k./ 1990 Oxford University Press
4610 Nucleic Acids Research, Vol. 18, No. 15
cDNA deduced amino-acid sequence of a new phospholipase from Bungarus multicinctus Jean-Marc Danse*, Jean-Marie Garnier' and Jules Kempf INSERM U-184, Institut de Chimie Biologique, Faculte de M6decine, 11 Rue Humann, 67085 Strasbourg Cedex, France and 1LGME du CNRS, 11 Rue Humann, 67085 Strasbourg Cedex, France Submitted June 26, 1990
EMBL accession
Most snake venoms contain one or more phospholipases A2 (PLA2), some of which are potent toxins (1). Six different PLA2 have been described in Bungarus multicinctus (2), one non-toxic PLA2 and five isoforms of fl-toxins, flu to j35. These fl-toxins are composed of three variant Al to A3-chains homologous to other snake PLA2 (3). A cDNA library has been prepared from poly(A+) RNA isolated from the venom glands of one specimen of Bungarus multicinctus. The figure presents a nucleotide sequence whose longest open reading frame encodes a signal peptide (underlined) of 27 residues followed by a 120 residues polypeptide chain (A4-chain). The signal peptide is quite identical to those described for the precursor forms of snake PLA2 (4-7). A4-chain is highly homologous to other snake PLA2 (3) in particular Bungarus multicinctus A3-chain. However, we observe the following differences as seen elsewhere (8), positions 66 and 67 (Gln-Ser) are inversed with regard to Al, A2 or A3-chains (2), positions 1 to 6 correspond to the same positions of non toxic Bungarus multicintus PLA2 (2) and the other positions are similar to Bungarus multicinctus A3-chain with the following exceptions position 23 corresponds to Al or A2-chain (2); position 36 corresponds to non toxic PLA2 (2); one aspartic acid is deleted at position 55 and one asparagine is inserted at position 57 which results in a better Al to A3 amino-acids sequence alignment; -1
80
+1
ACKNOWLEDGEMENTS We wish to thank M.-J.Osvald and J.L.Toussaint for their excellent technical assistance and E.Russell-Toussaint for typing this manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Karlsson,E. et al. (1985) Pharmac. Ther. 30, 259-276. Kondo,K. et al. (1982) J. Biochem. 91, 1531-1548. Kini,R.M. and Evans,H.J. (1987) J. Biol. Chem. 262, 14402-14407. Guignery-Frelat,G. et al. (1987) Nucl. Acids Res. 15, 5892. Ducancel,F. et al. (1988) Nucl. Acids Res. 16, 9048. Ducancel,F. et al. (1988) Nucl. Acids Res. 16, 9049. Danse,J.M. et al. (1990) Nucl. Acids Res. 18, 4608. Danse,J.M. et al. (1990) Nucl. Acids Res. 18, 4609. Dijkstra,B.W. et al. (1981) Nature 289, 604-606. -10 TTA GGA G L
30 GAA TAT GCG GAC TAC GGT TGC TAC TGC GGC GCA C G A Y E Y A Y G C D
121
181
50
TTG GAT AGG TGC TGC TAT GTT CAT GAC AAC TGC C L V H N C C Y D D R 70 TGC GAC CCC AAA ACG CAG TCA TAT TCA TAC AAA Q S Y S Y K T C K D P
241
301
90
TCACCTGCGCGTGGCAATTCTCTGCACGGGCGTCTATTATATATATATATAATAGAAAATTATATGTATACATACATAA TTTTGAAAAAACAAAAGGAACCATTTCCTGAACAATAAAGTGAGGCACCGAAACCCAAAAAAAAAAAAAAAAAA
To whom correspondence should be addressed
61
10
GAT CTC TAT CAG TTC AAG GAG ATG ATT CGA TAT Y 0 I F K E R M Y L D
TTG ACT AAA CGC ACG ATC ATC TGC TAT GGT GCC GCA GGT ACT TGT GCA CGT GTT GTC TGT C C C A V V Y G A A G T R L T K T I I R 110 100 GAT TGC GAC CGC ACA GCA GCC CTC TGC TTC GGC AAT TCC GAA TAC ATC GAG GGG CAC AAG K H Y S E I G C F G N E C D A A L R T D 120 AAT ATC GAC ACC GCG AGA TTT TGC CAA TGA TATTTGAGAGGCTTCAGCGCAAGGACTGTGGCACTTAC C A F Q I N R D T
*
X53408
positions 62, 103 and 105 are mutated. The same mutations are observed at positions 103 and 105 for A2-chain (8). Finally the 5' non coding sequence is highly conserved with regards to prePLA2 cDNA (4-8). The A4-chain mature sequence revealed that the ten residues (bold characters) presumed to be involved in the active site of mammalian PLA2 (9) are present in the reptile PLA2 described here.
-20 -27 GGACAAA ATG AAT CCT GCT CAC CTT CTT GTC CTG TCG GCA GTT TGT GTC TCC CTC L S V C N A V S V L L M H L P A
GCC GCC AAC ATT CCT CCT CAG CAT CTC I P P H A A N L O 20 ACC ATC CCC TGC GAA AAA ACT TGG GGT W G K T T I C E P 40 GGA GGT AGC GGG ACA CCG ATA GAT GCC A S I D G G G T P 60 TAT GGT GAT GCC GCA AAT ATT CGT GAT Y G A A N I R D D
no.
361
421
489
568 642
4610 Nucleic Acids Research, Vol. 18, No. 15
cDNA deduced amino-acid sequence of a new phospholipase from Bungarus multicinctus Jean-Marc Danse*, Jean-Marie Garnier' and Jules Kempf INSERM U-184, Institut de Chimie Biologique, Faculte de M6decine, 11 Rue Humann, 67085 Strasbourg Cedex, France and 1LGME du CNRS, 11 Rue Humann, 67085 Strasbourg Cedex, France Submitted June 26, 1990
EMBL accession
Most snake venoms contain one or more phospholipases A2 (PLA2), some of which are potent toxins (1). Six different PLA2 have been described in Bungarus multicinctus (2), one non-toxic PLA2 and five isoforms of fl-toxins, flu to j35. These fl-toxins are composed of three variant Al to A3-chains homologous to other snake PLA2 (3). A cDNA library has been prepared from poly(A+) RNA isolated from the venom glands of one specimen of Bungarus multicinctus. The figure presents a nucleotide sequence whose longest open reading frame encodes a signal peptide (underlined) of 27 residues followed by a 120 residues polypeptide chain (A4-chain). The signal peptide is quite identical to those described for the precursor forms of snake PLA2 (4-7). A4-chain is highly homologous to other snake PLA2 (3) in particular Bungarus multicinctus A3-chain. However, we observe the following differences as seen elsewhere (8), positions 66 and 67 (Gln-Ser) are inversed with regard to Al, A2 or A3-chains (2), positions 1 to 6 correspond to the same positions of non toxic Bungarus multicintus PLA2 (2) and the other positions are similar to Bungarus multicinctus A3-chain with the following exceptions position 23 corresponds to Al or A2-chain (2); position 36 corresponds to non toxic PLA2 (2); one aspartic acid is deleted at position 55 and one asparagine is inserted at position 57 which results in a better Al to A3 amino-acids sequence alignment; -1
80
+1
ACKNOWLEDGEMENTS We wish to thank M.-J.Osvald and J.L.Toussaint for their excellent technical assistance and E.Russell-Toussaint for typing this manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Karlsson,E. et al. (1985) Pharmac. Ther. 30, 259-276. Kondo,K. et al. (1982) J. Biochem. 91, 1531-1548. Kini,R.M. and Evans,H.J. (1987) J. Biol. Chem. 262, 14402-14407. Guignery-Frelat,G. et al. (1987) Nucl. Acids Res. 15, 5892. Ducancel,F. et al. (1988) Nucl. Acids Res. 16, 9048. Ducancel,F. et al. (1988) Nucl. Acids Res. 16, 9049. Danse,J.M. et al. (1990) Nucl. Acids Res. 18, 4608. Danse,J.M. et al. (1990) Nucl. Acids Res. 18, 4609. Dijkstra,B.W. et al. (1981) Nature 289, 604-606. -10 TTA GGA G L
30 GAA TAT GCG GAC TAC GGT TGC TAC TGC GGC GCA C G A Y E Y A Y G C D
121
181
50
TTG GAT AGG TGC TGC TAT GTT CAT GAC AAC TGC C L V H N C C Y D D R 70 TGC GAC CCC AAA ACG CAG TCA TAT TCA TAC AAA Q S Y S Y K T C K D P
241
301
90
TCACCTGCGCGTGGCAATTCTCTGCACGGGCGTCTATTATATATATATATAATAGAAAATTATATGTATACATACATAA TTTTGAAAAAACAAAAGGAACCATTTCCTGAACAATAAAGTGAGGCACCGAAACCCAAAAAAAAAAAAAAAAAA
To whom correspondence should be addressed
61
10
GAT CTC TAT CAG TTC AAG GAG ATG ATT CGA TAT Y 0 I F K E R M Y L D
TTG ACT AAA CGC ACG ATC ATC TGC TAT GGT GCC GCA GGT ACT TGT GCA CGT GTT GTC TGT C C C A V V Y G A A G T R L T K T I I R 110 100 GAT TGC GAC CGC ACA GCA GCC CTC TGC TTC GGC AAT TCC GAA TAC ATC GAG GGG CAC AAG K H Y S E I G C F G N E C D A A L R T D 120 AAT ATC GAC ACC GCG AGA TTT TGC CAA TGA TATTTGAGAGGCTTCAGCGCAAGGACTGTGGCACTTAC C A F Q I N R D T
*
X53408
positions 62, 103 and 105 are mutated. The same mutations are observed at positions 103 and 105 for A2-chain (8). Finally the 5' non coding sequence is highly conserved with regards to prePLA2 cDNA (4-8). The A4-chain mature sequence revealed that the ten residues (bold characters) presumed to be involved in the active site of mammalian PLA2 (9) are present in the reptile PLA2 described here.
-20 -27 GGACAAA ATG AAT CCT GCT CAC CTT CTT GTC CTG TCG GCA GTT TGT GTC TCC CTC L S V C N A V S V L L M H L P A
GCC GCC AAC ATT CCT CCT CAG CAT CTC I P P H A A N L O 20 ACC ATC CCC TGC GAA AAA ACT TGG GGT W G K T T I C E P 40 GGA GGT AGC GGG ACA CCG ATA GAT GCC A S I D G G G T P 60 TAT GGT GAT GCC GCA AAT ATT CGT GAT Y G A A N I R D D
no.
361
421
489
568 642
