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Nucleotide sequence and deduced amino acid sequence of a putative asparagine synthetase in the mosquito Aedes aegypti (L.)1
Biochimica et Biophysica Acta 1383 Ž1998. 179–182
Short sequence-paper
Nucleotide sequence and deduced amino acid sequence of a putative asparagine synthetase in the mosquito Aedes aegypti žL. / 1 Urs Ackermann 2 , Rolf Graf
)
Zoologisches Institut, UniÕersitat Winterthurerstrasse 190, 8057 Zurich, Switzerland ¨ Zurich-Irchel, ¨ ¨ Received 30 October 1997; revised 18 December 1997; accepted 9 January 1998
Abstract A cDNA was cloned from a Aedes aegypti head cDNA library, containing the complete coding sequence for an asparagine synthetase homolog. The predicted polypeptide sequence exhibits high homology with different proteins of the ‘purF’ glutamine amidotransferase enzyme family. The aminoterminal region, containing Cys-1 which is crucial to perform the glutaminase reaction, was highly conserved among the asparagine synthetase family. Subsequent expression of the cDNA yielded a 54 000 Da protein corresponding to the molecular weight of other asparagine synthetases. q 1998 Elsevier Science B.V. Keywords: ‘purF’ glutamine amidotransferase; Glutaminase activity; Asparagine synthesis; cDNA sequence; cDNA expression; Baculovirus expression system
We have isolated a putative asparagine synthetase cDNA sequence from a head library Ž Clone pB52. . L-Asparagine synthetase Ž EC 6.3.1.1. which is a housekeeping enzyme, belongs to the glutamine amidotransferase enzyme family of the ‘purF’-type w1x and is responsible for asparagine production. The
Abbreviations: aa, amino acidŽs.; bp, base pairŽs.; kDa, kiloDalton ) Corresponding author. Dept. Chirurgie DL36, Universitats¨ spital Zurich, 8091 Zurich, Switzerland. Fax: q41-1-255-4393; ¨ ¨ E-mail: [email protected] 1 The sequence was deposited at the Genbank sequence library and assigned the accession number U84118. 2 Present address: Hofmattweg 44, 4710 Balsthal, Switzerland.
enzyme uses either glutamine or ammonia to catalyse asparagine synthesis as follows. L-Asp q ATP q L-Gln ™ L-Asn q AMP
q PPi q L-Glu L-Asp q ATP q NH 3 ™ L-Asn q AMP q PPi
Ž1. Ž2.
In different mammals an additional function, the cleavage of L-Gln to L-Glu and NH 3 Ži.e., glutaminase reaction. is reported in the absence of L-Asp and ATP w1x. The possibility to use either glutamine or NH 3 is typical for enzymes of the amidotransferase family Že.g., GMP synthetase, glutamine phosphoribosylpyrophosphate amidotransferase.. Sequence comparison of different amidotransferases pointed to two functional domains: the aminatordomain catalyses the NH 3-dependent reaction whereas the glutami-
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 0 8 - 9
180
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
namide transfer domain is responsible for glutamine binding and amide transfer to the aminator domain. Size estimation of asparagine synthetases from different species, using SDS-PAGE, indicated an average molecular weight of approximately 54 kDa. Asparagine synthetase is found in almost all eukaryotic cells. Some tumor cells have little or no asparagine synthetase activity compared to normal cells. The tumor cells are dependent on asparagine and can be selectively killed by the chemotherapeutic drug L-asparginase which hydrolyses asparagine w2,3x. In some insect species, e.g., Culex pipiens, larvae are dependent on external asparagine w4x. It is unclear whether in this species the asparagine synthetase is dysfunctional or absent. To be able to investigate whether asparagine synthetase is present in these insects, we set out to characterise a putative asparagine synthetase cDNA which was isolated from the closely related yellow-fever mosquito w5x. Here we report the sequence, its presence as a single gene and the molecular weight of the recombinant asparagine synthetase expressed in baculovirus. Clone pB52 from a Aedes aegypti head library was restriction digested with EcoRI. Two different fragments, a long one with a length of 5300 bp ŽpB52L. and a short one, approximately 260 bp long pB52S were resolved. The shorter one was subcloned into M13mp18 and sequenced using the dideoxy chain termination method ŽUS Biochemical, Cleveland, OH. and the primer sites of the vector. Subsequent computer translation and comparison with the ‘Sequence Analysis Software Package’ Ž Genetics Computer Group, WI. exhibited high homologies with a central portion of the enzyme asparagine synthetase of different species Ž e.g., asparagine synthetase B Escherichia coli: 61.6% w6x; Homo sapiens: 50% w1,2x. Sequencing primers were then designed at the 5X and 3X end. With further primers and suitable deletions, sequencing on both strands was carried out. The resulting nucleotide and deduced protein sequence is shown in Fig. 1. The 2171 nucleotides long cDNA has a 5X untranslated region of
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the A. aegypti asparagine synthetase. Conserved domains are underlined, indicated in bold letters ŽA, B, C. and explained in the text.
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
181
96 nucleotides. A single open reading frame of 1689 nucleotides was found. The deduced protein sequence contains 562 Žaa., including the initiation codon. Southern blot analysis was used to determine the number of genes encoding asparagine synthetase in A. aegypti w7x. Aedes genomic DNA was digested with six restriction enzymes Ž EcoRI, BamHI, HindIII, BglI, PÕuII, SacI. and fractionated on a 1% agarose gel. Southern blots were probed with the 32 P-labeled pB52S cDNA fragment. Results are shown in Fig. 2 and demonstrate that asparagine synthetase is present as a single gene. Theoretically, there should be only one EcoRI fragment labelled. We have to assume that the corresponding region of
Fig. 3. Estimation of relationship between various asparagine synthetases from prokaryotes and eukaryotes. As a control, unrelated sequences ŽASNA_ECOLI, VS09_ROTEL. were included. Distances along the horizontal axis are inversely proportional to the degree of relatedness. The dendrogram was generated using the ‘pileup’ program of the Genetics Computer Group software package. ASN1_PEA: asparagine synthetase, Pisum satiÕum; ASNS_ARATH: asparagine synthetase, Arabidopsis thalliana; ASNB_ECOLI: asparagine synthetase B, E. coli; ASN1_YEAST: asparagine synthetase, Saccharomyces cereÕisiae; ASN_AAE: asparagine synthetase, A. aegypti; ASNS_HUMAN: asparagine synthetase, H. sapiens; ASNA_ECOLI: asparagine synthetase A, E. coli; VS09_ROTEL: rotavirus glycoprotein.
Fig. 2. Southern blot analysis of the AS gene A. aegypti. Twenty microgram genomic DNA each were digested with BamHI, HindIII, EcoRI, SacI, BglII or PÕuII. The DNA was separated by agarose electrophoresis, denatured and transferred to a Genescreen plus membrane. After prehybridization, the blot was probed with 300 000 cpmrml of the randomly primed EcoRI fragment ŽpB52S. at 658C. The blot was then washed at a high stringency Ž0.2=SSC, 658C. and exposed to a X-ray film.
the EcoRI fragment contains an intron with an internal EcoRI site. A comparison of asparagine synthetases from various species, performed by the FASTA computer program w8x, pointed to several domains with high homology of which the N-terminus seems the most conserved. The cysteine following the first methionine ŽA in Fig. 1. is thought to be critical for the glutamineamide transfer, i.e., the binding of glutamine and the transfer of the NH 3 to the aminatordomain w9x. Other domains, although highly conserved ŽB,C in Fig. 1., have not been assigned definitive functions. The overall homology as shown by a dendrogram depicting molecular relationships was best with lower eukaryotes and prokaryotes. An inclusion of two unrelated proteins Žrotavirus glyco-
182
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
apparent molecular weight of recombinant protein expressed with the help of the baculovirus expression system was approximately 54 000 Da as determined by SDS-PAGE Ž Fig. 4. . This finding corresponds with the size of different asparagine synthetases Že.g., for the enzyme of H. sapiens the calculated weight was 64 400 Da while the observed size by SDS-PAGE was 54 000 Da w1x.. The protein will have to be tested in functional assays. Conclusions from these studies will have to be integrated with the analysis of the conservation of the various domains when comparing enzymatic properties and structures of asparagine synthetases from different species. We would like to thank Dr. E. Kubli and Y. Choffat for the synthesis of oligonucleotides. We are also grateful to Dr. H. Briegel for generously providing us with mosquitoes. This work was in part funded by the Swiss National Foundation and the ‘Kommission zur Forderung des wissenschaftlichen Nach¨ wuchses des Kantons Zurich’. ¨ Fig. 4. Molecular weight determination of the recombinant A. aegypti asparagine synthetase. Cell cultures were infected with recombinant baculovirus and harvested 3 days later. The cells were lysed and analysed by SDS-PAGE. Marker: marker-proteins with the molecular weight indicated at left. A dominant protein band at 54 kDa can be seen in lane 2 and lane 3, indicated by arrow. Lane 1: cells prior to infection; Lane 2: lysate from cells with multiplicity of infection of 2; Lane 3: lysate from cells with multiplicity of infection of 10.
protein ŽVS09_ROTEL. and asparagine synthetase A from E. coli Ž ASNA_ECOLI.. demonstrated that the mosquito enzyme belongs to the asparagine synthetase B family ŽFig. 3.. Particularly, the functionally related asparagine synthetase A from E. coli w10x, although not of the ‘trp-G’ glutamine amidotransferase type, shows only weak sequence homology. While the calculated molecular weight of the mosquito asparagine synthetase is 63 701 Da, the
References w1x N.C. Pfeiffer, P.M. Mehlhaff, D.E. Wylie, S.M. Schuster, J. Biol. Chem. 262 Ž1987. 11565–11570. w2x I.L. Andrulis, J. Chen, P.N. Ray, Mol. Cell. Biol. 7 Ž1987. 2435–2443. w3x J.W. Ortega, M.E. Nisbit Jr., M.H. Donaldson, R.H. Hittle, J. Weiner, M. Karon, D. Hammond, Cancer Res. 37 Ž1977. 535–540. w4x R.H. Dadd, J. Insect Physiol. 24 Ž1978. 25–30. w5x U. Ackermann, Diplomarbeit, Zoologisches Institut der Universitat 1993. ¨ Zurich, ¨ w6x M.A. Scofield, W.S. Lewis, S.M. Schuster, J. Biol. Chem. 265 Ž1990. 12895–12902. w7x E.M. Southern, J. Mol. Biol. 98 Ž1975. 503–517. w8x D.J. Lipman, W.R. Pearson, Science 237 Ž1985. 1435–1441. w9x S. Sheng, D.A. Moraga-Amador, G. van-Heeke, R.D. Allison, N.G. Richards, S.M. Schuster, J. Biol. Chem. 268 Ž1993. 16771–16780. w10x M. Weng, H. Zalkin, J. Bacteriol. 169 Ž1987. 3023–3028.
Short sequence-paper
Nucleotide sequence and deduced amino acid sequence of a putative asparagine synthetase in the mosquito Aedes aegypti žL. / 1 Urs Ackermann 2 , Rolf Graf
)
Zoologisches Institut, UniÕersitat Winterthurerstrasse 190, 8057 Zurich, Switzerland ¨ Zurich-Irchel, ¨ ¨ Received 30 October 1997; revised 18 December 1997; accepted 9 January 1998
Abstract A cDNA was cloned from a Aedes aegypti head cDNA library, containing the complete coding sequence for an asparagine synthetase homolog. The predicted polypeptide sequence exhibits high homology with different proteins of the ‘purF’ glutamine amidotransferase enzyme family. The aminoterminal region, containing Cys-1 which is crucial to perform the glutaminase reaction, was highly conserved among the asparagine synthetase family. Subsequent expression of the cDNA yielded a 54 000 Da protein corresponding to the molecular weight of other asparagine synthetases. q 1998 Elsevier Science B.V. Keywords: ‘purF’ glutamine amidotransferase; Glutaminase activity; Asparagine synthesis; cDNA sequence; cDNA expression; Baculovirus expression system
We have isolated a putative asparagine synthetase cDNA sequence from a head library Ž Clone pB52. . L-Asparagine synthetase Ž EC 6.3.1.1. which is a housekeeping enzyme, belongs to the glutamine amidotransferase enzyme family of the ‘purF’-type w1x and is responsible for asparagine production. The
Abbreviations: aa, amino acidŽs.; bp, base pairŽs.; kDa, kiloDalton ) Corresponding author. Dept. Chirurgie DL36, Universitats¨ spital Zurich, 8091 Zurich, Switzerland. Fax: q41-1-255-4393; ¨ ¨ E-mail: [email protected] 1 The sequence was deposited at the Genbank sequence library and assigned the accession number U84118. 2 Present address: Hofmattweg 44, 4710 Balsthal, Switzerland.
enzyme uses either glutamine or ammonia to catalyse asparagine synthesis as follows. L-Asp q ATP q L-Gln ™ L-Asn q AMP
q PPi q L-Glu L-Asp q ATP q NH 3 ™ L-Asn q AMP q PPi
Ž1. Ž2.
In different mammals an additional function, the cleavage of L-Gln to L-Glu and NH 3 Ži.e., glutaminase reaction. is reported in the absence of L-Asp and ATP w1x. The possibility to use either glutamine or NH 3 is typical for enzymes of the amidotransferase family Že.g., GMP synthetase, glutamine phosphoribosylpyrophosphate amidotransferase.. Sequence comparison of different amidotransferases pointed to two functional domains: the aminatordomain catalyses the NH 3-dependent reaction whereas the glutami-
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 8 . 0 0 0 0 8 - 9
180
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
namide transfer domain is responsible for glutamine binding and amide transfer to the aminator domain. Size estimation of asparagine synthetases from different species, using SDS-PAGE, indicated an average molecular weight of approximately 54 kDa. Asparagine synthetase is found in almost all eukaryotic cells. Some tumor cells have little or no asparagine synthetase activity compared to normal cells. The tumor cells are dependent on asparagine and can be selectively killed by the chemotherapeutic drug L-asparginase which hydrolyses asparagine w2,3x. In some insect species, e.g., Culex pipiens, larvae are dependent on external asparagine w4x. It is unclear whether in this species the asparagine synthetase is dysfunctional or absent. To be able to investigate whether asparagine synthetase is present in these insects, we set out to characterise a putative asparagine synthetase cDNA which was isolated from the closely related yellow-fever mosquito w5x. Here we report the sequence, its presence as a single gene and the molecular weight of the recombinant asparagine synthetase expressed in baculovirus. Clone pB52 from a Aedes aegypti head library was restriction digested with EcoRI. Two different fragments, a long one with a length of 5300 bp ŽpB52L. and a short one, approximately 260 bp long pB52S were resolved. The shorter one was subcloned into M13mp18 and sequenced using the dideoxy chain termination method ŽUS Biochemical, Cleveland, OH. and the primer sites of the vector. Subsequent computer translation and comparison with the ‘Sequence Analysis Software Package’ Ž Genetics Computer Group, WI. exhibited high homologies with a central portion of the enzyme asparagine synthetase of different species Ž e.g., asparagine synthetase B Escherichia coli: 61.6% w6x; Homo sapiens: 50% w1,2x. Sequencing primers were then designed at the 5X and 3X end. With further primers and suitable deletions, sequencing on both strands was carried out. The resulting nucleotide and deduced protein sequence is shown in Fig. 1. The 2171 nucleotides long cDNA has a 5X untranslated region of
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the A. aegypti asparagine synthetase. Conserved domains are underlined, indicated in bold letters ŽA, B, C. and explained in the text.
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
181
96 nucleotides. A single open reading frame of 1689 nucleotides was found. The deduced protein sequence contains 562 Žaa., including the initiation codon. Southern blot analysis was used to determine the number of genes encoding asparagine synthetase in A. aegypti w7x. Aedes genomic DNA was digested with six restriction enzymes Ž EcoRI, BamHI, HindIII, BglI, PÕuII, SacI. and fractionated on a 1% agarose gel. Southern blots were probed with the 32 P-labeled pB52S cDNA fragment. Results are shown in Fig. 2 and demonstrate that asparagine synthetase is present as a single gene. Theoretically, there should be only one EcoRI fragment labelled. We have to assume that the corresponding region of
Fig. 3. Estimation of relationship between various asparagine synthetases from prokaryotes and eukaryotes. As a control, unrelated sequences ŽASNA_ECOLI, VS09_ROTEL. were included. Distances along the horizontal axis are inversely proportional to the degree of relatedness. The dendrogram was generated using the ‘pileup’ program of the Genetics Computer Group software package. ASN1_PEA: asparagine synthetase, Pisum satiÕum; ASNS_ARATH: asparagine synthetase, Arabidopsis thalliana; ASNB_ECOLI: asparagine synthetase B, E. coli; ASN1_YEAST: asparagine synthetase, Saccharomyces cereÕisiae; ASN_AAE: asparagine synthetase, A. aegypti; ASNS_HUMAN: asparagine synthetase, H. sapiens; ASNA_ECOLI: asparagine synthetase A, E. coli; VS09_ROTEL: rotavirus glycoprotein.
Fig. 2. Southern blot analysis of the AS gene A. aegypti. Twenty microgram genomic DNA each were digested with BamHI, HindIII, EcoRI, SacI, BglII or PÕuII. The DNA was separated by agarose electrophoresis, denatured and transferred to a Genescreen plus membrane. After prehybridization, the blot was probed with 300 000 cpmrml of the randomly primed EcoRI fragment ŽpB52S. at 658C. The blot was then washed at a high stringency Ž0.2=SSC, 658C. and exposed to a X-ray film.
the EcoRI fragment contains an intron with an internal EcoRI site. A comparison of asparagine synthetases from various species, performed by the FASTA computer program w8x, pointed to several domains with high homology of which the N-terminus seems the most conserved. The cysteine following the first methionine ŽA in Fig. 1. is thought to be critical for the glutamineamide transfer, i.e., the binding of glutamine and the transfer of the NH 3 to the aminatordomain w9x. Other domains, although highly conserved ŽB,C in Fig. 1., have not been assigned definitive functions. The overall homology as shown by a dendrogram depicting molecular relationships was best with lower eukaryotes and prokaryotes. An inclusion of two unrelated proteins Žrotavirus glyco-
182
U. Ackermann, R. Graf r Biochimica et Biophysica Acta 1383 (1998) 179–182
apparent molecular weight of recombinant protein expressed with the help of the baculovirus expression system was approximately 54 000 Da as determined by SDS-PAGE Ž Fig. 4. . This finding corresponds with the size of different asparagine synthetases Že.g., for the enzyme of H. sapiens the calculated weight was 64 400 Da while the observed size by SDS-PAGE was 54 000 Da w1x.. The protein will have to be tested in functional assays. Conclusions from these studies will have to be integrated with the analysis of the conservation of the various domains when comparing enzymatic properties and structures of asparagine synthetases from different species. We would like to thank Dr. E. Kubli and Y. Choffat for the synthesis of oligonucleotides. We are also grateful to Dr. H. Briegel for generously providing us with mosquitoes. This work was in part funded by the Swiss National Foundation and the ‘Kommission zur Forderung des wissenschaftlichen Nach¨ wuchses des Kantons Zurich’. ¨ Fig. 4. Molecular weight determination of the recombinant A. aegypti asparagine synthetase. Cell cultures were infected with recombinant baculovirus and harvested 3 days later. The cells were lysed and analysed by SDS-PAGE. Marker: marker-proteins with the molecular weight indicated at left. A dominant protein band at 54 kDa can be seen in lane 2 and lane 3, indicated by arrow. Lane 1: cells prior to infection; Lane 2: lysate from cells with multiplicity of infection of 2; Lane 3: lysate from cells with multiplicity of infection of 10.
protein ŽVS09_ROTEL. and asparagine synthetase A from E. coli Ž ASNA_ECOLI.. demonstrated that the mosquito enzyme belongs to the asparagine synthetase B family ŽFig. 3.. Particularly, the functionally related asparagine synthetase A from E. coli w10x, although not of the ‘trp-G’ glutamine amidotransferase type, shows only weak sequence homology. While the calculated molecular weight of the mosquito asparagine synthetase is 63 701 Da, the
References w1x N.C. Pfeiffer, P.M. Mehlhaff, D.E. Wylie, S.M. Schuster, J. Biol. Chem. 262 Ž1987. 11565–11570. w2x I.L. Andrulis, J. Chen, P.N. Ray, Mol. Cell. Biol. 7 Ž1987. 2435–2443. w3x J.W. Ortega, M.E. Nisbit Jr., M.H. Donaldson, R.H. Hittle, J. Weiner, M. Karon, D. Hammond, Cancer Res. 37 Ž1977. 535–540. w4x R.H. Dadd, J. Insect Physiol. 24 Ž1978. 25–30. w5x U. Ackermann, Diplomarbeit, Zoologisches Institut der Universitat 1993. ¨ Zurich, ¨ w6x M.A. Scofield, W.S. Lewis, S.M. Schuster, J. Biol. Chem. 265 Ž1990. 12895–12902. w7x E.M. Southern, J. Mol. Biol. 98 Ž1975. 503–517. w8x D.J. Lipman, W.R. Pearson, Science 237 Ž1985. 1435–1441. w9x S. Sheng, D.A. Moraga-Amador, G. van-Heeke, R.D. Allison, N.G. Richards, S.M. Schuster, J. Biol. Chem. 268 Ž1993. 16771–16780. w10x M. Weng, H. Zalkin, J. Bacteriol. 169 Ž1987. 3023–3028.