0041-0101/92 $5 .00 + A) OD 1992 Pergamon Pree Ltd
Tackimr, Vol . 30, No. 11, pp. 1331-1341, 1992. Printed in Great Britain .
SEQUENCE DETERMINATION AND CHARACTERIZATION OF A PHOSPHOLIPASE A2 ISOZYME FROM TRIMERESURUS GRAMINEUS (GREEN HABU SNAKE) VENOM TOYOKA FUKAGAWA, l Hmosm MATSUMOTO,' YASUYUKI SHIMOHIGASHI,' TOMOHISA OGAWA,' NAOKO ODA,' CHUN-CHANG CHAN& and MOTONORI OHNOI
'Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812, Japan ; and 'Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan, R .O.C . (Received
26 May 1992 ;
accepted I 1 July
1992)
T . FUKAGAWA, H. MATSUMOTO, Y . SHIMOHIGASHI, T . OGAWA, N . ODA, C.-C . CHANG and M . OHNo. Sequence determination and characterization of a
phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom. Toxicon 30, 1331-1341, 1992.-In addition to phospholipase A2-I (PLA 2-,) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA,-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-,, PLA2II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLAA and PLA2-II for Ca 2+ . Secondary structure compositions of PLA2I, were similar to those of PLAA and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-,, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLAA and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLAA and Asn for PLA2-II). INTRODUCTION
A2 (PLA 2s) [EC 3.1 .1.4] catalyse the hydrolysis of the 2-acyl ester bond in 3-sn-phosphoglycerides. They play a central role in metabolism of phospholipid (vAN DEENAN, 1965) and function as a trigger of the arachidonate cascade (McKEAN et al., 1981 ; VADAs and PRUZANSKI, 1986). For stabilization of a conformation essential for the PHOSPHOLIPASFS
" Author to whom correspondence should be addressed . 1331
1332
T. FUKAGAWA et al.
catalytic function, PLAZs require Ca t+ and possess a binding site for this divalent ion (VERHEu et al., 1980). The fl-carboxyl group of aspartic acid-48 is one of key constituents of the site, and in addition to this group the a-carbonyl groups of Tyr-28, Gly-30, and lily-32 form a part of Ca' binding loop (FLEER et al., 1981). A single amino acid replacement often causes a drastic change in function and therefore offers a good chance to develop a novel study of the structure-function relationships. For instance, a PLA 2 from Notechis scutatus scutatus (Australian tiger snake) venom contains Ser-30 (LIND and EAKER, 1980). Inactivity of this PLAZ was substantiated by the fact that a mutant protein of porcine pancreas PLAZ in which Gly-30 was replaced by serine diminished dramatically the ability to bind to Ca2+ (BEKKERS et al., 1991). We have recently isolated a PLA Z named PLA 2-I from Trimeresurus gramineus (green habu snake) venom, and determined its sequence (ODA et al., 1991). PLAZ-I was characteristic of containing phenylalanine at the 28th position instead of commonly conserved tyrosine residue. Besides PLAZ-I, T. gramineus venom contained some other PLAZ isozymes (ODA et al., 1987, 1991). We have intended to isolate these isozymes which might be useful for structure-function studies. In the work reported here, we isolated one of isozymes, named PLA Z -II, and determined its amino acid sequence. PLAZ-II contained aspargine instead of aspartic acid for PLA2-I at the 58th position that has been assumed to a lipid recognition site (DuKSTRA et al., 1981). A great difference was found between them in contractile activity in guinea-pig ileum (GPI), although other properties have appeared to be similar. MATERIALS AND METHODS Materials Achromobacter protease I [EC 3.4.21 .50] and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine were purchased from Wako Pure Chemical Industries (Osaka, Japan) . Clostripain [EC 3.4 .22.8] was obtained from Funakoshi (Tokyo, Japan) . The chemicals used for gas-phase sequencing were supplied by Applied Biosystems Japan (Tokyo). All other chemicals were of the best grade available. Purification A fraction containing PLA2s obtained after DEAE-cellulose column chromatography (ODA et al., 1987, 1991) was subjected to high-performance liquid chromatography (HPLQ under the conditions described in the legend for Fig. 1 . Polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDSPAGE) was carried out according to the method of LAmaru (1970) . Polyacrylamide isoelectric focusing (IEF) was conducted in the range of pH 2.5-5 .5 with Pharmalyte (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the instruction manual for a miniIEF kit (Bio-Rad Japan, Tokyo) . Determination of phospholipase As activity The PLA2 activity was routinely determined using an egg-yolk emulsion as a substrate on a Radiometer RTS5 titration assembly (pH 8.0 and 37°C) as described by IstfleuRU et al. (1980). Enzymatically released fatty acids were tîtrated with 10 mM NaOH, and the unit of enzyme activity was defined in terms of the uptake of alkali in pmole/min. Specific activity was expressed as units per mg of protein. Effect of calcium ion on phospholipase A2 activity Ten microlitres of aqueous solution of PLA2 (0 .2 mg/ml) was added to a buffer solution (50mM Tris-HC1, pH 8.5) (2.5 ml) containing 1,2-dilauroyl-sn-glycero-3-phosphoryl choline (3 .0 mM) and Triton X-100 (20 mM). CaCl 2 was then added to prepare the solutions with various concentrations (0, 0.3, 0.8, 1.6, 3.3 and 6.4 mM) of
Trimeresurus gramineus PLA2 Isozyme
1333
Cal* . Liberated lauric acid was titrated with lOmM NaOH . The dissociation constants for binding of Cat* to PLA2 isozymes were calculated by Lineweaver-Burk plot (L1NEwEAvER and BURK, 1934) . Amino acid analysis Proteins and peptides were hydrolysed with 5 .7 M HC I at IIO'C for 24 hr in evacuated and sealed tubes (Pierce, Rockford, IL, U.S .A .) . Amino acid analyses were carried out on a Hitachi 835 amino acid analyser by the method of SPACKMAN et al. (1958) . S-Carboxamidomethylation of phospholipase A= The protein (500 ug) was reduced by 4 mM dithiothereitol in 0 .4 M NH4HCO 3 (1001íl) containing 8 M urea. According to the method of STONE et al. (1989), the reaction mixture was treated with 100mM iodoacetamide (101íl) to convert freshly generated cysteine residues into S-carboxamidomethyl (CAM)-cysteine. After purification on HPLC, CAM-PLA2-1 was employed as starting material for the sequence study. Enzymatic cleavages CAM-PLA2-II (1401íg) was digested with Achromobacter protease I (I% by weight) in 0.2 M NH4 HCO3 (pH 8 .0) containing 4 M urea, 1 .9 mM dithiothreitol and 4 .1 mM iodoacetamide at 37°C for l l hr as described (STONE et al., 1989). The digest was fractionated by HPLC. For Arg-X cleavage, CAM-PLA2-II (1001íg) was digested with clostripain (2% by weight) in 0.05 M Tris-HC1 (pH 7 .6) containing 4 M urea and 2% 2mercaptoethanol at 37°C for 4 hr. The digest was fractionated by HPLC . Sequence analysis The amino acid sequence of the native protein and the enzymatically cleaved fragments of CAM-PLA2-II were analysed on an Applied 11iosystems 470A gas-phase sequencer equipped with a model 120A phenylthiohydantoin (PTH) analyser for the on-line detection of PTH-amino acids. Designation of peptides All peptides were designated by letters and numbers. Letters indicate the type of cleavages: K, Achromobacter protease I ; and R, clostripain . Peptide fragments obtained by HPLC were numbered by Arabic numerals in the order of their elution. Estimatlon of secondary structures The secondary structure compositions were analysed on the basis of the primary structure according to the method of CHaU FAsmAN and FAsmAN (1978) . The values of < P, > , < P p > and < P, > were calculated for every tetrapeptide span starting from the N-terminus . The hydropathic indexes were calculated according to the method of KYTE and Doot.n-rtE (1982). The spans of nine consecutive residues were analysed to depict a hydropathic profile . Guineapig ileum contraction assay The GPI assay was carried out essentially as described previously (MATSUMaro et al., 1991). From male guinea-pigs (350-450 g) the ileum was taken out rapidly to remove longitudinal strips . The strip (1 .0-1 .5 cm) was hung in a 5 ml organ bath containing Krebs-Ringer bicarbonate buffer (pH 7 .4) gassed with the 95% 0 2/5% C02 mixture at 37°C. The enzyme solution was injected cumulatively to the reaction bath at the concentration range of l0- "-10ßM . The results of each assay were expressed as percentages for the maximal contraction obtained with IOuM carbachol. RESULTS
A fraction containing PLA2 obtained from DEAE-column chromatography (ODA et al ., 1987) gave three peaks on HPLC (ODA et al., 1991). In the previous study a protein named PLA2-I was isolated from the first peak and its sequence was determined (ODA et al., 1991). An enzyme from the third peak was denoted as PLA2-II, because the second peak was less intense. When another pool of T. gramineus venom was purified in the present
1334
T. FUKAGAWA et al.
65
m 40 0
10 a Q
O
10
20
30
Retention time (min) FIG. 1 . ELUTION PROFILE ON HPLC OF PROTEINS FROM DEAF .-ceLLULOSE . TSK gel ODS-120T column (0.46 x 25 cm). Solvent system : 0.1% trifluoroacetic acid (A)-acetonitrile containing 20% A (B) . Elution was done with a linear concentration gradient of B (40-65%) for 30 min at a flow rate of 1 ml/min .
work, the second peak became about twice intense when compared with the former venom and exhibited a lypolytic activity similarly as the first and third peaks. The second peak gave a single protein band on SDS-PAGE (data not shown) . Consequently, this second peak was designated as PLAZ-II and the third peak was renamed as PLAZIII (Fig. 1.). In the present work, PLAZII from the second peak was sequenced and characterized . The mol. wt of PLAZII was estimated to be approximately 14,000 on SDS-PAGE . From its mobility relative to those of the pl-marker proteins, its isoelectric point was determined to be 4.5 (data not shown). The specific activity of PLAZII for egg-yolk emulsion was calculated to be 1400 units/mg, which was about 73% of that of PLAZI. The amino acid composition of PLAZ-II, which was calculated in accord with its mol. wt of 14,000, was in good agreement with that from the sequence data (data not shown). Figure 2 summarizes the strategy employed to establish the complete amino acid sequence of PLAZ-II. The N-terminal sequence analysis of native PLA Z-II identified the first 19 residues. The digest of CAM-PLAZ-II with Achromobacter protease I was fractionated by HPLC (Fig. 3a). The sequence determinations were conducted for almost all the peptides purified. The N-terminal sequence of K15 was identical to that of the native protein (Fig. 2). Successive sequence analyses of K15, K6, K1, K11, K4, K7, K3 and K8 covered almost the whole amino acid residues except for residues 22-30. The peptides necessary for overlaps were prepared by digesting CAM-protein with clostripain. Figure 3b shows an HPLC fractionation profile of the digest of CAM-PLAZII with clostripain. The peptides indicated in Fig. 2 were sequenced. The sequence of peptide R1 covered the entire region of K3 and K8 and the C-terminal region of K7. The sequence determination of R2 established the connections between peptides K 11 and K4 and between K4 and K7. The sequence of R4 followed by R3 covered the entire regions of K6
Trlmeresurus gramineus PLA= Isozyme
Pt t}1I
1335
a 10 la m Asa Lau Leu Glu Pha Glu Am met Ile Arg Asn val Ala Gly Arg sur Gly il* Trp Trp Tyr Bar xls- -. -. -. -. -. -. - -. -. -. -. - - -. A6
40 m 70 ]6 45 Asp Tyr Gly Cya iyr Cya Gly Lys Gly Gly Ris Gly Arg Pro Gln Asp Ala Bar Amp Arg Cys Cys Phe Val His
as as 60 70 50 Asp Cys Cys Tyr Gly Lys Val Asn Gly Cys Asa Pro Lys Lys Ala Val Tyr Il* iyr tar Leu Glu Aan Gly lup t=- xl
-+
---
a3
Kil ----------
73 so as go as Ils Val Cys Gly Gly Asp Asp Pro Cys Arq Lys Glu Val Cys Glu Cys Asp Lys Ala Ala Ala Ile Cya Phe Arg rC1 ' bx7 -s -i -i -s -s -s -i ~ -~ -i -i a--i -s -s -s --r -i -i -s -i -i --~ -s -s ~ R2
100 105 110 na 120 Aap Aan Lys Asp Thr Tyr Aap Asn Lys Tyr Trp Asn Ila Pro Bar Glu Kun Cys Gln Glu Glu Bar Glu Pro Cys
FIG . 2 . AMINO ACID sEQuENcE OF PLA,-II . The arrows (+s) indicate the amino acid residues identified by automated Edman degradation of the native protein . The arrows (-r) represent the amino acid residues identified for the fragments.
Rat~ time FIG . 3 .
HPLC
(min)
Rete~ time
FRACTIONATION PROMLP3 OF THE DIGEST OF CAM-PLA,-II PROTEASE I (a) AND CLOSPRIPAIN (b) .
(min)
wrrrl Acromobacter
TSK gel ODS-120T column (0 .46 x 25 cm) . Solvent system : 0.06% trifluroacetic acid (A)-0.052% trifluroacetic acid, 80% aeetonitrile (B) (STONE et al., 1989) . Elution was done with a linear gradient of B (0-70%) for 110 min at a flow rate of 1 ml/min.
133 6
T . FUKAGAWA et al.
and K1, N-terminal portion of K11, and the intermediate portion of peptide K15 (Fig. 2). R4 covered the residues 22-30. R6 was identical to the N-terminal moiety of K 15. Thus, the connections of all peptides from PLAZ-II were confirmed and the positions of 122 amino acid residues have been determined as shown in Fig . 2. Its mol . wt was calculated to be 13,793 . The sequence similarity between PLAZ I and PLA ZII was 79% . Their sequence differences were found for 26 amino acid residues and concentrated in the three major regions: i.e. the N-terminal (1-33), intermediate (58-72), and C-terminal (109-113) . The region that is assumed to construct a part of the active site (DUKSTRA et al., 1981), namely residues 34-57, was completely conserved between these isozymes . The positions of all 14 half-cystine residues were identical. For mixed micelles of 1,2-dilauroyl-sn-glycero-3-phosphorylcholine and Triton-100, PLAZ-1 and PLAZII showed enhanced activity with increase of CaZ+ -concentration in a hyperbolic manner . The estimated dissociation constants for the binding of Ca Z+ were essentially comparable between PLAZI and PLAZII: 5.2 x 10' M for the former and 3.7 x 10' M for the latter . These values are almost comparable to that of T. flavoviridis PLAZ (OHNo et al., 1984). The secondary structure components of PLA ZII, < PQ > , < Pfl > , and < P, > were very similar to those of PLA Z-II and also of Crotalus atrox PLAZ, whose tertiary structure had been established (BRUME et al., 1985; RENETSEDER et al., 1985) (Fig. 4). The overall profiles of hydropathic indexes were also essentially similar between PLAZ-I and PLAZ-II . These observations suggested that the tertiary structure of PLAZ-II might resemble those of PLA Z-1 and Crotalus atrox PLAZ. When PLAZI and PLAZII were administered to GPI at the concentration range of 10'-10'M, contraction occurred in a concentration-dependent manner (Fig. 5). It is evident that from the dose-response curves that PLAZ-II is approximately 100 times more active than PLAZ-1 at the lower concentration range (10'"-l0' M) and about 10 times at the higher concentration range (10-10M). The results indicate that there is a distinct difference between PLA ZI and PLA Z-II in terms of contractile activity for GPI . DISCUSSION
The amino acid sequence of PLA Z-II from T. gramineus venom has been determined (Fig. 2). The peptide fragments obtained by Achromobacter protease I covered almost the entire sequence. Clostripain provided the peptides which served for covering a portion which remained unidentified and for obtaining overlaps between the major peptides by Achromobacter protease 1. PLAZII consisted of 122 amino acid residues similarly to PLAZI, and His-47, Asp-48, and Asp-98 essential for PLAZ activity were conserved . PLAZII was thus judged to be classified into Group II (HEINRncSON et al., 1977) . One of the most prominent sequence differences between PLAZI and PLAZ-II exists in their N-terminal portion . It is notable that the amino acid sequence of PLAZ-II at this portion is very similar to that of taipoxin a (ß,-bungarotoxin), a Group I PLAZ from Bungarus multicinctus (KoNDo et al., 1978) (Fig. 6). Agkistrodotoxin, a PLAZ from Agkistrodon hallys pallas, also has a similar N-terminal sequence (KONDO et al., 1989) . Although the sequence homology among these PLAZs is not high, their similar N-terminal portions may share a common functional significance. Group II PLAZs have been classified further into two sub-groups, i.e. Asp-49-PLAZs and Lys-49-PLAZs. More than 60 kinds of Asp-49-PLAZs have been sequenced, whereas only
Trimeresurua grmnbreus PLAZ Isozyme
la
1337
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1
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.
05
4
Lo
as
3
20
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69
89
100
Residue number
Fio. 4. Am oF rnE PARA mE renes < P > < Pp > , Arm
Fox PLAZ-I (--)
120 AND
PLAZ-II
T.
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et al.
0 0
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0.
-12
FIG .
5.
-11 -10 -9 -8 -7 -6 Log[Protein concentration (M))
2
-5
DosE-mpoNsE CURVES of PLA -1 (0-0) AND PLA2711 CONTRACTU.E ACl'IVrrY IN GUINEA-PIO ILEUM .
(0
0)
FOR
1 2 3 4 5 6 7 8 9
10 20 30 40 50 60 70 80 90 100 RISAFEQlIRllVAG-RSGIIMYSDYGCYCGRGGSGRPQDASDRCCPVRDCCY--G-RVäW -----RPRIUVYIYSLE--NG-DIVCGG-DDPCRREVCE HLAQDETLINRVAG-RSGVNYYGSYGCrCGAGGOGRPODASDRCCrVROCCY--G-RVIIGC ----- DPIQfDPYTYSEE--NG-AIVCGG-DDPCRREICE GLNQFENNIIRVVRR-SGILSYSAYGCYCGNGGRGRPRDATDRCCrVHDCCY--G-RVTGC -----NPRLGEYTYSWQG-N---IVCEG-DGPC-REVCE BLVQLIHOIIFQETGRE-AARRYGLYGCRCGVGRRf'RPRDATDSCCYVBRCCYR---RVTGC ----- DPRKDSYSYSNR--NR-AIVCGERNPPCLRQVCE BLVOLWEMIFQETGRE-AARIFYGLYGCNCGVGRRGRPKDATDSCCYVBI000YR ---RVTGC -----NPRND8YSYBWR--NR-AIVCGERNPPCLRQVCE BLVQFFTLIIIRIAG-RSGLLNYSAYGCYCGWGGBGLPODATDRCCFVBDCCY--G-RATDC -----NPRTVSYTYBEE--11G-EIICGG-DDPCGTQICE S7MEYSIRRIITGR-SGNLIRSAYGCYCGNGGQGRPRDATDRCCFVSDCCY--G-RVTGC -----NPRNDIYTYSVE--NG-AIICGG-NTPCRRQICE NLINFNFNIRYTIPCERTIIGETADYGCYCGAGGBGRPIDALDRCCYVSDNCYGDAERRRRC -----NPRTSQYSYRLT ---RTTIICYGAAGGCRI-VCE ALNOFEWIRCRIPBSEPLLDFNNYGCYCGLGGSGTPVDDLDR000THDNCYRQARRLDSCRVLVDNPYTNNYSYS-C-SNNE-ITCSSENNACEAFICN
1 2 3 4 5 6 7 8 9
120 130 140 110 CDRDAAICrRONR-DTYD-N--RYNFFPARNCQ-EESEPC CDRAAAICFRDIM-DTYD-N--RYNNIPBENCQ-EESEPC CDRAAAICFRDNL-DTYDRN--RYNRYPASNCQ-EDSEPC CDRAVAICLRENL-GTYNR--- RYTIYPRPFCRR - -ADTC CDRAVAICLRENL-GTYNR--- RYTIYPRPFCRR - -ADTC CDRAARICFRDNI-PSYD-NR-YNLFPPRD-C-REEPEPC CDRAAAICFRDNL-LTYDS-RTYNRY-PRN-CTIGIESEPC CDRTAALCFG08--D-YIE-EER-QIDTARrM CDRNAAICF--S-RVPYIIK-EBR-NM-RRNC
T . gramineus II T . gramineos I T . llavoviridis Asp-49 T . flavovirldis Basic protein I T . flavoviridis Basic protein II C . atrox A . piscivores piscivores Asp-49 B . multicinctue bovine pancreas
Ref . 1 2 3 4 5 6 7 8 9
T. grami»ew, T. flavoviridis, Crotalus atrox, A . piscivores piscivores . Bungarus multicinctus AND BovINE PANCREAS. Reference: (1) present work ; (2) ODA et al. (1991) ; (3) TANAYA et al. (1986) and ODA et al. (1990); (4) YOsmzu1N1 et al. (1989); (5) Llu et at. (1990); (6) RANDOLPH et al. (1980); (7) KoNDo et al. (1978); (8) KoNDo et al. (1989) ; (9) FL EER et al. (1978) . Flo .
6.
TI1g AuGNED AMINo Acm sEQuENcEs oF PLA2s FRom
five Lys-49-PLA 2s are known (MARAGANoRE et al., 1984; MARAGANoRE and HEINRIKSON, 1986 ; Yosmzumi et al ., 1990; Liu et al., 1990; FRANCIS et al., 1991). The Lys-49-PLA2 family contains aspargine at the 28th position, while the Asp-49-PLA2 family has tyrosine at this position . The a-carbonyl group of the amino acid residue at this position is one of the constituents of Cat+ binding loop. Although the residue at the 28th position of PLA2I from T. gramineus is phenylalanine (ODA et al., 1991), PLA2-II was found to contain tyrosine there like most Asp-49-PLA2s. In the previous study (ODA et al., 1991), it was shown that replacement of Tyr-28 by phenylalanine in PLA2-I gives rise to no large effect on the binding of Ca2+ . In fact, this phenomenon was also confirmed by the fact that there is no significant difference in the dissociation constant for the binding of Ca2+ between PLA2-I and PLA2-II. Snake venom PLA2s have been known to show a wide variety of pharmacological activities . For example, Asp-49-PLA2 from T. favoviridis venom (TANAKA et al., 1986 ; ODA et al., 1990) was reported to have an activity to contract the longitudinal muscle
Trimeresurus gramineus PLAT Isozyme
133 9
preparation of GPI (MATSUMOTO et al., 1991). This effect is caused by prostaglandins derived from arachidonic acid, which is released by the enzymatic action of PLA2 on biomembranes . Recently, we found that basic protein II from T. flavoviridis, a Lys-49-PLA Z with low lipolytic activity (Liu et al ., 1990), elicited a strong contractile activity for GPI which was comparable with that induced by Asp-49-PLA, (SHIMOHIGASHI et al., unpublished) . However, another Lys-49-PLA Z isozyme named basic protein I was almost completely inactive in GPI. The difference between basic proteins I and II is only one amino acid at the 67th position in the aligned sequence (Fig. 6). Basic protein I has aspartic acid, while basic protein II aspargine at this position . Interestingly, the same residual replacement was found to occur between PLAZ I and PLAZ II. The amino acid residues at the 58th position, which corresponds to the 67th in the aligned sequence, are aspartic acid for PLA Z -1 and aspargine for PLA Z-II. As observed for a pair of basic proteins I and II, PLA ZII was considerably stronger than PLA2-I in contractile activity (Fig. 5). These observations indicate that the amino acid residue at the 58th (or 67th in the aligned sequence) position of PLA2s is very important for contractile activity in GPI, and PLA2 with Asp-58 is less effective than ones with Asn-58 . The region around position 58 has been assumed to be involved in a lipid-recognizing site (DIJKSTRA et al., 1981). A negative charge of Asp-58 might be unfavourable in the interaction of PLA2 with membrane lipids, resulting in a decrease in contractile activity for GPI. It is not so usual to find more than two isospecific PLAZs in a single source . In general, such isozymes induce similar pathological symptoms with variations in intensity (HARVEY and TAMIYA, 1980 ; ROWAN et al., 1985) and have high homology in structures among themselves (SCHMIDT and MIDDLEBROOK, 1989 ; NISHIDA et al., 1982; TAKASAKI et al ., 1988). Therefore, they might be very useful for studies of the structure-function relationships, and PLA2 isozymes from T. gramineus are definitely invaluable for such studies. Acknowledgements--We thank Prof. assistance in biological assays .
Hullo-o K.wuyA
and
DR Yuxto TAtuxo
of Fukuoka University for their
REFERENCES A. C . A . P. A., FRANKEN, P . A . ToxopFus, E., VERHI7J, H . M . and DE HAAS, G . H . (1991) The importance of glycine-30 for enzymatic activity of phospholipase A2 Biochim . biophys . Acts 1076, 374-378 . BRUME, S., BoLnv, J ., GEwmnt, D. and $IGLER, P . B . (1985) The refined crystal structure of dimeric phospholipase A2 at 2 .5 A. J. biol. Chem . 260, 9742-9749. CHou FAsuAN, P. Y . and FASMAN, G . D. (1978) Prediction of the secondary structure of proteins from their amino acid sequence . In: Advances in Enzymology, Vol. 47, pp . 45-148 (MaRsm, A., Ed .). New York: Wiley. DuesrRA, B . W., DRENTH, J . and KALR, K . H . (1981) Active site and catalytic mechanism of phospholipase A 2 . Nature 289, 604-606 . Fi.EER, E . A . M ., VERHEu, H . M . and DE HAAS, G . H . (1978) The primary structure of bovine pancreatic phospholipase A2 . Eur. J. Biochem. 82, 261-269 . MEErt, E. A . M ., VERHEu, H . M . and DE HAAS, G. H . (1981) Modification of carboxylate groups in bovine pancreatic phospholipase A2 . Eur . J. Blochem . 113, 283-288 . FRANcI% B ., GurmRREz, J . M ., LomoNTE, B. and KAtnR, I . I . (1991) Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49-phospholilipaw A2 . Archs Biochem. Biophys . 284, 352-359. HARvEY, A . L. and TAMiyA, N . (1980) Role of phospholipase A activity in the neuromuscular paralysis produced by some components isolated from the venom of the seasnake . Laticauda semifasciata . Toxicon 18, 65-69. HEINRIKSON, R . L ., KRUEGER, E . T. and KEUt, P. S . (1977) Amino acid sequence of phospholipaw A2-'a form the venom of C"otalus adamanteus. A new classification of the phospholipases A2 based upon structural determinants . J. biol. Chem. 252, 4913-6921 . ISHIMARu, K ., KniARA, H . and OHNo, M. (1980) Purification and properties of phospholipase A 2 from venom of Trimeresurm flavovirldis (habu snake). J. Blochem ., Tokyo 88, 443-451 . BEtc~
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KoNDo, K., NARrrA, K. and Lm, C. Y. (1978) Amino acid sequences of the two polypeptide chains in fit-bungarotoxin from the venom of Bungarus multicinctus . J. Biochem., Tokyo 83, 101-115 . KoNDo, K., ZHANG, J., Xu, K. and KAOAtuyAuA, H. (1989) . Amino acid sequence of a presynaptic neurotoxin, agkistrodotoxin, from the venom of Agkistrodon hallys pallas . J. Biochem., Tokyo 105, 196-203. KYTE, J. and DootsmE, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. mol. Biol. 137, 105-132. LAnumu, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680. LIND, P. and EAxEIt, D. (1980) Complete amino-acid sequence of a non-neurotoxic, non-enzymatic phosphofipase A2 homolog from the venom of the Australian tiger snake Notechis scutatus scutatus. Eur. J. Biochem. 111, 403-409. LINEWEAvER, H. and BuRK, D. (1934) The determination of enzyme dissociation constants. J. Am . them. Soc. 56, 658-666. Lau, S.Y., Yosmzuhn, K., ODA, N., OHNo, M ., ToKuNAGA, F., IWANAGA, S. and KmARA, H. (1990) Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trinteresurus flavovirldts venom. J. Blochem., Tokyo 107, 400-408. MARAGANORE, J. M. and HEINRDtSON, R. L. (1986) The lysine-49 phospholipase A2 from the venom of Agkistrodon píicivorus plscivorus. Relation of structure and function to other phospholipases A2. J. biol. Chem . 261,4797-4804. MARAGANORE, J. M., MERuTKA, G., CHO, W., WELcms, W., KEZDY, F. J. and HnNRtKsoN, R. L. (1984) A new class of phospholipase A= with lysine in place of aspartate 49 . Functional consequences for calcium and substrate binding. J. Not. Chem. 299, 13,839-13,843. MATsuetoTo, H., $iDIAOHIGAsm, Y., NONAKA, S., SArro, R., TAKANO, Y., KAAUYA, H. and OHNo, M. (1991) Contractile activity of Trimeresurusflavoviridis phospholipase A2 on guinea pig ileum and artery. Biochem. Int. 24, 181-186. MMEAN, M. L., ShuTH, J. B. and SILVER, M. J. (1981) Formation of lysophosphadidylcholine by human platelets in response to thrombin ; support for the phospholipase A2 pathway for the liberation of arachidonic acid . J. biol. Chem. 256, 1522-1524 . NtatmA, S., Kim, H. S. and TAnayA, N. (1982) Amino acid sequences of three phospholipases A2 I, III and IV from the venom of the sea snake Laticatala semifasclata. Biochem. J. 207, 589-594. ODA, N., SAKAI, H., SHEH, T.-C., NAKAmuiLA, H., SAKAIIO'rO, S., KniARA, H., CluNG, C.-C. and OHNo, M. (1987) Purification and characterization of phospholipase A2 from Trimeresurus gramineus venom. J. Biochem., Tokyo 102, 1441-1449. ODA, N., OGAWA, T., OHNo, M., SAsAIu, H., SAKAKI, Y. and KmARA, H. (1990) Cloning and sequence analysis of cDNA for Trimeresurus flavoviridis phospholipase A2, and consequent revision of the amino acid sequence . J. Biochem., Tokyo 106, 816-821. ODA, N., NAKAMuRA, H., SAKAStoTo, S., Liu, S.Y., KIHARA, H., GANG, C.-C. and OHNo, M. (1991) Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake) . Toxicon 29,157-166 . OHNo, M., HONDA, A., TANAKA, S., MoHw, N., SHmH, T.-C. and KIHARA, H. (1984) Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion. J. Biochem., Tokyo 96, 1183-1191 . RANDOLPH, A., SAKMAR, T. P. and HErNwKSON, R. L. (1980) Phospholipase A2: structure, function and evolution. In : Frontiers in Protein Chemistry, pp . 297-322 (Lm, T.Y., MAmYA, G. and YAsuNoeu, K. T., Eds). Amsterdam: Elsevier/North-Holland . RENEIswER, R., BRUNIE, S., DUKSrRA, B. W., DRENTH, J. and SIGIER, P. B. (1985) A comparison of the crystal structures of phosphilipase A2 from bovine pancreas and Crotalus atrox venom. J. biol. Chem . 260, 11,627-11,634. ROWAN, E. G., HARVEY, A. L. and TAMIYA, N. (1985) Effects of two from the Australian king brown snake (Pseudrchis australls) on isolated nerve-muscle preparations . Toxicon 23, 606. SctmDT, J. J. and MmDLtiBRODK, J. L., (1989) Purification,, sequencing and characterization of pseudexin phospholipase A2 from Pseudechis porphyriacus (Australian red-bellied black snake). Toxicon 27, 805-818. SPAcKMAN, D. H., STEIN, W. H. and MOORE, S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt . Chem . 30, 1190-1206. STONE, K. L., LoPREsTi, M. B., CRAwFoND, J. M., DEANOEms, R. and Wu-LIAws, K. R. (1989) Enzymatic digestion of proteins and HPLC peptide isolation. In: A PracticalGuide to Protein and Peptide Purification for Microsequencing, pp . 42-47 (MATsmAmA, P. T., Ed.). New York: Academic Press. TAKAsAm , C., KmuRA, S., KOKuBuN, Y. and TAuryA, N. (1988) Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda cohibrina, from the Solomon islands . Biochem. J. 253, 869-875. TANAKA, S., MoFm, N., KniARA, H. and OHNO, M. (1986) Amino acid sequence of Trimeresurm flavovirldis phospholipase A2. J. Biochem., Tokyo 99, 281-289. VADAs, P. and PRuzANsiu, W. (1986) Biology of disease: role of secretory phospholipase A2 in the pathobiology of disease. Lab. Invest 55, 391-404.
Trimeresurus gramineus PLAZ Isozyme
134 1
VAN DEENAN, L . L . M . (1965) Phospholipids and biomembranes. In: Progress in Chemistry of Fats and Other Lipids, Vol . 8 (HOLMAN, R. T ., F.d.) Oxford: Pergamon Press. VERHEU, H. M., VOLWERK, J. J., JANSEN, E. H. J. M., PuYK, W. C., DuKSrRA, B. W., DR~, J. and DE HAAS, G. H. (1980) Methylation of histidine-48 in pancreatic phospholipase AZ . Rok of histidine and calcium ion in
the catalytic mechanism . Biochemistry 19, 743-750. Yossnzum, K., LIu, S.Y., MIYATA, T., SAITA, S., OHM, M., IWANAGA, S. and KmARA, H. (1990) Purification and amino acid sequence of basic protein I, a lysine-49-phospholipase Az with low activity, from the venom of Trimeresurus Jlavoviridis (habu snake) . Toxicon 4$, 43-54.
Tackimr, Vol . 30, No. 11, pp. 1331-1341, 1992. Printed in Great Britain .
SEQUENCE DETERMINATION AND CHARACTERIZATION OF A PHOSPHOLIPASE A2 ISOZYME FROM TRIMERESURUS GRAMINEUS (GREEN HABU SNAKE) VENOM TOYOKA FUKAGAWA, l Hmosm MATSUMOTO,' YASUYUKI SHIMOHIGASHI,' TOMOHISA OGAWA,' NAOKO ODA,' CHUN-CHANG CHAN& and MOTONORI OHNOI
'Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812, Japan ; and 'Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan, R .O.C . (Received
26 May 1992 ;
accepted I 1 July
1992)
T . FUKAGAWA, H. MATSUMOTO, Y . SHIMOHIGASHI, T . OGAWA, N . ODA, C.-C . CHANG and M . OHNo. Sequence determination and characterization of a
phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom. Toxicon 30, 1331-1341, 1992.-In addition to phospholipase A2-I (PLA 2-,) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA,-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-,, PLA2II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLAA and PLA2-II for Ca 2+ . Secondary structure compositions of PLA2I, were similar to those of PLAA and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-,, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLAA and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLAA and Asn for PLA2-II). INTRODUCTION
A2 (PLA 2s) [EC 3.1 .1.4] catalyse the hydrolysis of the 2-acyl ester bond in 3-sn-phosphoglycerides. They play a central role in metabolism of phospholipid (vAN DEENAN, 1965) and function as a trigger of the arachidonate cascade (McKEAN et al., 1981 ; VADAs and PRUZANSKI, 1986). For stabilization of a conformation essential for the PHOSPHOLIPASFS
" Author to whom correspondence should be addressed . 1331
1332
T. FUKAGAWA et al.
catalytic function, PLAZs require Ca t+ and possess a binding site for this divalent ion (VERHEu et al., 1980). The fl-carboxyl group of aspartic acid-48 is one of key constituents of the site, and in addition to this group the a-carbonyl groups of Tyr-28, Gly-30, and lily-32 form a part of Ca' binding loop (FLEER et al., 1981). A single amino acid replacement often causes a drastic change in function and therefore offers a good chance to develop a novel study of the structure-function relationships. For instance, a PLA 2 from Notechis scutatus scutatus (Australian tiger snake) venom contains Ser-30 (LIND and EAKER, 1980). Inactivity of this PLAZ was substantiated by the fact that a mutant protein of porcine pancreas PLAZ in which Gly-30 was replaced by serine diminished dramatically the ability to bind to Ca2+ (BEKKERS et al., 1991). We have recently isolated a PLA Z named PLA 2-I from Trimeresurus gramineus (green habu snake) venom, and determined its sequence (ODA et al., 1991). PLAZ-I was characteristic of containing phenylalanine at the 28th position instead of commonly conserved tyrosine residue. Besides PLAZ-I, T. gramineus venom contained some other PLAZ isozymes (ODA et al., 1987, 1991). We have intended to isolate these isozymes which might be useful for structure-function studies. In the work reported here, we isolated one of isozymes, named PLA Z -II, and determined its amino acid sequence. PLAZ-II contained aspargine instead of aspartic acid for PLA2-I at the 58th position that has been assumed to a lipid recognition site (DuKSTRA et al., 1981). A great difference was found between them in contractile activity in guinea-pig ileum (GPI), although other properties have appeared to be similar. MATERIALS AND METHODS Materials Achromobacter protease I [EC 3.4.21 .50] and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine were purchased from Wako Pure Chemical Industries (Osaka, Japan) . Clostripain [EC 3.4 .22.8] was obtained from Funakoshi (Tokyo, Japan) . The chemicals used for gas-phase sequencing were supplied by Applied Biosystems Japan (Tokyo). All other chemicals were of the best grade available. Purification A fraction containing PLA2s obtained after DEAE-cellulose column chromatography (ODA et al., 1987, 1991) was subjected to high-performance liquid chromatography (HPLQ under the conditions described in the legend for Fig. 1 . Polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDSPAGE) was carried out according to the method of LAmaru (1970) . Polyacrylamide isoelectric focusing (IEF) was conducted in the range of pH 2.5-5 .5 with Pharmalyte (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the instruction manual for a miniIEF kit (Bio-Rad Japan, Tokyo) . Determination of phospholipase As activity The PLA2 activity was routinely determined using an egg-yolk emulsion as a substrate on a Radiometer RTS5 titration assembly (pH 8.0 and 37°C) as described by IstfleuRU et al. (1980). Enzymatically released fatty acids were tîtrated with 10 mM NaOH, and the unit of enzyme activity was defined in terms of the uptake of alkali in pmole/min. Specific activity was expressed as units per mg of protein. Effect of calcium ion on phospholipase A2 activity Ten microlitres of aqueous solution of PLA2 (0 .2 mg/ml) was added to a buffer solution (50mM Tris-HC1, pH 8.5) (2.5 ml) containing 1,2-dilauroyl-sn-glycero-3-phosphoryl choline (3 .0 mM) and Triton X-100 (20 mM). CaCl 2 was then added to prepare the solutions with various concentrations (0, 0.3, 0.8, 1.6, 3.3 and 6.4 mM) of
Trimeresurus gramineus PLA2 Isozyme
1333
Cal* . Liberated lauric acid was titrated with lOmM NaOH . The dissociation constants for binding of Cat* to PLA2 isozymes were calculated by Lineweaver-Burk plot (L1NEwEAvER and BURK, 1934) . Amino acid analysis Proteins and peptides were hydrolysed with 5 .7 M HC I at IIO'C for 24 hr in evacuated and sealed tubes (Pierce, Rockford, IL, U.S .A .) . Amino acid analyses were carried out on a Hitachi 835 amino acid analyser by the method of SPACKMAN et al. (1958) . S-Carboxamidomethylation of phospholipase A= The protein (500 ug) was reduced by 4 mM dithiothereitol in 0 .4 M NH4HCO 3 (1001íl) containing 8 M urea. According to the method of STONE et al. (1989), the reaction mixture was treated with 100mM iodoacetamide (101íl) to convert freshly generated cysteine residues into S-carboxamidomethyl (CAM)-cysteine. After purification on HPLC, CAM-PLA2-1 was employed as starting material for the sequence study. Enzymatic cleavages CAM-PLA2-II (1401íg) was digested with Achromobacter protease I (I% by weight) in 0.2 M NH4 HCO3 (pH 8 .0) containing 4 M urea, 1 .9 mM dithiothreitol and 4 .1 mM iodoacetamide at 37°C for l l hr as described (STONE et al., 1989). The digest was fractionated by HPLC. For Arg-X cleavage, CAM-PLA2-II (1001íg) was digested with clostripain (2% by weight) in 0.05 M Tris-HC1 (pH 7 .6) containing 4 M urea and 2% 2mercaptoethanol at 37°C for 4 hr. The digest was fractionated by HPLC . Sequence analysis The amino acid sequence of the native protein and the enzymatically cleaved fragments of CAM-PLA2-II were analysed on an Applied 11iosystems 470A gas-phase sequencer equipped with a model 120A phenylthiohydantoin (PTH) analyser for the on-line detection of PTH-amino acids. Designation of peptides All peptides were designated by letters and numbers. Letters indicate the type of cleavages: K, Achromobacter protease I ; and R, clostripain . Peptide fragments obtained by HPLC were numbered by Arabic numerals in the order of their elution. Estimatlon of secondary structures The secondary structure compositions were analysed on the basis of the primary structure according to the method of CHaU FAsmAN and FAsmAN (1978) . The values of < P, > , < P p > and < P, > were calculated for every tetrapeptide span starting from the N-terminus . The hydropathic indexes were calculated according to the method of KYTE and Doot.n-rtE (1982). The spans of nine consecutive residues were analysed to depict a hydropathic profile . Guineapig ileum contraction assay The GPI assay was carried out essentially as described previously (MATSUMaro et al., 1991). From male guinea-pigs (350-450 g) the ileum was taken out rapidly to remove longitudinal strips . The strip (1 .0-1 .5 cm) was hung in a 5 ml organ bath containing Krebs-Ringer bicarbonate buffer (pH 7 .4) gassed with the 95% 0 2/5% C02 mixture at 37°C. The enzyme solution was injected cumulatively to the reaction bath at the concentration range of l0- "-10ßM . The results of each assay were expressed as percentages for the maximal contraction obtained with IOuM carbachol. RESULTS
A fraction containing PLA2 obtained from DEAE-column chromatography (ODA et al ., 1987) gave three peaks on HPLC (ODA et al., 1991). In the previous study a protein named PLA2-I was isolated from the first peak and its sequence was determined (ODA et al., 1991). An enzyme from the third peak was denoted as PLA2-II, because the second peak was less intense. When another pool of T. gramineus venom was purified in the present
1334
T. FUKAGAWA et al.
65
m 40 0
10 a Q
O
10
20
30
Retention time (min) FIG. 1 . ELUTION PROFILE ON HPLC OF PROTEINS FROM DEAF .-ceLLULOSE . TSK gel ODS-120T column (0.46 x 25 cm). Solvent system : 0.1% trifluoroacetic acid (A)-acetonitrile containing 20% A (B) . Elution was done with a linear concentration gradient of B (40-65%) for 30 min at a flow rate of 1 ml/min .
work, the second peak became about twice intense when compared with the former venom and exhibited a lypolytic activity similarly as the first and third peaks. The second peak gave a single protein band on SDS-PAGE (data not shown) . Consequently, this second peak was designated as PLAZ-II and the third peak was renamed as PLAZIII (Fig. 1.). In the present work, PLAZII from the second peak was sequenced and characterized . The mol. wt of PLAZII was estimated to be approximately 14,000 on SDS-PAGE . From its mobility relative to those of the pl-marker proteins, its isoelectric point was determined to be 4.5 (data not shown). The specific activity of PLAZII for egg-yolk emulsion was calculated to be 1400 units/mg, which was about 73% of that of PLAZI. The amino acid composition of PLAZ-II, which was calculated in accord with its mol. wt of 14,000, was in good agreement with that from the sequence data (data not shown). Figure 2 summarizes the strategy employed to establish the complete amino acid sequence of PLAZ-II. The N-terminal sequence analysis of native PLA Z-II identified the first 19 residues. The digest of CAM-PLAZ-II with Achromobacter protease I was fractionated by HPLC (Fig. 3a). The sequence determinations were conducted for almost all the peptides purified. The N-terminal sequence of K15 was identical to that of the native protein (Fig. 2). Successive sequence analyses of K15, K6, K1, K11, K4, K7, K3 and K8 covered almost the whole amino acid residues except for residues 22-30. The peptides necessary for overlaps were prepared by digesting CAM-protein with clostripain. Figure 3b shows an HPLC fractionation profile of the digest of CAM-PLAZII with clostripain. The peptides indicated in Fig. 2 were sequenced. The sequence of peptide R1 covered the entire region of K3 and K8 and the C-terminal region of K7. The sequence determination of R2 established the connections between peptides K 11 and K4 and between K4 and K7. The sequence of R4 followed by R3 covered the entire regions of K6
Trlmeresurus gramineus PLA= Isozyme
Pt t}1I
1335
a 10 la m Asa Lau Leu Glu Pha Glu Am met Ile Arg Asn val Ala Gly Arg sur Gly il* Trp Trp Tyr Bar xls- -. -. -. -. -. -. - -. -. -. -. - - -. A6
40 m 70 ]6 45 Asp Tyr Gly Cya iyr Cya Gly Lys Gly Gly Ris Gly Arg Pro Gln Asp Ala Bar Amp Arg Cys Cys Phe Val His
as as 60 70 50 Asp Cys Cys Tyr Gly Lys Val Asn Gly Cys Asa Pro Lys Lys Ala Val Tyr Il* iyr tar Leu Glu Aan Gly lup t=- xl
-+
---
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73 so as go as Ils Val Cys Gly Gly Asp Asp Pro Cys Arq Lys Glu Val Cys Glu Cys Asp Lys Ala Ala Ala Ile Cya Phe Arg rC1 ' bx7 -s -i -i -s -s -s -i ~ -~ -i -i a--i -s -s -s --r -i -i -s -i -i --~ -s -s ~ R2
100 105 110 na 120 Aap Aan Lys Asp Thr Tyr Aap Asn Lys Tyr Trp Asn Ila Pro Bar Glu Kun Cys Gln Glu Glu Bar Glu Pro Cys
FIG . 2 . AMINO ACID sEQuENcE OF PLA,-II . The arrows (+s) indicate the amino acid residues identified by automated Edman degradation of the native protein . The arrows (-r) represent the amino acid residues identified for the fragments.
Rat~ time FIG . 3 .
HPLC
(min)
Rete~ time
FRACTIONATION PROMLP3 OF THE DIGEST OF CAM-PLA,-II PROTEASE I (a) AND CLOSPRIPAIN (b) .
(min)
wrrrl Acromobacter
TSK gel ODS-120T column (0 .46 x 25 cm) . Solvent system : 0.06% trifluroacetic acid (A)-0.052% trifluroacetic acid, 80% aeetonitrile (B) (STONE et al., 1989) . Elution was done with a linear gradient of B (0-70%) for 110 min at a flow rate of 1 ml/min.
133 6
T . FUKAGAWA et al.
and K1, N-terminal portion of K11, and the intermediate portion of peptide K15 (Fig. 2). R4 covered the residues 22-30. R6 was identical to the N-terminal moiety of K 15. Thus, the connections of all peptides from PLAZ-II were confirmed and the positions of 122 amino acid residues have been determined as shown in Fig . 2. Its mol . wt was calculated to be 13,793 . The sequence similarity between PLAZ I and PLA ZII was 79% . Their sequence differences were found for 26 amino acid residues and concentrated in the three major regions: i.e. the N-terminal (1-33), intermediate (58-72), and C-terminal (109-113) . The region that is assumed to construct a part of the active site (DUKSTRA et al., 1981), namely residues 34-57, was completely conserved between these isozymes . The positions of all 14 half-cystine residues were identical. For mixed micelles of 1,2-dilauroyl-sn-glycero-3-phosphorylcholine and Triton-100, PLAZ-1 and PLAZII showed enhanced activity with increase of CaZ+ -concentration in a hyperbolic manner . The estimated dissociation constants for the binding of Ca Z+ were essentially comparable between PLAZI and PLAZII: 5.2 x 10' M for the former and 3.7 x 10' M for the latter . These values are almost comparable to that of T. flavoviridis PLAZ (OHNo et al., 1984). The secondary structure components of PLA ZII, < PQ > , < Pfl > , and < P, > were very similar to those of PLA Z-II and also of Crotalus atrox PLAZ, whose tertiary structure had been established (BRUME et al., 1985; RENETSEDER et al., 1985) (Fig. 4). The overall profiles of hydropathic indexes were also essentially similar between PLAZ-I and PLAZ-II . These observations suggested that the tertiary structure of PLAZ-II might resemble those of PLA Z-1 and Crotalus atrox PLAZ. When PLAZI and PLAZII were administered to GPI at the concentration range of 10'-10'M, contraction occurred in a concentration-dependent manner (Fig. 5). It is evident that from the dose-response curves that PLAZ-II is approximately 100 times more active than PLAZ-1 at the lower concentration range (10'"-l0' M) and about 10 times at the higher concentration range (10-10M). The results indicate that there is a distinct difference between PLA ZI and PLA Z-II in terms of contractile activity for GPI . DISCUSSION
The amino acid sequence of PLA Z-II from T. gramineus venom has been determined (Fig. 2). The peptide fragments obtained by Achromobacter protease I covered almost the entire sequence. Clostripain provided the peptides which served for covering a portion which remained unidentified and for obtaining overlaps between the major peptides by Achromobacter protease 1. PLAZII consisted of 122 amino acid residues similarly to PLAZI, and His-47, Asp-48, and Asp-98 essential for PLAZ activity were conserved . PLAZII was thus judged to be classified into Group II (HEINRncSON et al., 1977) . One of the most prominent sequence differences between PLAZI and PLAZ-II exists in their N-terminal portion . It is notable that the amino acid sequence of PLAZ-II at this portion is very similar to that of taipoxin a (ß,-bungarotoxin), a Group I PLAZ from Bungarus multicinctus (KoNDo et al., 1978) (Fig. 6). Agkistrodotoxin, a PLAZ from Agkistrodon hallys pallas, also has a similar N-terminal sequence (KONDO et al., 1989) . Although the sequence homology among these PLAZs is not high, their similar N-terminal portions may share a common functional significance. Group II PLAZs have been classified further into two sub-groups, i.e. Asp-49-PLAZs and Lys-49-PLAZs. More than 60 kinds of Asp-49-PLAZs have been sequenced, whereas only
Trimeresurua grmnbreus PLAZ Isozyme
la
1337
,.
1
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.
05
4
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as
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69
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Residue number
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120 AND
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T.
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0 0
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-12
FIG .
5.
-11 -10 -9 -8 -7 -6 Log[Protein concentration (M))
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-5
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(0
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FOR
1 2 3 4 5 6 7 8 9
10 20 30 40 50 60 70 80 90 100 RISAFEQlIRllVAG-RSGIIMYSDYGCYCGRGGSGRPQDASDRCCPVRDCCY--G-RVäW -----RPRIUVYIYSLE--NG-DIVCGG-DDPCRREVCE HLAQDETLINRVAG-RSGVNYYGSYGCrCGAGGOGRPODASDRCCrVROCCY--G-RVIIGC ----- DPIQfDPYTYSEE--NG-AIVCGG-DDPCRREICE GLNQFENNIIRVVRR-SGILSYSAYGCYCGNGGRGRPRDATDRCCrVHDCCY--G-RVTGC -----NPRLGEYTYSWQG-N---IVCEG-DGPC-REVCE BLVQLIHOIIFQETGRE-AARRYGLYGCRCGVGRRf'RPRDATDSCCYVBRCCYR---RVTGC ----- DPRKDSYSYSNR--NR-AIVCGERNPPCLRQVCE BLVOLWEMIFQETGRE-AARIFYGLYGCNCGVGRRGRPKDATDSCCYVBI000YR ---RVTGC -----NPRND8YSYBWR--NR-AIVCGERNPPCLRQVCE BLVQFFTLIIIRIAG-RSGLLNYSAYGCYCGWGGBGLPODATDRCCFVBDCCY--G-RATDC -----NPRTVSYTYBEE--11G-EIICGG-DDPCGTQICE S7MEYSIRRIITGR-SGNLIRSAYGCYCGNGGQGRPRDATDRCCFVSDCCY--G-RVTGC -----NPRNDIYTYSVE--NG-AIICGG-NTPCRRQICE NLINFNFNIRYTIPCERTIIGETADYGCYCGAGGBGRPIDALDRCCYVSDNCYGDAERRRRC -----NPRTSQYSYRLT ---RTTIICYGAAGGCRI-VCE ALNOFEWIRCRIPBSEPLLDFNNYGCYCGLGGSGTPVDDLDR000THDNCYRQARRLDSCRVLVDNPYTNNYSYS-C-SNNE-ITCSSENNACEAFICN
1 2 3 4 5 6 7 8 9
120 130 140 110 CDRDAAICrRONR-DTYD-N--RYNFFPARNCQ-EESEPC CDRAAAICFRDIM-DTYD-N--RYNNIPBENCQ-EESEPC CDRAAAICFRDNL-DTYDRN--RYNRYPASNCQ-EDSEPC CDRAVAICLRENL-GTYNR--- RYTIYPRPFCRR - -ADTC CDRAVAICLRENL-GTYNR--- RYTIYPRPFCRR - -ADTC CDRAARICFRDNI-PSYD-NR-YNLFPPRD-C-REEPEPC CDRAAAICFRDNL-LTYDS-RTYNRY-PRN-CTIGIESEPC CDRTAALCFG08--D-YIE-EER-QIDTARrM CDRNAAICF--S-RVPYIIK-EBR-NM-RRNC
T . gramineus II T . gramineos I T . llavoviridis Asp-49 T . flavovirldis Basic protein I T . flavoviridis Basic protein II C . atrox A . piscivores piscivores Asp-49 B . multicinctue bovine pancreas
Ref . 1 2 3 4 5 6 7 8 9
T. grami»ew, T. flavoviridis, Crotalus atrox, A . piscivores piscivores . Bungarus multicinctus AND BovINE PANCREAS. Reference: (1) present work ; (2) ODA et al. (1991) ; (3) TANAYA et al. (1986) and ODA et al. (1990); (4) YOsmzu1N1 et al. (1989); (5) Llu et at. (1990); (6) RANDOLPH et al. (1980); (7) KoNDo et al. (1978); (8) KoNDo et al. (1989) ; (9) FL EER et al. (1978) . Flo .
6.
TI1g AuGNED AMINo Acm sEQuENcEs oF PLA2s FRom
five Lys-49-PLA 2s are known (MARAGANoRE et al., 1984; MARAGANoRE and HEINRIKSON, 1986 ; Yosmzumi et al ., 1990; Liu et al., 1990; FRANCIS et al., 1991). The Lys-49-PLA2 family contains aspargine at the 28th position, while the Asp-49-PLA2 family has tyrosine at this position . The a-carbonyl group of the amino acid residue at this position is one of the constituents of Cat+ binding loop. Although the residue at the 28th position of PLA2I from T. gramineus is phenylalanine (ODA et al., 1991), PLA2-II was found to contain tyrosine there like most Asp-49-PLA2s. In the previous study (ODA et al., 1991), it was shown that replacement of Tyr-28 by phenylalanine in PLA2-I gives rise to no large effect on the binding of Ca2+ . In fact, this phenomenon was also confirmed by the fact that there is no significant difference in the dissociation constant for the binding of Ca2+ between PLA2-I and PLA2-II. Snake venom PLA2s have been known to show a wide variety of pharmacological activities . For example, Asp-49-PLA2 from T. favoviridis venom (TANAKA et al., 1986 ; ODA et al., 1990) was reported to have an activity to contract the longitudinal muscle
Trimeresurus gramineus PLAT Isozyme
133 9
preparation of GPI (MATSUMOTO et al., 1991). This effect is caused by prostaglandins derived from arachidonic acid, which is released by the enzymatic action of PLA2 on biomembranes . Recently, we found that basic protein II from T. flavoviridis, a Lys-49-PLA Z with low lipolytic activity (Liu et al ., 1990), elicited a strong contractile activity for GPI which was comparable with that induced by Asp-49-PLA, (SHIMOHIGASHI et al., unpublished) . However, another Lys-49-PLA Z isozyme named basic protein I was almost completely inactive in GPI. The difference between basic proteins I and II is only one amino acid at the 67th position in the aligned sequence (Fig. 6). Basic protein I has aspartic acid, while basic protein II aspargine at this position . Interestingly, the same residual replacement was found to occur between PLAZ I and PLAZ II. The amino acid residues at the 58th position, which corresponds to the 67th in the aligned sequence, are aspartic acid for PLA Z -1 and aspargine for PLA Z-II. As observed for a pair of basic proteins I and II, PLA ZII was considerably stronger than PLA2-I in contractile activity (Fig. 5). These observations indicate that the amino acid residue at the 58th (or 67th in the aligned sequence) position of PLA2s is very important for contractile activity in GPI, and PLA2 with Asp-58 is less effective than ones with Asn-58 . The region around position 58 has been assumed to be involved in a lipid-recognizing site (DIJKSTRA et al., 1981). A negative charge of Asp-58 might be unfavourable in the interaction of PLA2 with membrane lipids, resulting in a decrease in contractile activity for GPI. It is not so usual to find more than two isospecific PLAZs in a single source . In general, such isozymes induce similar pathological symptoms with variations in intensity (HARVEY and TAMIYA, 1980 ; ROWAN et al., 1985) and have high homology in structures among themselves (SCHMIDT and MIDDLEBROOK, 1989 ; NISHIDA et al., 1982; TAKASAKI et al ., 1988). Therefore, they might be very useful for studies of the structure-function relationships, and PLA2 isozymes from T. gramineus are definitely invaluable for such studies. Acknowledgements--We thank Prof. assistance in biological assays .
Hullo-o K.wuyA
and
DR Yuxto TAtuxo
of Fukuoka University for their
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Trimeresurus gramineus PLAZ Isozyme
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