino acid sequences corresponding to those determined from active site peptide (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and 29Janes S.M. Palcic M.M. Scaman C.H. Smith A.J. Brown D.E. Dooley D.M. Mure M. Klinman J.P. Biochemistry. 1992; 31: 12147-12154Crossref PubMed Scopus (183) Google Scholar Biochemistry 31, 12147-12154) and N-terminal sequencing experiments. The latter reveals the protein to have a 25-amino acid leader sequence with characteristics of a secretion signal peptide, as expected for this extracellular enzyme. Comparisons of the amino acid sequence of the mature pea enzyme (649 amino acids) with that of the mature lentil enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agr, A.(1992) FEBS Lett. 301, 253-257) reveal important and unexpected differences particularly with regard to protein length. Sequencing of part of the lentil gene identified several frameshift differences within the coding region resulting in a mature lentil protein of exactly the same length, 649 amino acids, as the pea enzyme. Multiple alignments of 10 copper amine oxidase sequences reveal 33 completely conserved residues of which 10 are found within 41 aligned residues at the C-terminal tails, the region missing from the original lentil sequence. One of only four conserved histidines is found in this region and may represent the third ligand to the copper. The pea enzyme contains around 3-4% carbohydrate as judged by deglycosylation experiments. We have also demonstrated by hybridization analysis that copper amine oxidase genes are present in a range of mono- and dicotyledonous plants. A pea seedling amine oxidase cDNA has been isolated and sequenced. A single long open reading frame has amino acid sequences corresponding to those determined from active site peptide (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and 29Janes S.M. Palcic M.M. Scaman C.H. Smith A.J. Brown D.E. Dooley D.M. Mure M. Klinman J.P. Biochemistry. 1992; 31: 12147-12154Crossref PubMed Scopus (183) Google Scholar Biochemistry 31, 12147-12154) and N-terminal sequencing experiments. The latter reveals the protein to have a 25-amino acid leader sequence with characteristics of a secretion signal peptide, as expected for this extracellular enzyme. Comparisons of the amino acid sequence of the mature pea enzyme (649 amino acids) with that of the mature lentil enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agr, A.(1992) FEBS Lett. 301, 253-257) reveal important and unexpected differences particularly with regard to protein length. Sequencing of part of the lentil gene identified several frameshift differences within the coding region resulting in a mature lentil protein of exactly the same length, 649 amino acids, as the pea enzyme. Multiple alignments of 10 copper amine oxidase sequences reveal 33 completely conserved residues of which 10 are found within 41 aligned residues at the C-terminal tails, the region missing from the original lentil sequence. One of only four conserved histidines is found in this region and may represent the third ligand to the copper. The pea enzyme contains around 3-4% carbohydrate as judged by deglycosylation experiments. We have also demonstrated by hybridization analysis that copper amine oxidase genes are present in a range of mono- and dicotyledonous plants. Copper amine oxidases (EC 1.4.3.6) have been identified in a wide range of microbial, plant, and animal systems (for reviews see 39McIntire W.S. Hartmann C. Davidson V.L. Principles and Applications of Quinoproteins. Marcel Dekker, New York1993: 97-171Google Scholar; Knowles and 32Knowles P.F. Dooley D.M. Sigel H. Sigel A. Metal Ions in Biological Systems. Marcel Dekker, New York1994: 361-403Google Scholar). In microbes they allow utilization of unusual amine substrates as nitrogen and/or carbon sources, whereas in plants and animals their functions are less clear. The copper amine oxidases catalyze the oxidation of a wide variety of biogenic amines, including mono-, di-, and polyamines to the corresponding aldehyde with release of NH3 and H2O2. The pea seedling enzyme preferentially catalyzes oxidation of the diamine substrates putrescine (R = NH2(CH2)3) and cadaverine (R = NH2(CH2)4) at the primary amino group according to Reaction 1, Reaction 1 yielding 4-aminobutyraldehyde and 5-aminovaleraldehyde, respectively. This enzyme is relatively abundant comprising at least 0.1% total soluble protein in etiolated pea seedling epicotyls (37McGowan R.E. Muir R.M. Plant Physiol. 1971; 47: 644-648Crossref PubMed Google Scholar; 38McGuirl M.A. McCahon C.D. McKeown K.A. Dooley D.M. Plant Physiol. 1994; 106: 1205-1211Crossref PubMed Scopus (53) Google Scholar) but despite extensive study its function(s) remain unclear (53Smith T.A. Biochem. Soc. Trans. 1985; 13: 319-322Crossref PubMed Scopus (87) Google Scholar). Plant copper amine oxidases are generally found in the apoplast, loosely associated with the cell wall (50Scalet M. Federico R. Angelini R. Plant Physiol. 1991; 137: 571-575Crossref Scopus (34) Google Scholar; 16Federico R. Angelini R. Planta (Heidelb.). 1986; 167: 300-302Crossref PubMed Scopus (77) Google Scholar; 1Angelini R. Federico R. Calderera C.M. Clo C. Guarnieri C. Biochemical Studies of Natural Polyamines. CLUEB, Bologna1985: 183-189Google Scholar, 17Federico R. Angelini R. Planta (Heidelb.). 1988; 173: 317-321Crossref PubMed Scopus (51) Google Scholar; 52Slocum R.D. Furey M.J. Planta (Heidelb.). 1991; 183: 443-450Crossref PubMed Scopus (57) Google Scholar). Di- and polyamines are also present within the apoplast, and the H2O2 formed by their oxidation may be important in lignosuberization and cross-linking of extracellular macromolecules such as extensins, both during normal growth and in response to stress and wounding (18Federico R. Angelini R. Ceru M.P.A. Manes F Cell. Mol. Biol. 1985; 31: 171-174PubMed Google Scholar, 2Angelini R. Federico R. J. Plant Physiol. 1989; 135: 212-217Crossref Scopus (119) Google Scholar; 19Federico R. Alisi C. Angelini R. Crane F.L. Morre D.J. Low H. Plasma Membrane Oxidoreductases in Control of Animal and Plant Growth. Plenum Press, New York1988: 333-337Crossref Google Scholar; 3Angelini R. Manes F. Federico R. Planta (Heidelb.). 1990; 182: 89-96Crossref PubMed Scopus (151) Google Scholar; Slocum and Furey, 1991). Recently a correlation has been demonstrated between copper amine oxidase and peroxidase levels in chickpea tissues undergoing wound healing (3Angelini R. Manes F. Federico R. Planta (Heidelb.). 1990; 182: 89-96Crossref PubMed Scopus (151) Google Scholar, 4Angelini R. Bragloni M. Federico R. Infantino A. Porta-Puglia A. J. Plant Physiol. 1993; 142: 704-709Crossref Scopus (80) Google Scholar; 50Scalet M. Federico R. Angelini R. Plant Physiol. 1991; 137: 571-575Crossref Scopus (34) Google Scholar). Differential induction levels of amine oxidase and peroxidase have also been observed in susceptible and resistant cultivars of chickpea upon infection by Ascochyta rabiei, suggesting that they play a role in the defense response (4Angelini R. Bragloni M. Federico R. Infantino A. Porta-Puglia A. J. Plant Physiol. 1993; 142: 704-709Crossref Scopus (80) Google Scholar). The copper amine oxidases are homodimers of subunit size approximately 70-95 kDa depending on the source. Each subunit contains one Cu(II) and a quinone cofactor (for review see 31Klinman J.P. Mu D Annu. Rev. Biochem. 1994; 63: 299-344Crossref PubMed Scopus (301) Google Scholar) which has been identified as 2,4,5-trihydroxyphenylalanine quinone (TPQ 1The abbreviations used are: TPQ2,4,5-trihydroxyphenylalanine quinonePCRpolymerase chain reactionMOPS4-morpholinepropanesulfonic acid. or TOPA quinone) in the bovine serum enzyme (28Janes S.M. Mu D. Wemmer D. Smith A.J. Kaur S. Maltby D. Burlingame A.L. Klinman J.P. Science. 1990; 248: 981-987Crossref PubMed Scopus (635) Google Scholar). Subsequently TPQ has been identified biochemically or its presence inferred from amino acid sequence homology in other copper-containing amine oxidases from Hansenula polymorpha (8Bruinenberg P.G. Evers M. Waterham H.R. Kuipers J. Arnberg A.C. Ab G. Biochim. Biophys. Acta. 1989; 1008: 157-167Crossref PubMed Scopus (71) Google Scholar; 41Mu D. Janes S.M. Smith A.J. Brown D.E. Dooley D.M. Klinman J.P. J. Biol. Chem. 1992; 267: 7979-7982Abstract Full Text PDF PubMed Google Scholar), Klebsiella aerogenes (56Sugino H. Sasaki M. Azakami H. Yamashita M. Murooka Y. J. Bacteriol. 1992; 174: 2485-2492Crossref PubMed Google Scholar), Escherichia coli K12 (12Cooper R.A. Knowles P.F. Brown D.E. McGuirl M.A. Dooley D.M. Biochem. J. 1992; 288: 337-340Crossref PubMed Scopus (78) Google Scholar; 47Roh J.H. Suzuki H. Kumagai H. Yamashita M. Azakami H. Murooka Y. Mikami B. J. Mol. Biol. 1994; 238: 635-637Crossref PubMed Scopus (17) Google Scholar), lentil (44Pedersen J.Z. El-Sherbini S. Finazzi-Agro A. Rotilio G. Biochemistry. 1992; 31: 8-12Crossref PubMed Scopus (34) Google Scholar; 48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar), pea (41Mu D. Janes S.M. Smith A.J. Brown D.E. Dooley D.M. Klinman J.P. J. Biol. Chem. 1992; 267: 7979-7982Abstract Full Text PDF PubMed Google Scholar), Arthrobacter strain P1 (63Zhang X. Fuller J.H. McIntire W.S. J. Bacteriol. 1993; 175: 5617-5627Crossref PubMed Google Scholar), Arthrobacter globiformis (58Tanizawa K. Matsuzaki R. Shimizu E. Yorifuji T. Fukui T. Biochem. Biophys. Res. Commun. 1994; 199: 1096-1102Crossref PubMed Scopus (68) Google Scholar), human kidney (first identified as amiloride-binding protein; 6Barbry P. Champe M. Chassande O. Munemitsu S. Champigny G. Lingueglia E. Maes P. Frelin C. Tartar A. Ullrich A. Lazdunski M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7347-7351Crossref PubMed Scopus (65) Google Scholar), rat kidney (34Lingueglia E. Renard S. Voilley N. Waldmann R. Chassande O. Barbry P. Eur. J. Biochem. 1993; 216: 679-687Crossref PubMed Scopus (45) Google Scholar), and A. globiformis histaminase (11Choi Y.-H. Matsuzaki R. Fukui T. Shimizu E. Yorfuji T. Sato H. Ozaki Y. Tanizawa K. J. Biol. Chem. 1995; 270: 4712-4720Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Preliminary structural studies have been reported for the copper amine oxidases from pea seedlings (61Vignevich V. Dooley D.M. Guss J.M. Harvey I. McGuirl M.A. Freeman H.C. J. Mol. Biol. 1993; 229: 243-245Crossref PubMed Scopus (20) Google Scholar) and from E. coli (47Roh J.H. Suzuki H. Kumagai H. Yamashita M. Azakami H. Murooka Y. Mikami B. J. Mol. Biol. 1994; 238: 635-637Crossref PubMed Scopus (17) Google Scholar). 2,4,5-trihydroxyphenylalanine quinone polymerase chain reaction 4-morpholinepropanesulfonic acid. We are interested in the biological functions of plant copper amine oxidases, and we report here the molecular cloning and sequence analysis of the pea seedling amine oxidase gene. Important discrepancies between the translated pea and corresponding lentil sequence (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar) are considered, and a corrected lentil sequence is presented. The implications of this revision for interpretation of comparative amino acid sequence alignments to highlight important residues are discussed. We have also examined the level of glycosylation of pea seedling amine oxidase. Finally, we demonstrate the presence of copper amine oxidase gene homologs in a range of plant species. E. coli strain LE 392 (supE44, supF58, hsdR514, galK2, galT22, metB1, trpR55, lacY1) was used as host for cDNA library screening, and DH1 (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used for routine plasmid growth. A 10-μg sample of pea seedling amine oxidase, kindly provided by Professor D. M. Dooley (Montana State University), was coupled to p-phenylene diisothiocyanate glass and was subjected to N-terminal amino acid sequencing by Edman degradation. Released amino acids were assigned following high performance liquid chromatography reverse phase analysis against suitable standards. Amino acid sequencing was performed by the Biotechnology and Biological Sciences Research Council protein microsequencing facility in the Department of Biochemistry and Molecular Biology, University of Leeds. Routine DNA manipulations were performed according to 49Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar. Restriction endonuclease, ligase, and DNA polymerase reactions were performed in the buffers and under the conditions recommended by their manufacturers. PCR fragments were cloned into the pCR vector according to the protocols supplied with the TA cloning system (Invitrogen). A cDNA library prepared from mRNA isolated from 6-day-old etiolated pea seedlings with a Stratagene cDNA synthesis kit was a gift from Dr. D. Bucke (Schering Agrochemicals Ltd.). The cDNA was cloned into EcoRI/XhoI digested Uni-ZAP (Stratagene) to give a library of 1.5 × 106 independent clones, which was amplified to a titer of 3.8 × 1011 plaque-forming units/ml. Eight- to 10-day postgermination lentil and pea etiolated seedlings were dissected and the epicotyls frozen in liquid N2 and stored at −70°C. Frozen tissue was powdered in liquid N2 using a pestle and mortar, and mRNA was isolated using a Pharmacia Microprep reagent kit according to the manufacturer's instructions. First-strand cDNA was prepared from poly(A)+ RNA with a Pharmacia first-strand cDNA synthesis kit using either a 5′-tailed oligo(dT) primer (Pharmacia) or a sequence specific primer. PCR primers were designed to amplify the lentil seedling amine oxidase cDNA coding region (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar) for use as a heterologous probe to screen the pea cDNA library. The PCR mix contained 100 pg of lentil cDNA, 100 pmol (2 μM) of each primer (5′-TTTACACCATTGCATACTCAACATC-3′) and (5′-CCACACAGCCAAAGTATCGTCTCC-3′), and a 50 μM concentration of each dNTP in a 50-μl reaction. The reaction mix was overlaid with 50 μl of mineral oil and subjected to a "hot-start" by adding 1 unit of SuperTaq (HT Biotechnology) during the last minute of the initial 5-min denaturation (94°C) step. Amplification was achieved by 30 cycles of 94°C, 1 min; 55°C for 1 min; 72°C for 3 min. The reaction products were separated through a 1.5% NuSieve agarose gel (FMC), and a DNA band of the expected size of 1.74 kilobase pairs was recovered. Phage dilutions were plated on LE392 to give 15,000 plaques/9-cm-diameter plate. Duplicate plaque lifts were taken onto Hybond N+ membranes (Amersham) according to the manufacturer's instructions. A 30-ng aliquot of the lentil seedling amine oxidase fragment in NuSieve agarose was radiolabeled with [α-32P]dCTP by random hexamer labeling (20Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5193) Google Scholar), and the probe was separated from unincorporated label by spermine precipitation (26Hoopes B.C. McClure W.R. Nucleic Acids Res. 1981; 9: 5493-5504Crossref PubMed Scopus (172) Google Scholar). The membranes were prehybridized at 56°C for 4 h in 5 × SSPE (1 × SSPE, 150 mM NaCl, 10 mM NaH2PO4· H2O, 1 mM Na2 EDTA, pH 7.4), 6% polyethylene glycol, 0.5% skimmed milk powder (Marvel), 1% SDS, 0.1% sodium pyrophosphate, and 250 μg/ml sonicated and denatured calf thymus DNA (22Gurr S.J. McPherson M.J. Gurr S.J. McPherson M.J. Bowles D.J. in Molecular Plant Pathology: A Practical Approach. IRL Press, Oxford1992: 109-121Google Scholar). Hybridization at 56°C was performed overnight in the same buffer after the addition of the radiolabeled probe. The membranes were finally washed in 0.1% SSC (1 × SSC, 150 mM NaCl, 15 mM trisodium citrate), 0.1% SDS at 65°C. Positive plaques were purified by successive rounds of screening at lower plaque densities. cDNA clones were rescued as pBluescript SK(-) plasmids by in vivo excision from Uni-ZAP as detailed by Stratagene. DNA sequence analysis was performed with Sequenase V2.0 kits (Amersham) with product analysis on 6% wedge gels. Initial sequencing reactions were performed using vector-specific T3 and T7 primers and degenerate primers. Forward and reverse orientation primers (primers a and b, respectively) were designed by back-translation of the amino acid sequence of the TPQ-containing peptide (41Mu D. Janes S.M. Smith A.J. Brown D.E. Dooley D.M. Klinman J.P. J. Biol. Chem. 1992; 267: 7979-7982Abstract Full Text PDF PubMed Google Scholar). These primers were designed prior to the definitive identification of Tyr as the residue modified to TPQ (41Mu D. Janes S.M. Smith A.J. Brown D.E. Dooley D.M. Klinman J.P. J. Biol. Chem. 1992; 267: 7979-7982Abstract Full Text PDF PubMed Google Scholar) and so included coding potential for both Tyr and Phe at the codon corresponding to the TPQ site. Primer c was designed from the N-terminal amino acid sequence of the mature pea seedling amine oxidase determined during the course of the present study. The primer a sequence was 5′-GTI GGI AA(TC) T(TA) (TC) GA(TC) AA(TC) GT-3′. The primer b sequence was 5′-ATI AC(AG) TT(AG) TC(AG) (AT)A(AG) TT(AGCT) CC-3′. The primer c sequence was 5′-CA(TC) GTI CA(GA) CA(TC) CCI (TC)T-3′. Parentheses indicate degenerate positions. A progressive sequencing strategy was used with design of further primers to complete the sequence of both strands of the cDNA insert. Sequence data were compiled and analyzed on a MicroVax 3600 computer using 55Staden R. Nucleic Acids Res. 1986; 14: 217-231Crossref PubMed Scopus (240) Google Scholar software. Amino acid sequence alignments were performed using CLUSTALV and SOMAP (25Higgins D.G. Bleasby A.J. Fuchs R. Comput. Appl. Biosci. 1992; 8: 189-191PubMed Google Scholar; 43Parry-Smith D.J. Attwood T.K. Comput. Appl. Biosci. 1991; 7: 233-235PubMed Google Scholar). Lentil cDNA clones were sequenced in a similar manner using primers selected from the pea seedling sequencing project. Genomic DNAs were prepared from lentil and pea according the method of 14Dellaporta S.L. Wood J. Hicks J.B. Plant Mol. Biol. Rep. 1983; 1: 19-21Crossref Scopus (6355) Google Scholar with additional purification by cesium chloride density gradient centrifugation (49Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Genomic DNAs from barley, rice, soybean, sugar beet, tomato, vine, wheat, and Arabidopsis were kindly provided by Claire Scollan, Yi Li, and Ruth Turnbull (Centre for Plant Biochemistry and Biotechnology, Leeds). Ten-μg aliquots of DNA were digested with restriction enzymes according to the manufacturer's recommendations and were separated through a 1% agarose gel (15 × 20 cm) by electrophoresis at 1.5 V/cm for 16 h before transfer to Hybond N+, followed by prehybridization and hybridization as described for the cDNA library screen. The filter was successively washed in 5 × SSC, 0.1% SDS at 56°C; then 1 × SSC, 0.1% SDS at 65°C; and finally in 0.1% SSC, 0.1% SDS at 65°C with autoradiographic exposure between each wash. Poly(A)+ mRNA was isolated from 6-day-old etiolated pea and lentil epicotyl tissue using a Pharmacia Microprep reagent kit according to the manufacturer's instructions. The mRNA was electrophoresed at 5 V/cm in a 1% agarose, 1 × MOPS (49Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) gel containing 0.66 M formaldehyde and ethidium bromide together with 0.24-9.5-kilobase RNA markers (Life Technologies, Inc.). The gel was capillary blotted onto a Hybond N+ membrane and fixed according to the manufacturer's instructions. The membrane was prehybridized in 5 × Denhardt's, 6 × SSC, 100 μg/ml sonicated and denatured calf thymus DNA at 65°C overnight and hybridized overnight under the same conditions with a pea seedling amine oxidase cDNA random hexamer-labeled probe prepared as described previously. The membrane was washed to 0.1 × SSC, 0.1 × SDS at 65°C before autoradiography. Protein samples of approximately 1 mg were denatured by boiling for 5 min and were then deglycosylated with N-glycosidase in 20 mM sodium phosphate buffer, pH 7.2, at 37°C for 16 h. Control reactions containing no N-glycosidase were also performed. Proteins were analyzed on 0.1% SDS, 10% polyacrylamide gel and stained with Coomassie Brilliant Blue (Hames, 1981). Of 150,000 plaques of the cDNA library screened, 102 gave positive hybridization signals after the primary screen using a radiolabeled lentil amine oxidase gene probe (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar). Several clones were taken through two further rounds of screening, and pBluescript clones were rescued from Uni-ZAP by in vivo excision. One clone, pPSAO1, contained an insert of 2.3 kilobases which probably represents a full-length cDNA based on three lines of experimental evidence. First, Northern analysis of poly(A)+ RNA from 8-10-day-old pea and lentil seedlings reveal a single transcript of approximately 2.3 kilobases (data not shown). Second, rescreening of the cDNA library with a 740-base pair fragment from the 5′-proximal half of the pPSAO1 insert (bases 490-1230) failed to identify clones with inserts longer than that of pPSAO1. Finally, PCR screening of the cDNA library with a vector primer and internal PSAO-specific primers failed to amplify any sequences longer than those amplified from pPSAO1. The DNA sequence of the cDNA insert in pPSAO1 is shown in Fig. 1 and was determined for both strands using a progressive primer design strategy. A long open reading frame extends from position 74 to 2098 (Fig. 1), and the translated amino acid sequence shows good overall homology with the published lentil amine oxidase cDNA sequence (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar). The identity of the translated pea seedling amine oxidase was confirmed by comparison with two regions of peptide sequence data. First, the amino acid sequence of the TPQ-containing peptide determined by 41Mu D. Janes S.M. Smith A.J. Brown D.E. Dooley D.M. Klinman J.P. J. Biol. Chem. 1992; 267: 7979-7982Abstract Full Text PDF PubMed Google Scholar matches a region of coding sequence between 1298 and 1331 (Fig. 1). X indicates an unassigned residue which corresponds to Tyr387, the residue proposed to form the TPQ cofactor. In the lentil sequence this residue is found at an identical position in the polypeptide chain (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar). Second, the N-terminal region of pea seedling amine oxidase was determined to be X-T-P-L-H-V-Q-H-P-L-D. This latter sequence matches the amino acids encoded by bases 149-181 (Fig. 1), and the unassigned residue (X) can be identified as valine. This region of amino acid sequence does not correspond to the start of the open reading frame but is preceded by a putative signal sequence of 25 residues with characteristics expected of a secretion signal (62von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3694) Google Scholar). This finding is consistent with evidence that amine oxidase is an extracellular enzyme in the Leguminoseae (Federico and Angelini, 1986; 19Federico R. Alisi C. Angelini R. Crane F.L. Morre D.J. Low H. Plasma Membrane Oxidoreductases in Control of Animal and Plant Growth. Plenum Press, New York1988: 333-337Crossref Google Scholar; Slocum and Furey, 1991). It also agrees with the interpretation of the lentil cDNA sequence (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar), although in this case the start of the open reading frame was not present. The ATG codon at nucleotides 74-76 (Fig. 1) most probably represents the amine oxidase translation initiation site. It should be noted that a second in-frame ATG (position 92-96; Fig. 1) occurs six codons into the coding region and could represent the translation start site. However, in eukaryotes the first ATG encountered by the translation machinery is usually selected as the initiation site. The sequence context of the first ATG (CTCACGATGGCT) matches well the consensus translation start site (TAAACAATGGCT) defined by 30Joshi C.P. Nucleic Acids Res. 1987; 15: 6643-6653Crossref PubMed Scopus (686) Google Scholar for plant genes. Although polyadenylation signals are less well defined for plant than for mammalian genes, the 3′-untranslated region contains two potential polyadenylation signals. The first (2152-2157; Fig. 1) exactly matches the AATAAA consensus; however, the second AATGAA (2223-2228; Fig. 1) lies only 21 residues from the poly(A) tail. It seems likely that this second signal represents a near-upstream element associated with polyadenylation of the 3′-end of the mRNA (27Hunt A.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1994; 45: 47-60Crossref Scopus (92) Google Scholar) Multiple sequence alignment of amine oxidase protein sequences by 63Zhang X. Fuller J.H. McIntire W.S. J. Bacteriol. 1993; 175: 5617-5627Crossref PubMed Google Scholar revealed the lentil sequence to be significantly shorter at the C-terminal end than any other amine oxidase. Since the lentil diamine oxidase sequence was the first of plant origin to be reported, this difference in length suggested some functional significance. However, the sequence of the pea seedling amine oxidase, presented here, contradicts this view. Over the first 555 residues of the mature plant proteins there is very good sequence conservation between the pea and lentil sequences with only 30 amino acid differences, many of which are conservative changes. The region corresponding to residues 474-478 (-G-S-S-K-R-; Fig. 1) is not a good match, and there is an extra amino acid in the pea seedling sequence in this region compared with the lentil sequence (-E-V-Q-E-). More significantly, the predicted lentil mature protein contains 569 amino acids with a calculated molecular mass of 64.3 kDa (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar), whereas the pea protein has 649 residues and a calculated molecular mass of 73.7 kDa. This represents a difference in length of 80 amino acids. One residue difference occurs within coding region around amino acid 476 as discussed above, but the extra 79 amino acids in the pea protein represent a major difference between the C-terminal ends of the two polypeptide chains. Such a gross difference in size seemed surprising given the phylogenetic similarity of the two plants: both Leguminoseae, the similar physiological and biochemical properties of the two enzymes, and the good sequence identity within the rest of the coding regions. To investigate these differences further we first resequenced the pea seedling amine oxidase gene from a second clone, but this proved to be identical to the original sequence determination. We then isolated mRNA from lentil seedlings and prepared cDNA. Using pea gene sequencing primers we PCR amplified the region of the lentil amine oxidase gene corresponding to nucleotides 179-2149 (Fig. 1). The PCR fragment was cloned into the pCR vector (Invitrogen) and was sequenced. Only a limited number of differences was identified between this lentil sequence and that of 48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar; nonetheless, the differences are very important, as shown in Fig. 2. The most significant of the differences were several single base insertions and a deletion which had the effect of altering, several times, the reading frame for the encoded protein. Three independent insertions lead to frameshift alterations in the region encoding residues 474-478 (positions 1568-1582; Fig. 1) and result in a sequence with strong identity to the pea gene including the presence of the "missing" residue. Most significant are insertions of a C at the positions corresponding to nucleotides 1814 and 1859 in the pea sequence (Fig. 1) and deletion of a G which occurs in the lentil sequence between nucleotides 2033 and 2034 in the pea sequence (Fig. 1). These result in a sequence that matches the pea seedling amine oxidase sequence by extending the lentil coding region to encode a mature protein of 649 amino acids, identical in size to the pea protein. Fig. 2 shows a comparison of part of the revised (this study) and the previous (48Rossi A. Petruzzelli R. Finazzi Agr A. FEBS Lett. 1992; 301: 253-257Crossref PubMed Scopus (69) Google Scholar) lentil amine oxidase cDNA sequence, highlighting the observed differences and the consequences for the protein coding region. In the pea sequence there are four potential N-glycosylation sites (N-X-S/T where X is any residue) at amino acids 131, 334, 364, and 558. Three of these sites are also found in the corrected lentil sequence at 131, 364, and 558; however, the site at 334 is a D-G-T in lentil. On the basis of hydropathy profiles Rossi et al.(1992) proposed that the site at 364 would be unlikely to be modified, leaving two potential sites at 131 and 558. We analyzed the extent of glycosylation in pea seedling amine oxidase and compared this with the porcine plasma amine oxidase, a protein known to be extensively N-glycosy
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