ARCHIVES
OF BIOCHEMISTRY
Vol. 284, No. 2, February
AND
BIOPHYSICS
1, pp. 3522359, 1991
Myotoxin II from Bothrops asper (Terciopelo) Venom Is a Lysine-49 Phospholipase A, Brian Francis,* Jose Maria Gutierrez,? Bruno Lomonte,? and Ivan I. Kaiser*‘] *Department of Molecular Riology, University of Wyoming, Laramie, Wyoming 82071; and tlnstituto
Clodomiro Picardo, Universidad
de Costa Rica, San Jose, Costa Rica
Received July 9, 1990, and in revised form September 26, 1990
A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases AZ, except that it shows only 0.1% of the phospholipase activity against L-a-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase AZ. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca”-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys49 proteins from different snake venoms (Agkistrodon piscivoruspiscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase Az active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities. ea is91 Academic Press,
Inc.
Bothrops asper venom contains a number of different toxic proteins including basic phospholipases and phospholipase-like molecules which have been shown to promote myonecrosis by affecting the integrity of the plasma membrane of muscle fibers (l-4). A myotoxic phospholipase (myotoxin I) has been isolated and characterized (5,6) but not sequenced and another myotoxic phosphoi To whom correspondence
should be addressed. FAX: (307) 766-5098.
lipase (which will be referred to as myotoxin III) has been isolated, characterized, and sequenced (7). Myotoxin II is a myotoxic phospholipase-like protein which has low phospholipase activity (4). It is unknown which amino acids in these and other myotoxic phospholipase A2 proteins are responsible for the myotoxicity. Amino acid comparisons initially suggested involvement of a basic region between residues 79 and 87 (30), but such a region was not found in myotoxin III (7). Amino acid sequencing and other studies of myotoxin II were carried out to determine the reasons for the low phospholipase activity, whether this enzymatic activity was required for myotoxicity, and which amino acids could be implicated in myotoxicity. Comparison of this sequence with those of phospholipase-active proteins shows that the main differences are in the Ca2+ binding loop. MATERIALS
AND METHODS
Borhrops asper venom was obtained from adult specimens collected in the Atlantic region of Costa Rica and kept at the serpentarium of the Instituto Clodomiro Picado. Myotoxin II was purified by the procedure of Gutierrez et al. (1) with modifications described in Ref. (4). Reversed phase chromatography was performed on an FPLC system using a Vydac 0.46 X 15-cm Cis column (5 pm) for peptide fractionation. The equilibration buffer was 0.1% trifluoroacetic acid and the eluting buffer 0.1% trifluoroacetic acid-75% acetonitrile. Peptides were detected by ultraviolet absorption at 214 nm. SDSPAGE’ (15%) gels and ureaPAGE (10%) gels were prepared as described (8, 9) and stained with Coomassie blue. Molecular weight standards were from Bio-Rad. Phospholipase assays followed previously described methods (10, 11). Lyophylized myotoxin II (2 mg) was reduced and carboxyamidomethylated using a 12.fold excess of DTT over protein disulfide and a 5fold excess of iodoacetamide over DTT (12). After dialysis against 0.1% trifluoroacetic acid at 4°C for 48 h the solution was divided into five portions (400 pg each) and lyophylized. Fragmentation of the reduced and alkylated protein was accomplished using glutamate- and arginine-
’ Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT, dithiothreitol; PTH, phenylthiohydantoin.
352 All
Copyright (6, 1991 rights of reproduction
0003.9861/91 $3.00 by Academic Press, Inc. in any form reserved.
MYOTOXIC
LYSINE-49
PHOSPHOLIPASE
specific endoproteases (Glu-C and Arg-C), which cleave at the C-terminal sides of their respective amino acids and an endoprotease which cleaves specifically on the N-terminal side of aspartate (Asp-N). Proteases were obtained from Boehringer-Mannheim, with Glu-C and Asp-N representing their sequencing grade reagents. Cleavage with Glu-C was in 25 mM ammonium bicarbonate (pH 7.8)-l M guanidine hydrochloride for 16 h at 23”C, at an enzyme to substrate ratio of 1:40 by weight. Asp-N cleavage (1:200 by weight) was in 50 mM sodium phosphate (pH 8.0)0.5 M guanidine hydrochloride for 16 h at 37°C. Arg-C cleavages were in 100 mM ammonium acetate (pH 7.8) containing 80 @g/ml enzyme for 3 or 16 h at 37°C. Sequencing employed a Applied Biosystems 470A Protein Sequencer with an Applied Biosystems Model 120A on-line analyzer for the analyzer of the phenylthiohydantoin (PTH) amino acid derivatives (8).
A, FROM 1
353
B. asper
2
3
4
5
6
42700
31000
RESULTS
Myotoxin II from B. asper venom is the last major eluting protein during chromatography of crude venom from a CM-Sephadex C-25 column buffered at pH 7 and developed with a NaCl gradient [see fraction 7 in Fig. 1 of Ref. (4)]. It appeared homogeneous, based on a number of criteria, including its initial elution as an individual peak from CM-Sephadex C-25. When rerun on either the same column or a reversed phase Cl8 column it gave a single, symmetrical peak. Electrophoresis on either an acid-urea gel or an SDS-gel (Fig. l), before and after reduction with DTT, produced single bands. Finally, it yielded a single N-terminal sequence of amino acids with no evidence of heterogeneity. Myotoxin I, dissolved in solubilizing mixture, was subjected to various treatments prior to electrophoresis on an SDS-polyacrylamide gel and stained with Coomassie blue (Fig. 1). In the absence of reducing agent or heating, myotoxin II ran as a single band with an apparent molecular weight of 26,000 (lane 2). Heating the sample or adding 2 M urea did not affect the migration on the gel (lanes 3 and 6), but addition of DTT caused the protein to run as a single band with an M, of 16,000 (lanes 4 and 5). Thus the nonreduced form of myotoxin II appears to exist as a homodimer. This molecular weight is similar to those of myotoxins I and III. Myotoxin III migrates on SDS-PAGE with an J4, of 16,000 in the absence of reducing agent but myotoxins II and III elute together from a gel filtration column, suggesting that myotoxin III may also normally exist as a dimer (7). Phospholipase activity of myotoxins II and III from B. asper were determined using L-ol-phosphatidylcholine from egg yolk as a substrate. When added to the reaction mixture to give a final concentration of 10 pg/ml, myotoxins II and III gave activities of 0.3 and 266 pmol of fatty acid released min-’ mg-‘, respectively. By comparison, intact crotoxin, basic, and acidic subunits of crotoxin gave values of 80, 147, and 0 pmol of fatty acid released mini mg-‘, respectively. B. asper myotoxin II showed only 0.1% of the phospholipase activity of myotoxin III. When an equal concentration of myotoxin II was added to myotoxin III there was no change in the activity of
21500
FIG. 1. Coomassie blue-stained SDS-polyacrylamide gel of B. asper myotoxin II. Prior to electrophoresis, myotoxin II was dissolved in protein solubilizing solution at room temperature followed by no further treatment (lane 2), heating at 85°C for 5 min (lane 3), addition of 10 mM DTT (lane 4), addition of 10 mM DTT and heating for 5 min at 85°C (lane 51, and addition of 2 M urea (lane 6). Protein standards were run in lane 1 with molecular weights as shown on the left of the figure.
myotoxin III. Intact crotoxin displayed a sigmoidal relationship between fatty acids released vs time, whereas the basic subunit, as well as myotoxins II and III from B. asper, showed hyperbolic curves with no initial lag period. Rates of hydrolysis for intact crotoxin were determined between 4 and 6 min into the run and for the basic subunit of crotoxin during the first 2 min. The acidic subunit of crotoxin gave no evidence of phospholipase activity (10). Bothrops asper myotoxin II (2 mg) was reduced wit,h DTT and the cysteine sulfhydryls carboxyamidomethylated. After dialysis, the alkylated myotoxin II was transferred to several microfuge tubes and lyophylized to give about 400 ygltube for sequencing. Initial sequencing of the undigested protein yielded the first 55 amino acids at the N-terminus (Fig. 3). Peptides were generated by cleavage with Asp-N protease and separated by reversed phase column chromatography (Fig. 2A). Sequencing of peptide DN-1 confirmed residues 41-55 and extended the N-terminal sequence five residues over that obtained by
354
FRANCIS
0
10
20
30
40
50
60
ET AL.
70
MINUTES 60 0.6.
c
/ 0
/
, 10
20
30
1 40
50
64"
MINUTES
0
10
20
34
40
50
Ml"
MINUTES
FIG. 2. Elution profile myotoxin II peptides from a C,, reverse phase column after the indicated endoprotease treatments. (A) Endoprotease Asp-N fragments of B. asper myotoxin II. (B) Endoprotease Glu-C fragments of B. asper myotoxin II. (C) Endoprotease Arg-C fragments of peptide EC-4 recovered from Glu-C digest (Fig. 2B). (D) Endoprotease Arg-C fragments of peptides EC-5 + EC-6 recovered from Glu-C digest (Fig. 2B).
direct sequencing in from the N-terminus, through amino acid 60 (Fig. 3). By using the previously sequenced myotoxin III (7) as a guide and aligning conserved cysteine residues, provisional peptide assignments were made as follows: DN-2,70-83; DN-3 was a 5 amino acid-containing peptide starting with Glu instead of the expected Asp, 84-88; and DN-4,89-117. Not shown in Fig. 3 are peptides DN-5, 62-69, and DN-6 (89-92), the latter of which started with the same sequence as DN-4 and was aborted after four residues. Myotoxin II was next hydrolyzed with Glu-C protease and the resulting peptides were separated as shown in Fig. 2B. Peptide EC-l, 88-91 (aborted after four residues) provided the overlap between fragments DN-3 and DN-4. The major peak in Fig. 2B, EC-2 C EC3, appeared heterogeneous and upon sequencing yielded
the sequence of two peptides, with one (EC-2) present in a 5:2 molar ratio over the other (EC-3). The EC-4 peptide showed the same N-terminus as EC-3, and corresponded to 99-109+, when sequencing was aborted. The EC-2 peptide started at residue 13 and was aborted after three cycles; the EC-5 peptide corresponded to the N-terminus; and the EC-6 peptide corresponded to residues 5-12. Peptides in peaks containing EC-4 and EC-2 + EC-3 were further digested with Arg-C at low protease to peptide ratios for 3 to 16 h, respectively, giving the chromatograms shown in Figs. 2C and 2D. Low protease to peptide ratios were used to minimize anomalous cleavage sometimes observed with this protease. RC-(4)-l peptide (Fig. 2C) was sequenced and corresponded to the C-terminus of the protein, or residues 109 to 121. A nine amino
*
MYOTOXIC
Ser
5 Leu Phe Glu Leu Gly Lys Met
Leu Gly Arg
35 Gly Lys
Pro
65 Ser
Tyr
10 15 Leu Gin Glu Thr Gly Lys Asn Pro N-terminus __
Val
Ala
Ile
-~---~-__--__-
Ser Trp
70 Lys Asp Lys Thr
95 Cys Leu Arg
100 Glu Asn Leu Asn Thr -----
Ile
20 Ala Lys Ser Tyr
B. a-per
355
Gly Ala Tyr
25 Gly Cys Asn Cys Gly
50 Cys Tyr
Lys Lys
55 Leu Thr
60 Gly Cys Asn Pro Lys
DN- 1 ____-___-~_____ ~~_____~_______ 85 90 Glu Leu Cys Glu Cys Asp Lys
~-DN-2----.-.-__~_--_ -__105 Tyr Asn Lys Lys Tyr DN- 4
30 Val
--p-v------
75 80 Val Cys Gly Glu Asn Asn Ser Cys Leu Lys
EC-1
121 Ala Cys ----RC(
A2 FROM
45 Asp Arg Cys Cys Tyr Val His Lys Cys _ N-terminus ___ ----__ ---__--_____---RC (2+3) - 1
-___--___ fx ( 2+3 ) - 1 __-_-__--______
Ala
PHOSPHOLIPASE
40 Lys Asp Ala Thr
-
Lys Asp Arg Tyr
Ile
LYSINE-49
110 Tyr
Arg Tyr
Leu Lys Pro LeulPhe
__-----.--__
DN- 3 115 Cys Lys
_---DN-4 .--. EC-1 .____ __-_
Lys
Ala
119 Asp
__--____RC ( 4 ) _ I.----------_
4)-l
FIG. 3.
The amino acid sequence of myotoxin
acid overlap with peptide DN-4 completed the sequence of the C-terminus. This left a gap in our sequence near the middle of the molecule (61-69) and the lack of an overlap with fragment DN-2. Fragment RC-(2+3)-l was sequenced and provided the necessary data. An alternative sequence for 109-116, present in small amounts relative to that found in RC(4)-l, was present in RC-(2+3)-2. Peaks labeled RC(2+3)-3 through RC-(2+3)-7 (Fig. 2D) were partially sequenced in an effort to locate amino acids 117-121 of this alternative sequence, but the peptide was not found. The first few amino acids of peaks RC-(2+3)-4 through RC(2+3)-7 revealed that they came from cleavages of the long Glu-C peptide 13-77 (EC-2), and were aborted. Peptides RC-(2+3)-3 and RC-(4)-2 corresponded to amino acids 99-108. From the N-terminal sequence and the peptide sequences the overall amino acid sequence of B. asper myotoxin II was constructed as shown in Fig. 3. There were two isoforms of myotoxin II with different C-terminal sequences in the ratio of 13:1, with leucine and phenylalanine occupying the variable position at 114 and representing the major and minor isoforms, respectively. Myotoxin II contains 121 amino acids, the same as the Lys-49 protein from Agkistrodonpisciuoruspisciuorus (15), rather than the 122 found for myotoxin III (7). The calculated molecular weight, 15,229, is consistent with the apparent molecular weight of 16,000 for the monomer of myotoxin II estimated from the SDS-gel (Fig. 1). Amino acid composition of myotoxin II, as determined from the sequence is also close to that reported previously from amino acid analyses (4). Sequencing revealed three ad-
II from B. asper.
ditional lysines and three fewer tryptophans, acid analyses.
than amino
DISCUSSION Lack of phospholipase activity in myotoxin II can be accounted for by the presence of asparagine at position 28, leucine at position 32, and lysine at position 49 (Fig. 4). In proteins expressing phospholipase AZ activity these highly conserved positions are occupied by tyrosine, glytine, and aspartate, respectively. Analysis by X-ray crystallography of the bovine phospholipase AZ has shown that the essential Ca2+ ion is liganded by the carboxylate side group of Asp-49 and the carbonyls of Tyr-28, Gly30, and Gly-32 in a Ca2+ binding loop (13). Thus three out of the four amino acids in the loop are changed in myotoxin II. Lysine at position 49 and asparagine at position 28 have been identified in the sequences of Lys-49 proteins isolated from A. p. piscivorus, Bothrops atrox and Trimeresurus flavoviridis (14-17). Sequences of these proteins are compared, along with that of myotoxin II in Fig. 4. Replacement of glycine by leucine at position 32 is a unique amino acid replacement in myotoxin II and has not been reported previously. While other sequenced Lys-49 proteins do not have a change at position 32, the highly conserved glycine at position 33 is substituted by histidine, serine, or arginine. Glycine-33 is present in myotoxin II. Amino acid 53 is structurally adjacent to amino acid 49 as a result of folding and has been proposed to be important in events leading to Ca2+ binding (18). In proteins from Viperidae venoms when position 49 is lysine, position 53 is also a lysine.
356
FRANCIS Species B. asper A.p.p. T. f. l&II 8. atrox B. asper Bovine Rat Human
Protein K-49 K-49 K-49 K-49 D-49 Pancrea Platelet Platelet
1 5 SLFEL SVLEL SLVQLWKMIFQ SLVEL SLIEFAKMILE ALWQF SLLEFGQMIL NLVNF
B.asper A.p.p. T. f. I811 B. atrox B. asper Bovine Rat Human
K-49 K-49 K-49 K-49 D-49 Pancrea Platelet Platelet
30 C G V L C G W G C G V G C G V G C G W G C G L G C G V G C G V G
G H R S G G G G
R R R R Q S R R
35 G G G H G G G G
60 G D G N S G G
C C C C C C C
K -
V -
B. asper K-49 A.p.p. K-49 T. f. l&II K-49 B.asper D-49 Bovine Pancrea Rat Platelet Human Platelet
K K K K K K K
L L V L L R R
T T T S D -
B. asper K-49 K-49 A.p.p. T. 1. l&II K-49 B.asper D-49 Pancrea Bovine Platelet Rat Human Platelet
a5 G S S -
GENN EEKNP E K N GEGT S E N T N Q AKQD
B. asper K-49 K-49 A.p.p. T. f. l&II K-49 B.asper D-49 Pancrea Bovine Platelet Rat Human Platelet
Y Y Y Y K Y Y
N N N K E S N
15 K K K K H L K
Q Q
GKMIL
Q
N
GMI
K
H
RMI
K
C F L
K P K 0 P K K P K K P K Q P K T P V S P K S P K
D D D D D D D D
90 S P N D
K Y K Y K Y R Y K N K Y K Y
15 ETGKNPAK ETGKNAI ETGKEAA ETGKNPL ETKRLPFP K I PSSEP K TGKRAD TTGKEAA
10 GKMIL GKMIL
L -
P P A S S
R K T M L Q Q
20 Y Y L A Y F I YPKP A Y P DKF Y L Y Y S
P H P P P F-F--
95 L Q F Q
K K
D L/F K N K N K
L V L
V V V V T T
70 KKDRYS KTDRYS K M D S KTDRYS YTNNYS LTYK LSYK
Y
S
75 Y Y Y Y Y F F
0
L/F K F L NC F H
A A A A A A A
25 C CCC -
K
CC-
KGKT RGST
-
20 25 S - YGAYGCN S - YGSYGCN N - YGLYGCN S-YGVYGCN Y - YTTYGCY L DFNNYGCY S -TGFYGCH S - YGFYGCH
45
C ECDKAV CECDKAV CECDKAV C ECDKAA C NCDRNA CQCDKAA CECDKAA
V I I L L
P L
T K T
40 ATDRCCY ATDRCCFV ATDSCCY DTDRCCY ATDRCCF D LDRCCQT ATDWCCV ATDRCCV
65 - N - N - - DIN - K V D N GTK GTK
CLKE CLKQM C L K CEKQ C E A C R K CRSQ
ET AL.
H H H H H H H H
50 KCCYKKCCYKKCCYKKCCY DCCYGDNCYKQAK DCCYNRLE DCCYKRLE 80
SWKDKTIVC SWKNKAI SWKNKAI S RKSGVI SCSNNEITC SYRGGQI SNSGSRITC 5 I CLRENLNT I CLRENLDT I CLRENLGT VCFRENLRT I CFSKVPYN E CFARNKKS T CFARNKTT
30 KKAD AC KKP D T C KKAD TC K P A E/D K/P C P P
55
I C VC I C SC IO
35
SC R C
FIG. 4. Comparison of the amino acid sequence of B. asper myotoxin II with proteins having related sequences. References for sequences 2 through 7 are Maraganore and Heinrikson (15), Yoshizumi et al. (16), Liu et al. (17), Maraganore et al. (14), Kaiser et al. (7), Fleer et al. (47), Komada et al. (45), and Kramer et al. (46), respectively. Only the sequence of the N-terminal 51 amino acids of the B. atrox protein is known.
After initial reports that the A. p. piscivorus protein with lysine at position 49 exhibited phospholipase activity (14), subsequent experiments demonstrated that it actually had very low enzymatic activity. In allied site-directed mutagenesis work, deI-Iaas and his group (19) demonstrated that conversion of Asp-49 in porcine phospholipase A2 to Lys or Glu resulted in loss of Cazi binding with the concomitant loss of all enzymatic activity. T. flavoviridis proteins with lysine replacing aspartate at position 49 have only 1.5-1.7% of the activity of the phospholipase-active protein isolated from the same venom (16, 17). We determined that the phospholipase activity of myotoxin II was about 0.1% of that of the phospholi-
pase-active B. asper protein myotoxin III. We assume that myotoxin II retains residual phospholipase activity, but the activity could represent a trace phospholipase contaminant. Myotoxin II shows high sequence identity with other Lys-49 proteins as illustrated in Fig. 4. T. flauoviridis and A. p. piscivorus proteins exhibited 78 and 75% sequence identity with myotoxin II, respectively, whereas the first 51 N-terminal residues from B. atrox phospholipase showed 84% identity. Thus, four different viperid snake venoms contain highly homologous Lys-49 proteins with significant differences in their Ca2+ binding sites, when compared with sequences from those found in phospho-
MYOTOXIC
LYSINE-49
PHOSPHOLIPASE
lipase AZ-active proteins. Myotoxin II and myotoxin III, two muscle damaging proteins from the same venom, showed only 61% amino acid identity. Myotoxin II showed even lower amino acid sequence identity with other phospholipase AZ-active proteins from other venoms such as those from A. p. pisciuorus PLA2 (53%) (15), T. flauouiridis (54%) (20, 21), Agkistrodon halys blomhofi (58%) (22), Crotalus durissus terrificus (49%) (23), and Vipera ammodytes ammodytes: ammodytoxin A (60%) (24), ammodytoxin B (59%) (25). Low levels of sequence identity between nonphospholipase Aa proteins and phospholipase AZ-active enzymes in the same venom are also seen in A. p. piscivorus (51%) and T. flavoviridis (54%). Thus there is less homology between the nonphospholipase A, and phospholipase Az proteins from the same snakes than there is between nonphospholipase A2 proteins or phospholipase Az proteins from different snakes, indicating that these different types of protein have been present in viperids for a long evolutionary period. In elapids the presence of non-phospholipase-active proteins is less well established. Notechis II-1 from Notechis scutatus scutatus venom has very low phospholipase activity and toxicity (26). Because serine replaces glycine at position 30 in notechis II-l, it also has a change in the Ca” binding loop, although aspartate is retained at position 49. There appear to be a number of other amino acid differences between the Asp-49 and Lys-49 proteins, besides those already mentioned. Generally, in Lys-49 proteins leucine replaces phenylalanine at position 5; glutamine and glycine replace several different amino acids at position 11 and 23, respectively; lysine replaces glycine at positions 53 and 78; leucine at position 92 replaces a hydrophilic amino acid; lysine replaces arginine at position 100; valine replaces alanine at position 102; and lysine replaces several different amino acids at position 122. The presence of leucine and glutamine at positions 5 and 11, near the N-terminus of Lys-49 proteins, may prove to be a useful identifier of these proteins. In fact, a phospholipase-like protein identified as bothropstoxin from Bothrops jararacussu venom (27) has the sequence S L F Z L G H M I L for its 10 amino terminal residues. This region shows at least 80% identity with the comparable region of myotoxin II (Fig. 4) and includes a Leu-5. Our work would predict that this protein, which has many phospholipase-like properties, but which lacks phospholipase activity (27), is a Lys-49 phospholipase AZ-like protein. Native myotoxin II appears to exist as a dimer in solution (Fig. l), just like phospholipase Az from C. atrox. Heinrikson’s group has suggested that phospholipases that exist as monomers in solution are mono- or diacylated autocatalytically, which promote enzyme dimerization and enhances enzyme activity (up to 200-fold). With phospholipase A2 from A. p. piscivorus covalent diacyla-
A2 FROM
B. asper
357
tion occurred on the c-amino groups of lysine at positions 7 and 10 (28). Porcine phospholipase As was monoacylated at position 56 (29). From limited studies, it appears that the number and location of added acyl groups will be variable, depending on the phospholipase, but will occur at positions which promote formation of more efficient catalytic dimers. It is presently unclear whether the differences in phospholipase activities reported for different Lys-49 proteins, which vary from 0 to 2% of Asp-49 phospholipases Aa, could be due in part to acylation altered activity. Lysine residues exist at positions 7 and 15, as well as 57, in all Lys-49 proteins sequenced to date, and are either identical or near those lysines known to be acylated in phospholipases AZ from A. p. piscivorus and pigs. It is also unknown whether the autocatalytic acylation of either Lys-49 or Asp-49 proteins alter myotoxic effects. Myotoxins II and III are myotoxic and not neurotoxic. Examination of their amino acid sequences and comparison with sequences in other proteins may eventually provide information on the regions of these molecules which are responsible for myotoxicity. Kini and Iwanaga (30) have suggested that a cationic site around residues 79-87 is involved in the myotoxic activity of presynaptic neurotoxic phospholipases AZ. This site is not found in either myotoxin II or III. The amino acid sequences of five myotoxic viperid proteins are known: myotoxins II and III from B. asper (7), phospholipase-active proteins from A. p. piscivorus and T. fkvoviridis (2, 15,20, al), and the phospholipase-active basic subunit of crotoxin from C. d. terrificus (23, 31). They can be compared with the sequences of mammalian pancreatic and C. atrox phospholipases Aa which do not exhibit myotoxicity. Lysine at position 38, threonine at position 112, and tyrosine at position 113 are found in all the myotoxic proteins and none of the nonmyotoxic proteins. The same amino acids are found in the sequences of the Lys-49 proteins from A. p. piscivorus and T. fluvoviridis (see Fig. 4). Another feature of the myotoxic protein sequences is a set of three or four tyrosines between residues 112 and 121. These tyrosines are not observed in the nonmyotoxic phospholipase A, sequences but are in those of the other Lys-49 proteins. Protein structures from X-ray crystallographic analysis of the bovine pancreatic (13) and C. atrox (32) phospholipases Az suggest that Lys-38 is probably located close to the sequences containing these tyrosines. Whether or not these amino acids are involved in the myotoxicity of the viperid proteins remains to be established. The almost complete lack of phospholipase activity of myotoxin II with the same quantitative myotoxicity as myotoxin I (1, 4) indicates that phospholipase activity is not necessary for the expression of myotoxicity by either Lys-49 or Asp-49 phospholipases A,. The same conclusion can be drawn for the edema-inducing activity found as-
358
FRANCIS
sociated with myotoxin II. On the other hand, the lack of anticoagulant effects on sheep, platelet-poor plasma (4) by myotoxin II suggests that phospholipase activity is necessary for this effect. It does not rule out the possibility however that some region of myotoxin II which affects anticoagulant properties of these molecules is altered. While myotoxin II has clearly been shown to be a Lys-49 phospholipase AZ in this study, other myotoxins with similar properties have been isolated from other Bothrops species that will most certainly prove to be Lys49 proteins. Examples are, bothropstoxin which was mentioned earlier (27) from the venom of B. jurarucussu and a myotoxin from the venom of Bothrops nummifer (33). Other nonphospholipase like proteins with myotoxic activity have also been isolated. These include the socalled “membrane toxins” (34) from elapid venoms and small, basic, crotalid proteins such as crotamine and myotoxin a (35-37). No enzymatic activity has been demonstrated for these two different toxin types. Some nonpancreatic mammalian phospholipases AZ are associated with pathologies involving infection, inflammation, and tissue damage (38-43). Myotoxins II and III are more related structurally to these phospholipases, since both are in class II (44), than they are to the class I pancreatic phospholipases. As expected from their structural similarity, myotoxin II and III have a greater level of amino acid identity with rat platelet phospholipase AZ (45) and the human phospholipase AZ found in platelets and rheumatoid synovial fluid (46), than with bovine pancreatic phospholipase AZ, as shown in Fig. 4. The regions of myotoxin II and III from Tyr-25 to Tyr-52 show a particularly high level (-80%) of sequence identity with the mammalian, nonpancreatic phospholipases A,. This includes the lysine at position 38 which is found in other myotoxic snake venom proteins. Another region with -60% sequence identity is located between Cys-96 and Tyr-120. Rat platelet phospholipase AZ has tyrosines at positions 113, 117, and 120 in this region like myotoxin III, and human nonpancreatic phospholipase AZ has tyrosines at positions 113, 117, 119, and 120 as does myotoxin II. Human phospholipase AZ contains threonine at position 112. Serine replaces threonine at this position in the rat enzyme. Thus, certain amino acids which are found in myotoxic snake venom proteins are also found in disease-associated mammalian phospholipases AZ. It will be of great interest to monitor the primary structures of future sequenced phospholipases AZ and determine whether this correlation between pathological activity and structure is maintained. ACKNOWLEDGMENTS We thank Dr. William Kruggel, manager of the Biotechnology Sequencing Facility at the University of Wyoming, for his expertise in sequence analysis. This work was supported in part by the U.S. Army Medical Research and Development Command under Contract DAMD 17-89-C-9007.
ET AL. REFERENCES 1. Gutierrez, J. M., Ownby, C. L., and Odell, G. V. (1984) Tozicon 22, 115-128. 2. Mebs, D., and Samejima, Y. (1986) Toxicon 24, 161-168. 3. Gutierrez, J. M., Ownby, C. L., and Odell, G. V. (1984) Exn. Molec. Pathol. 40, 367-379. 4 Lomonte, B., and Gutierrez, J. M. (1989) Toricon 27, 725-733. 5. Gutierrez, J. M., Lomonte, B., Chaves, F., Moreno, E., and Cerdas, L. (1986) Comp. Biochem. Physiol. 84C, 159-164. 6. Lomonte, B., and Kahan, L. (1988) Tozicon 26,675-689. 7. Kaiser, I. I., Gutierrez, J. M., Plummer, D., Aird, S. D., and Odell, G. V. (1990) Arch. Biochem. Biophys. 278,319-325. 8. Da Silva, N. J., Aird, S. D., Seebart, C., and Kaiser, I. I. (1989) Toxicon 27, 763-771. 9. Traub, P., Mizushima, S., Lowry, C. V., and Nomura, M. (1971) Methods in Enzymology 20, 391-407. 10. Aird, S. D., and Kaiser, I. I. (1985) Toricon 23, 361-374. 11. Aird, S. D., and Kaiser, I. I. (1985) Toxicon 23, 11-13. 12. Aird, S. D., Kaiser, I. I., Lewis, R. V., and Kruggel, W. G. (1985) Biochemistry 24, 7054-7058. 13. Dijkstra, 8. W., Kalk, K. H., Hol, W. G. J., and Drenth, J. (1981) J. Mol. Biol. 147, 97-123. 14. Maraganore, J. M., Merutka, G., Cho, W., Welches, W., Kezdy, F. J., and Heinrikson, R. L. (1984) J. Biol. Chem. 259, 13,83913,843. R. L. (1986) J. Biol. Chem. 15. Maraganore, J. M., and Heinrikson, 261, 4797-4804. 16. Yoshizumi, K., Liu, S-Y., Miyata, T., Saita, S., Ohno, M., Iwanaga, S., and Kihara, H. (1990) Toxicon 28, 43-54. 17. Liu, S-Y., Yoshizumi, K., Oda, N., Ohno, M., Tokunaga, F., Iwanaga, S., and Kihara, H. (1990) J. Biochem. (Tokyo) 107, 400-408. 18. Maraganore, J. M., and Heinrikson, R. L. (1985) Biochem. Biophys. Res. Commun. 131, 129-138. 19. van den Bergh, C. J., Slotboom, A. J., Verheij, H. M., and de Haas, G. H. (1988) Eur. J. Biochem. 176,353-357. 20. Tanaka, S., Mohri, N., Kihari, H., and Ohno, M. (1986) J. Biochem. (Tokyo) 99, 281-289. 21. Kini, R. M., Kawabata, S., and Iwanaga, S. (1986) Toxicon 24, 1117-1129. 22. Forst, S., Weiss, J., Blackburn, P., Fragione, B., Goni, F., and Elsbath, P. (1986) Biochemistry 25, 4309-4314. 23. Aird, S. D., Kaiser, I. I., Lewis, R. V., and Kruggel, W. G. (1986) Arch. Biochem. Biophys. 249, 296-300. 24. Ritonja, A., and Gubensek, F. (1985) Biochim. Biophys. Acta 828, 306-312. 25. Ritonja, A., Machleidt, W., Turk, V., and Gubensek, F. (1986) Biol. Chem. Hoppe-Seyler 367,919-923. 26. Lind, P., and Eaker, D. (1980) Eur. J. Biochem. 111, 403-409. M. I., Queiroz, L. S., Santo-Neto, H., Ro27. Homse-Brandeburgo, drigues-Simioni, L., and Giglio, J. R. (1988) Toxicon 26, 615-627. R. L., and Kezdy, F. J. 28. Cho, W., Tomasselli, A. G., Heinrikson, (1988) J. Biol. Chem. 263, 11,237-11,241. H., Reardon, 29. Tomasselli, A. G., Hui, J., Fisher, J., Zwcher-Neely, I. M., Driaku, E., Kezdy, F. J., and Heinrikson, R. L. (1989) J. Biol. Chem. 264, 10,041-10,047. 30. Kini, R. M., and Iwanaga, S. (1986) Toxicon 24, 895-905. P., Dempster, D. W., Hawgood, B. J., and Elder, 31. Gopalakrishnakone, H. Y. (1984) Toricon 22, 85-98. 32 Brunie, S., Bolin, J., Gewirth, D., and Sigler, P. B. (1985) J. Bid. Chen. 260, 9742-9749.
MYOTOXIC 33. Gutierrez, 885-894.
J. M., Lomonte,
LYSINE-49
PHOSPHOLIPASE
B., and Cerdas, L. (1986) Toricon
24,
34. Karlsson, E. (1979) in Snake Venoms (Lee, C.-Y., Ed.), pp 159-212, Springer-Verlag, Berlin. 35. Maeda, N., Tamiya, N., Pattabhiraman, (1978) Tonicon 16, 431-441. 36. Laure, C. J. (1975) Hoppe-Seyler’s
T. R., and Russell, F. E.
Z. Physiol. Chem. 356,213-217.
37. Fox, J. W., Elzinga, M., and Tu, A. T. (1979) Biochemistry 684. 38. Vadas, P., and Pruzanski,
18,678-
W. (1986) Lab. Inuest. 4, 391-404.
39. Forst, S., Weiss, J., Elsbach, P., Maraganore, J. M., Reardon, and Heinrikson, R. L. (1986) Biochemistry 25, 8381-8385. 40. Chang, J., Musser, J. H., and McGregor, macol. 36, 2429-2436.
I.,
H. (1987) Biochem. Phar-
A2 FROM
B. asper
359
41. Vadas, P., Pruzanski, W., Stefanski, E., Sternby, B., Mustard, R., Bohnen, J., Frazer, R., Farewell, V., and Bombardier, C. (1988) Crit. Care Med. 16, l-7. 42. Chang, H. W., Kudo, I., Tomita, M., and Inoue, K. (1988) J. Biochem. (Tokyo) 102, 147-154. 43. Hara, S., Kudo, I., Matsuta, K., Miyamoto, T., and Inoue, K. (1988) J. Biochem. Wokvo) 104.326-328. 44. Heinrikson, R. L., Trueger, E. T., and Kein, P. S. (1977) J. Biol. Chem. 252,4913-4921. 45. Komada, M., Kudo, I., and Inoue, K. (1990) Biochem. Biophys. Res. Commun. 168, 1059-1065. 46. Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R., and Pepinsky, R. B. (1989) J. Biol. Chem. 264, 5768-5775. 47. Fleer, E. A. M., Verhey, H. M., and deHaas, G. H. (1978) Eur. J. Biochem. 82, 261-270.
OF BIOCHEMISTRY
Vol. 284, No. 2, February
AND
BIOPHYSICS
1, pp. 3522359, 1991
Myotoxin II from Bothrops asper (Terciopelo) Venom Is a Lysine-49 Phospholipase A, Brian Francis,* Jose Maria Gutierrez,? Bruno Lomonte,? and Ivan I. Kaiser*‘] *Department of Molecular Riology, University of Wyoming, Laramie, Wyoming 82071; and tlnstituto
Clodomiro Picardo, Universidad
de Costa Rica, San Jose, Costa Rica
Received July 9, 1990, and in revised form September 26, 1990
A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases AZ, except that it shows only 0.1% of the phospholipase activity against L-a-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase AZ. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca”-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys49 proteins from different snake venoms (Agkistrodon piscivoruspiscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase Az active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities. ea is91 Academic Press,
Inc.
Bothrops asper venom contains a number of different toxic proteins including basic phospholipases and phospholipase-like molecules which have been shown to promote myonecrosis by affecting the integrity of the plasma membrane of muscle fibers (l-4). A myotoxic phospholipase (myotoxin I) has been isolated and characterized (5,6) but not sequenced and another myotoxic phosphoi To whom correspondence
should be addressed. FAX: (307) 766-5098.
lipase (which will be referred to as myotoxin III) has been isolated, characterized, and sequenced (7). Myotoxin II is a myotoxic phospholipase-like protein which has low phospholipase activity (4). It is unknown which amino acids in these and other myotoxic phospholipase A2 proteins are responsible for the myotoxicity. Amino acid comparisons initially suggested involvement of a basic region between residues 79 and 87 (30), but such a region was not found in myotoxin III (7). Amino acid sequencing and other studies of myotoxin II were carried out to determine the reasons for the low phospholipase activity, whether this enzymatic activity was required for myotoxicity, and which amino acids could be implicated in myotoxicity. Comparison of this sequence with those of phospholipase-active proteins shows that the main differences are in the Ca2+ binding loop. MATERIALS
AND METHODS
Borhrops asper venom was obtained from adult specimens collected in the Atlantic region of Costa Rica and kept at the serpentarium of the Instituto Clodomiro Picado. Myotoxin II was purified by the procedure of Gutierrez et al. (1) with modifications described in Ref. (4). Reversed phase chromatography was performed on an FPLC system using a Vydac 0.46 X 15-cm Cis column (5 pm) for peptide fractionation. The equilibration buffer was 0.1% trifluoroacetic acid and the eluting buffer 0.1% trifluoroacetic acid-75% acetonitrile. Peptides were detected by ultraviolet absorption at 214 nm. SDSPAGE’ (15%) gels and ureaPAGE (10%) gels were prepared as described (8, 9) and stained with Coomassie blue. Molecular weight standards were from Bio-Rad. Phospholipase assays followed previously described methods (10, 11). Lyophylized myotoxin II (2 mg) was reduced and carboxyamidomethylated using a 12.fold excess of DTT over protein disulfide and a 5fold excess of iodoacetamide over DTT (12). After dialysis against 0.1% trifluoroacetic acid at 4°C for 48 h the solution was divided into five portions (400 pg each) and lyophylized. Fragmentation of the reduced and alkylated protein was accomplished using glutamate- and arginine-
’ Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DTT, dithiothreitol; PTH, phenylthiohydantoin.
352 All
Copyright (6, 1991 rights of reproduction
0003.9861/91 $3.00 by Academic Press, Inc. in any form reserved.
MYOTOXIC
LYSINE-49
PHOSPHOLIPASE
specific endoproteases (Glu-C and Arg-C), which cleave at the C-terminal sides of their respective amino acids and an endoprotease which cleaves specifically on the N-terminal side of aspartate (Asp-N). Proteases were obtained from Boehringer-Mannheim, with Glu-C and Asp-N representing their sequencing grade reagents. Cleavage with Glu-C was in 25 mM ammonium bicarbonate (pH 7.8)-l M guanidine hydrochloride for 16 h at 23”C, at an enzyme to substrate ratio of 1:40 by weight. Asp-N cleavage (1:200 by weight) was in 50 mM sodium phosphate (pH 8.0)0.5 M guanidine hydrochloride for 16 h at 37°C. Arg-C cleavages were in 100 mM ammonium acetate (pH 7.8) containing 80 @g/ml enzyme for 3 or 16 h at 37°C. Sequencing employed a Applied Biosystems 470A Protein Sequencer with an Applied Biosystems Model 120A on-line analyzer for the analyzer of the phenylthiohydantoin (PTH) amino acid derivatives (8).
A, FROM 1
353
B. asper
2
3
4
5
6
42700
31000
RESULTS
Myotoxin II from B. asper venom is the last major eluting protein during chromatography of crude venom from a CM-Sephadex C-25 column buffered at pH 7 and developed with a NaCl gradient [see fraction 7 in Fig. 1 of Ref. (4)]. It appeared homogeneous, based on a number of criteria, including its initial elution as an individual peak from CM-Sephadex C-25. When rerun on either the same column or a reversed phase Cl8 column it gave a single, symmetrical peak. Electrophoresis on either an acid-urea gel or an SDS-gel (Fig. l), before and after reduction with DTT, produced single bands. Finally, it yielded a single N-terminal sequence of amino acids with no evidence of heterogeneity. Myotoxin I, dissolved in solubilizing mixture, was subjected to various treatments prior to electrophoresis on an SDS-polyacrylamide gel and stained with Coomassie blue (Fig. 1). In the absence of reducing agent or heating, myotoxin II ran as a single band with an apparent molecular weight of 26,000 (lane 2). Heating the sample or adding 2 M urea did not affect the migration on the gel (lanes 3 and 6), but addition of DTT caused the protein to run as a single band with an M, of 16,000 (lanes 4 and 5). Thus the nonreduced form of myotoxin II appears to exist as a homodimer. This molecular weight is similar to those of myotoxins I and III. Myotoxin III migrates on SDS-PAGE with an J4, of 16,000 in the absence of reducing agent but myotoxins II and III elute together from a gel filtration column, suggesting that myotoxin III may also normally exist as a dimer (7). Phospholipase activity of myotoxins II and III from B. asper were determined using L-ol-phosphatidylcholine from egg yolk as a substrate. When added to the reaction mixture to give a final concentration of 10 pg/ml, myotoxins II and III gave activities of 0.3 and 266 pmol of fatty acid released min-’ mg-‘, respectively. By comparison, intact crotoxin, basic, and acidic subunits of crotoxin gave values of 80, 147, and 0 pmol of fatty acid released mini mg-‘, respectively. B. asper myotoxin II showed only 0.1% of the phospholipase activity of myotoxin III. When an equal concentration of myotoxin II was added to myotoxin III there was no change in the activity of
21500
FIG. 1. Coomassie blue-stained SDS-polyacrylamide gel of B. asper myotoxin II. Prior to electrophoresis, myotoxin II was dissolved in protein solubilizing solution at room temperature followed by no further treatment (lane 2), heating at 85°C for 5 min (lane 3), addition of 10 mM DTT (lane 4), addition of 10 mM DTT and heating for 5 min at 85°C (lane 51, and addition of 2 M urea (lane 6). Protein standards were run in lane 1 with molecular weights as shown on the left of the figure.
myotoxin III. Intact crotoxin displayed a sigmoidal relationship between fatty acids released vs time, whereas the basic subunit, as well as myotoxins II and III from B. asper, showed hyperbolic curves with no initial lag period. Rates of hydrolysis for intact crotoxin were determined between 4 and 6 min into the run and for the basic subunit of crotoxin during the first 2 min. The acidic subunit of crotoxin gave no evidence of phospholipase activity (10). Bothrops asper myotoxin II (2 mg) was reduced wit,h DTT and the cysteine sulfhydryls carboxyamidomethylated. After dialysis, the alkylated myotoxin II was transferred to several microfuge tubes and lyophylized to give about 400 ygltube for sequencing. Initial sequencing of the undigested protein yielded the first 55 amino acids at the N-terminus (Fig. 3). Peptides were generated by cleavage with Asp-N protease and separated by reversed phase column chromatography (Fig. 2A). Sequencing of peptide DN-1 confirmed residues 41-55 and extended the N-terminal sequence five residues over that obtained by
354
FRANCIS
0
10
20
30
40
50
60
ET AL.
70
MINUTES 60 0.6.
c
/ 0
/
, 10
20
30
1 40
50
64"
MINUTES
0
10
20
34
40
50
Ml"
MINUTES
FIG. 2. Elution profile myotoxin II peptides from a C,, reverse phase column after the indicated endoprotease treatments. (A) Endoprotease Asp-N fragments of B. asper myotoxin II. (B) Endoprotease Glu-C fragments of B. asper myotoxin II. (C) Endoprotease Arg-C fragments of peptide EC-4 recovered from Glu-C digest (Fig. 2B). (D) Endoprotease Arg-C fragments of peptides EC-5 + EC-6 recovered from Glu-C digest (Fig. 2B).
direct sequencing in from the N-terminus, through amino acid 60 (Fig. 3). By using the previously sequenced myotoxin III (7) as a guide and aligning conserved cysteine residues, provisional peptide assignments were made as follows: DN-2,70-83; DN-3 was a 5 amino acid-containing peptide starting with Glu instead of the expected Asp, 84-88; and DN-4,89-117. Not shown in Fig. 3 are peptides DN-5, 62-69, and DN-6 (89-92), the latter of which started with the same sequence as DN-4 and was aborted after four residues. Myotoxin II was next hydrolyzed with Glu-C protease and the resulting peptides were separated as shown in Fig. 2B. Peptide EC-l, 88-91 (aborted after four residues) provided the overlap between fragments DN-3 and DN-4. The major peak in Fig. 2B, EC-2 C EC3, appeared heterogeneous and upon sequencing yielded
the sequence of two peptides, with one (EC-2) present in a 5:2 molar ratio over the other (EC-3). The EC-4 peptide showed the same N-terminus as EC-3, and corresponded to 99-109+, when sequencing was aborted. The EC-2 peptide started at residue 13 and was aborted after three cycles; the EC-5 peptide corresponded to the N-terminus; and the EC-6 peptide corresponded to residues 5-12. Peptides in peaks containing EC-4 and EC-2 + EC-3 were further digested with Arg-C at low protease to peptide ratios for 3 to 16 h, respectively, giving the chromatograms shown in Figs. 2C and 2D. Low protease to peptide ratios were used to minimize anomalous cleavage sometimes observed with this protease. RC-(4)-l peptide (Fig. 2C) was sequenced and corresponded to the C-terminus of the protein, or residues 109 to 121. A nine amino
*
MYOTOXIC
Ser
5 Leu Phe Glu Leu Gly Lys Met
Leu Gly Arg
35 Gly Lys
Pro
65 Ser
Tyr
10 15 Leu Gin Glu Thr Gly Lys Asn Pro N-terminus __
Val
Ala
Ile
-~---~-__--__-
Ser Trp
70 Lys Asp Lys Thr
95 Cys Leu Arg
100 Glu Asn Leu Asn Thr -----
Ile
20 Ala Lys Ser Tyr
B. a-per
355
Gly Ala Tyr
25 Gly Cys Asn Cys Gly
50 Cys Tyr
Lys Lys
55 Leu Thr
60 Gly Cys Asn Pro Lys
DN- 1 ____-___-~_____ ~~_____~_______ 85 90 Glu Leu Cys Glu Cys Asp Lys
~-DN-2----.-.-__~_--_ -__105 Tyr Asn Lys Lys Tyr DN- 4
30 Val
--p-v------
75 80 Val Cys Gly Glu Asn Asn Ser Cys Leu Lys
EC-1
121 Ala Cys ----RC(
A2 FROM
45 Asp Arg Cys Cys Tyr Val His Lys Cys _ N-terminus ___ ----__ ---__--_____---RC (2+3) - 1
-___--___ fx ( 2+3 ) - 1 __-_-__--______
Ala
PHOSPHOLIPASE
40 Lys Asp Ala Thr
-
Lys Asp Arg Tyr
Ile
LYSINE-49
110 Tyr
Arg Tyr
Leu Lys Pro LeulPhe
__-----.--__
DN- 3 115 Cys Lys
_---DN-4 .--. EC-1 .____ __-_
Lys
Ala
119 Asp
__--____RC ( 4 ) _ I.----------_
4)-l
FIG. 3.
The amino acid sequence of myotoxin
acid overlap with peptide DN-4 completed the sequence of the C-terminus. This left a gap in our sequence near the middle of the molecule (61-69) and the lack of an overlap with fragment DN-2. Fragment RC-(2+3)-l was sequenced and provided the necessary data. An alternative sequence for 109-116, present in small amounts relative to that found in RC(4)-l, was present in RC-(2+3)-2. Peaks labeled RC(2+3)-3 through RC-(2+3)-7 (Fig. 2D) were partially sequenced in an effort to locate amino acids 117-121 of this alternative sequence, but the peptide was not found. The first few amino acids of peaks RC-(2+3)-4 through RC(2+3)-7 revealed that they came from cleavages of the long Glu-C peptide 13-77 (EC-2), and were aborted. Peptides RC-(2+3)-3 and RC-(4)-2 corresponded to amino acids 99-108. From the N-terminal sequence and the peptide sequences the overall amino acid sequence of B. asper myotoxin II was constructed as shown in Fig. 3. There were two isoforms of myotoxin II with different C-terminal sequences in the ratio of 13:1, with leucine and phenylalanine occupying the variable position at 114 and representing the major and minor isoforms, respectively. Myotoxin II contains 121 amino acids, the same as the Lys-49 protein from Agkistrodonpisciuoruspisciuorus (15), rather than the 122 found for myotoxin III (7). The calculated molecular weight, 15,229, is consistent with the apparent molecular weight of 16,000 for the monomer of myotoxin II estimated from the SDS-gel (Fig. 1). Amino acid composition of myotoxin II, as determined from the sequence is also close to that reported previously from amino acid analyses (4). Sequencing revealed three ad-
II from B. asper.
ditional lysines and three fewer tryptophans, acid analyses.
than amino
DISCUSSION Lack of phospholipase activity in myotoxin II can be accounted for by the presence of asparagine at position 28, leucine at position 32, and lysine at position 49 (Fig. 4). In proteins expressing phospholipase AZ activity these highly conserved positions are occupied by tyrosine, glytine, and aspartate, respectively. Analysis by X-ray crystallography of the bovine phospholipase AZ has shown that the essential Ca2+ ion is liganded by the carboxylate side group of Asp-49 and the carbonyls of Tyr-28, Gly30, and Gly-32 in a Ca2+ binding loop (13). Thus three out of the four amino acids in the loop are changed in myotoxin II. Lysine at position 49 and asparagine at position 28 have been identified in the sequences of Lys-49 proteins isolated from A. p. piscivorus, Bothrops atrox and Trimeresurus flavoviridis (14-17). Sequences of these proteins are compared, along with that of myotoxin II in Fig. 4. Replacement of glycine by leucine at position 32 is a unique amino acid replacement in myotoxin II and has not been reported previously. While other sequenced Lys-49 proteins do not have a change at position 32, the highly conserved glycine at position 33 is substituted by histidine, serine, or arginine. Glycine-33 is present in myotoxin II. Amino acid 53 is structurally adjacent to amino acid 49 as a result of folding and has been proposed to be important in events leading to Ca2+ binding (18). In proteins from Viperidae venoms when position 49 is lysine, position 53 is also a lysine.
356
FRANCIS Species B. asper A.p.p. T. f. l&II 8. atrox B. asper Bovine Rat Human
Protein K-49 K-49 K-49 K-49 D-49 Pancrea Platelet Platelet
1 5 SLFEL SVLEL SLVQLWKMIFQ SLVEL SLIEFAKMILE ALWQF SLLEFGQMIL NLVNF
B.asper A.p.p. T. f. I811 B. atrox B. asper Bovine Rat Human
K-49 K-49 K-49 K-49 D-49 Pancrea Platelet Platelet
30 C G V L C G W G C G V G C G V G C G W G C G L G C G V G C G V G
G H R S G G G G
R R R R Q S R R
35 G G G H G G G G
60 G D G N S G G
C C C C C C C
K -
V -
B. asper K-49 A.p.p. K-49 T. f. l&II K-49 B.asper D-49 Bovine Pancrea Rat Platelet Human Platelet
K K K K K K K
L L V L L R R
T T T S D -
B. asper K-49 K-49 A.p.p. T. 1. l&II K-49 B.asper D-49 Pancrea Bovine Platelet Rat Human Platelet
a5 G S S -
GENN EEKNP E K N GEGT S E N T N Q AKQD
B. asper K-49 K-49 A.p.p. T. f. l&II K-49 B.asper D-49 Pancrea Bovine Platelet Rat Human Platelet
Y Y Y Y K Y Y
N N N K E S N
15 K K K K H L K
Q Q
GKMIL
Q
N
GMI
K
H
RMI
K
C F L
K P K 0 P K K P K K P K Q P K T P V S P K S P K
D D D D D D D D
90 S P N D
K Y K Y K Y R Y K N K Y K Y
15 ETGKNPAK ETGKNAI ETGKEAA ETGKNPL ETKRLPFP K I PSSEP K TGKRAD TTGKEAA
10 GKMIL GKMIL
L -
P P A S S
R K T M L Q Q
20 Y Y L A Y F I YPKP A Y P DKF Y L Y Y S
P H P P P F-F--
95 L Q F Q
K K
D L/F K N K N K
L V L
V V V V T T
70 KKDRYS KTDRYS K M D S KTDRYS YTNNYS LTYK LSYK
Y
S
75 Y Y Y Y Y F F
0
L/F K F L NC F H
A A A A A A A
25 C CCC -
K
CC-
KGKT RGST
-
20 25 S - YGAYGCN S - YGSYGCN N - YGLYGCN S-YGVYGCN Y - YTTYGCY L DFNNYGCY S -TGFYGCH S - YGFYGCH
45
C ECDKAV CECDKAV CECDKAV C ECDKAA C NCDRNA CQCDKAA CECDKAA
V I I L L
P L
T K T
40 ATDRCCY ATDRCCFV ATDSCCY DTDRCCY ATDRCCF D LDRCCQT ATDWCCV ATDRCCV
65 - N - N - - DIN - K V D N GTK GTK
CLKE CLKQM C L K CEKQ C E A C R K CRSQ
ET AL.
H H H H H H H H
50 KCCYKKCCYKKCCYKKCCY DCCYGDNCYKQAK DCCYNRLE DCCYKRLE 80
SWKDKTIVC SWKNKAI SWKNKAI S RKSGVI SCSNNEITC SYRGGQI SNSGSRITC 5 I CLRENLNT I CLRENLDT I CLRENLGT VCFRENLRT I CFSKVPYN E CFARNKKS T CFARNKTT
30 KKAD AC KKP D T C KKAD TC K P A E/D K/P C P P
55
I C VC I C SC IO
35
SC R C
FIG. 4. Comparison of the amino acid sequence of B. asper myotoxin II with proteins having related sequences. References for sequences 2 through 7 are Maraganore and Heinrikson (15), Yoshizumi et al. (16), Liu et al. (17), Maraganore et al. (14), Kaiser et al. (7), Fleer et al. (47), Komada et al. (45), and Kramer et al. (46), respectively. Only the sequence of the N-terminal 51 amino acids of the B. atrox protein is known.
After initial reports that the A. p. piscivorus protein with lysine at position 49 exhibited phospholipase activity (14), subsequent experiments demonstrated that it actually had very low enzymatic activity. In allied site-directed mutagenesis work, deI-Iaas and his group (19) demonstrated that conversion of Asp-49 in porcine phospholipase A2 to Lys or Glu resulted in loss of Cazi binding with the concomitant loss of all enzymatic activity. T. flavoviridis proteins with lysine replacing aspartate at position 49 have only 1.5-1.7% of the activity of the phospholipase-active protein isolated from the same venom (16, 17). We determined that the phospholipase activity of myotoxin II was about 0.1% of that of the phospholi-
pase-active B. asper protein myotoxin III. We assume that myotoxin II retains residual phospholipase activity, but the activity could represent a trace phospholipase contaminant. Myotoxin II shows high sequence identity with other Lys-49 proteins as illustrated in Fig. 4. T. flauoviridis and A. p. piscivorus proteins exhibited 78 and 75% sequence identity with myotoxin II, respectively, whereas the first 51 N-terminal residues from B. atrox phospholipase showed 84% identity. Thus, four different viperid snake venoms contain highly homologous Lys-49 proteins with significant differences in their Ca2+ binding sites, when compared with sequences from those found in phospho-
MYOTOXIC
LYSINE-49
PHOSPHOLIPASE
lipase AZ-active proteins. Myotoxin II and myotoxin III, two muscle damaging proteins from the same venom, showed only 61% amino acid identity. Myotoxin II showed even lower amino acid sequence identity with other phospholipase AZ-active proteins from other venoms such as those from A. p. pisciuorus PLA2 (53%) (15), T. flauouiridis (54%) (20, 21), Agkistrodon halys blomhofi (58%) (22), Crotalus durissus terrificus (49%) (23), and Vipera ammodytes ammodytes: ammodytoxin A (60%) (24), ammodytoxin B (59%) (25). Low levels of sequence identity between nonphospholipase Aa proteins and phospholipase AZ-active enzymes in the same venom are also seen in A. p. piscivorus (51%) and T. flavoviridis (54%). Thus there is less homology between the nonphospholipase A, and phospholipase Az proteins from the same snakes than there is between nonphospholipase A2 proteins or phospholipase Az proteins from different snakes, indicating that these different types of protein have been present in viperids for a long evolutionary period. In elapids the presence of non-phospholipase-active proteins is less well established. Notechis II-1 from Notechis scutatus scutatus venom has very low phospholipase activity and toxicity (26). Because serine replaces glycine at position 30 in notechis II-l, it also has a change in the Ca” binding loop, although aspartate is retained at position 49. There appear to be a number of other amino acid differences between the Asp-49 and Lys-49 proteins, besides those already mentioned. Generally, in Lys-49 proteins leucine replaces phenylalanine at position 5; glutamine and glycine replace several different amino acids at position 11 and 23, respectively; lysine replaces glycine at positions 53 and 78; leucine at position 92 replaces a hydrophilic amino acid; lysine replaces arginine at position 100; valine replaces alanine at position 102; and lysine replaces several different amino acids at position 122. The presence of leucine and glutamine at positions 5 and 11, near the N-terminus of Lys-49 proteins, may prove to be a useful identifier of these proteins. In fact, a phospholipase-like protein identified as bothropstoxin from Bothrops jararacussu venom (27) has the sequence S L F Z L G H M I L for its 10 amino terminal residues. This region shows at least 80% identity with the comparable region of myotoxin II (Fig. 4) and includes a Leu-5. Our work would predict that this protein, which has many phospholipase-like properties, but which lacks phospholipase activity (27), is a Lys-49 phospholipase AZ-like protein. Native myotoxin II appears to exist as a dimer in solution (Fig. l), just like phospholipase Az from C. atrox. Heinrikson’s group has suggested that phospholipases that exist as monomers in solution are mono- or diacylated autocatalytically, which promote enzyme dimerization and enhances enzyme activity (up to 200-fold). With phospholipase A2 from A. p. piscivorus covalent diacyla-
A2 FROM
B. asper
357
tion occurred on the c-amino groups of lysine at positions 7 and 10 (28). Porcine phospholipase As was monoacylated at position 56 (29). From limited studies, it appears that the number and location of added acyl groups will be variable, depending on the phospholipase, but will occur at positions which promote formation of more efficient catalytic dimers. It is presently unclear whether the differences in phospholipase activities reported for different Lys-49 proteins, which vary from 0 to 2% of Asp-49 phospholipases Aa, could be due in part to acylation altered activity. Lysine residues exist at positions 7 and 15, as well as 57, in all Lys-49 proteins sequenced to date, and are either identical or near those lysines known to be acylated in phospholipases AZ from A. p. piscivorus and pigs. It is also unknown whether the autocatalytic acylation of either Lys-49 or Asp-49 proteins alter myotoxic effects. Myotoxins II and III are myotoxic and not neurotoxic. Examination of their amino acid sequences and comparison with sequences in other proteins may eventually provide information on the regions of these molecules which are responsible for myotoxicity. Kini and Iwanaga (30) have suggested that a cationic site around residues 79-87 is involved in the myotoxic activity of presynaptic neurotoxic phospholipases AZ. This site is not found in either myotoxin II or III. The amino acid sequences of five myotoxic viperid proteins are known: myotoxins II and III from B. asper (7), phospholipase-active proteins from A. p. piscivorus and T. fkvoviridis (2, 15,20, al), and the phospholipase-active basic subunit of crotoxin from C. d. terrificus (23, 31). They can be compared with the sequences of mammalian pancreatic and C. atrox phospholipases Aa which do not exhibit myotoxicity. Lysine at position 38, threonine at position 112, and tyrosine at position 113 are found in all the myotoxic proteins and none of the nonmyotoxic proteins. The same amino acids are found in the sequences of the Lys-49 proteins from A. p. piscivorus and T. fluvoviridis (see Fig. 4). Another feature of the myotoxic protein sequences is a set of three or four tyrosines between residues 112 and 121. These tyrosines are not observed in the nonmyotoxic phospholipase A, sequences but are in those of the other Lys-49 proteins. Protein structures from X-ray crystallographic analysis of the bovine pancreatic (13) and C. atrox (32) phospholipases Az suggest that Lys-38 is probably located close to the sequences containing these tyrosines. Whether or not these amino acids are involved in the myotoxicity of the viperid proteins remains to be established. The almost complete lack of phospholipase activity of myotoxin II with the same quantitative myotoxicity as myotoxin I (1, 4) indicates that phospholipase activity is not necessary for the expression of myotoxicity by either Lys-49 or Asp-49 phospholipases A,. The same conclusion can be drawn for the edema-inducing activity found as-
358
FRANCIS
sociated with myotoxin II. On the other hand, the lack of anticoagulant effects on sheep, platelet-poor plasma (4) by myotoxin II suggests that phospholipase activity is necessary for this effect. It does not rule out the possibility however that some region of myotoxin II which affects anticoagulant properties of these molecules is altered. While myotoxin II has clearly been shown to be a Lys-49 phospholipase AZ in this study, other myotoxins with similar properties have been isolated from other Bothrops species that will most certainly prove to be Lys49 proteins. Examples are, bothropstoxin which was mentioned earlier (27) from the venom of B. jurarucussu and a myotoxin from the venom of Bothrops nummifer (33). Other nonphospholipase like proteins with myotoxic activity have also been isolated. These include the socalled “membrane toxins” (34) from elapid venoms and small, basic, crotalid proteins such as crotamine and myotoxin a (35-37). No enzymatic activity has been demonstrated for these two different toxin types. Some nonpancreatic mammalian phospholipases AZ are associated with pathologies involving infection, inflammation, and tissue damage (38-43). Myotoxins II and III are more related structurally to these phospholipases, since both are in class II (44), than they are to the class I pancreatic phospholipases. As expected from their structural similarity, myotoxin II and III have a greater level of amino acid identity with rat platelet phospholipase AZ (45) and the human phospholipase AZ found in platelets and rheumatoid synovial fluid (46), than with bovine pancreatic phospholipase AZ, as shown in Fig. 4. The regions of myotoxin II and III from Tyr-25 to Tyr-52 show a particularly high level (-80%) of sequence identity with the mammalian, nonpancreatic phospholipases A,. This includes the lysine at position 38 which is found in other myotoxic snake venom proteins. Another region with -60% sequence identity is located between Cys-96 and Tyr-120. Rat platelet phospholipase AZ has tyrosines at positions 113, 117, and 120 in this region like myotoxin III, and human nonpancreatic phospholipase AZ has tyrosines at positions 113, 117, 119, and 120 as does myotoxin II. Human phospholipase AZ contains threonine at position 112. Serine replaces threonine at this position in the rat enzyme. Thus, certain amino acids which are found in myotoxic snake venom proteins are also found in disease-associated mammalian phospholipases AZ. It will be of great interest to monitor the primary structures of future sequenced phospholipases AZ and determine whether this correlation between pathological activity and structure is maintained. ACKNOWLEDGMENTS We thank Dr. William Kruggel, manager of the Biotechnology Sequencing Facility at the University of Wyoming, for his expertise in sequence analysis. This work was supported in part by the U.S. Army Medical Research and Development Command under Contract DAMD 17-89-C-9007.
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