7

Biochimica et Biophysica Acta, 1037 (1990) 7-15 Elsevier BBAPRO 33518

Amino acid sequence and circular dichroism of Indian cobra ( Naja naja naja) venom acidic phospholipase A 2 F l o r e n c e F. D a v i d s o n a n d E d w a r d A. D e n n i s Department of Chemistry, University of California, San Diego, La Jolla, CA (U.S.A.) (Received 4 April 1989) (Revised manuscript received 21 July 1989)

Key words: Phospholipase A2; Amino acid sequence; CD; (N. naja naja)

The full amino acid sequence of the acidic phospholipase A 2 from Indian cobra (Naja naja naja) venom was determined and its tertiary structure examined by circular dichroism (CD). The sequence was aligned with other sequences of secreted phospholipase A 2 from snakes of the genus Naja, using the progressive alignment method of Feng and Doolittle (J. Mol. EvoL (1987) 25, 351-360). The primary sequence of Naja naja naja phospholipases A 2 shows up to 85% identity with the other acidic Naja phospholipase A 2- CD studies indicate a 40-50% a-helical content in a tertiary structure which resists denaturation at high temperature, with or without chaotropic salts.

Introduction

The acidic phospholipase A 2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) from the venom of the Indian Cobra (Naja naja naja) is one of the most completely characterized phospholipases A 2 in terms of its kinetics (review: Refs. 1 and 2). Yet, until recently, its structural characterization was limited to the amino acid composition and N-terminal sequence [3]. One of the most interesting facets of catalysis by the N. naja naja phospholipase A 2 is that this small, soluble enzyme recognizes and is specifically activated by phosphorylcholine-containing lipids in lipid/water interfaces [4-8]. In order to probe the nature of this specificity, more structural characterization of the N. naja naja phospholipase A 2 is needed. In this manuscript, the primary amino acid sequence of the N. naja naja acidic phospholipase A 2 is reported, as well as its stability at different temperatures and pH values, with and without chaotropic salts and divalent cations. This information should facilitate the solution of the tertiary structure by X-ray analysis, and also provide a new basis for ongoing analysis of the sites of protein and phospholipid interactions by chemical modification and N M R studies.

Correspondence: E.A. Dennis, Department of Chemistry, University of California, San Diego, La Jolla, CA 92093, U.S.A.

Materials and Methods

Materials Reagent grade sodium perchlorate, phenol, dithiothreitol, guanidine-HC1, HBr and dimethyl sulfoxide were from Aldrich. TPCK-treated trypsin immobilized on agarose beads, Ionate grade trifluoroacetic acid, heptafluorobutyric acid, and Sequenal grade HC1 and acetic acid were from Pierce. Iodo-l-[14C]acetamide was from ICN and Amersham. Staphylococcus aureus V8 proteinase was from Boehringer-Mannheim. Naja naja naja phospholipase A 2 (Pakistan) was purified according to previously published procedures [9] from lyophilized venom obtained from the Miami Serpentarium, Miami, Florida. Circular dichroism CD measurements were carried out on a Cary 61 spectropolarimeter which was modified as described previously [10]. CD spectra were obtained using a 0.05 cm cell, by signal averaging 20 scans. The a-helical content of phospholipase A 2 samples was calculated from curve-fitting of the CD spectra using the program of Hennessey and Johnson [11]. CD samples were prepared from lyophilized aliquots of 0.112 mg phospholipase A 2 which had been taken from a stock solution of Ca2+-free enzyme (0.373 mg/ml) that had been extensively dialyzed against 100 mM EGTA first, and then Milli-Q filtered water. The lyopbilized samples were

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

each resuspended in 3.0 ml of the solution in which their spectra were to be taken, and incubated overnight in a water bath at 20 °C before taking the first spectra at 20 ° C. The same samples were then incubated overnight at the next temperature to be tested, and their spectra taken the following day at that temperature. After the highest temperature spectra were recorded at 80 o C, the samples were allowed to cool to 20 ° C, and their spectra checked against the original ones to determine whether any observed denaturing effects were reversible or not.

Reduction and [14C]carboxyamidomethylation of N. naja naja phospholipase A 2 In a typical labeling reaction, 100 nmol of phospholipase A 2 was lyophilized and then resuspended in 100 /~1 of 0.1 M Tris-HC1 (pH 8) containing 8 M guanidineHC1, 10 mM dithiothreitol (DTT) and 10 mM ethylenediaminetetraacetic acid (EDTA) and the solution incubated for four hours at 40 ° C. A solution of 35.5 mM iodo-l-[14C]acetamide (spec. act. 10.6 /~Ci//~mol) was then prepared in 0.1 M Tris-HC1 (pH 8.0) containing 8 M guanidine-HC1 and 10 mM EDTA, and 140/~1 of it added to the protein solution. The mixture was incubated in the dark for 2 h at 40 ° C, and then a 60/~1 chase of the 35.5 mM iodo[14C]acetamide solution was added before incubating in the dark at room temperature overnight. The sample was removed from the labeling reagents by passage over a Pharmacia PD-10 column equilibrated either in buffer, if the next step was immediate proteolytic digestion, or 4 M guanidine-HC1 if the sample was to be stored for any length of time. Trypsin digests of 14C-labeled phospholipase A 2 and separation of tryptic peptides on HPLC TPCK-trypsin immobilized on beads (200/~1 in 50% glycerol) was added to approx. 1.5 ml of 14C-labeled phospholipase A 2 recovered from PD-10 chromatography in 10 mM ammonium bicarbonate, (pH 8.0), and the digestion allowed to proceed four hours at 36 ° C. To stop the reaction, the beads were spun down by centrifugation, and the supernatant containing the tryptic fragments-of phospholipase A 2 removed and subjected immediately to column chromatography. Tryptic peptides of phospholipase A 2 were separated into seven peaks on anion-exchange chromatography, each of which was then further purified on C18 HPLC. Anion-exchange chromatography was performed on a Waters protein-Pak DEAE-5PW analytical HPLC column. The column was preequilibrated in 10 mM ammonium bicarbonate (pH 8.0) and the peptides loaded in the same buffer and eluted with the reported ammonium bicarbonate gradient. Due to UV absorption interference from the buffer, peptides could not be detected by UV throughout the gradient, so aliquots of each fraction were assayed for radioactivity, and pools

made accordingly. Each pool was then injected directly onto an AUtech custom-packed Adsorbosphere HS18 5/~ column (250 × 10 mm) which was preequilibrated in 95% buffer A (0.1% trifluoroacetic acid in Milli-Q H20 ) and 5% buffer B (0.1% trifluoroacetic acid in HPLCgrade acetonitrile). Separations were effected with increasing gradients of buffer B.

H C I / H B r / D M S O cleavage of phospholipase A 2 at tryptophan and separation of tryptophanyl peptides on HPLC Phospholipase A 2 (100 nmol) was cleaved at the carboxy-terminal site of tryptophans using a slightly modified, previously published procedure [12]. Briefly, the salt-free lyophilized enzyme was resuspended in 300 /~1 of 59% acetic acid (v/v), 3.5 M HC1, 4.7 M guanidine-HC1, and 10/~1 of DMSO and 7 mg of phenol were added. The mixture was incubated 20 min at room temperature and then 30 /~1 of 48% HBr and 10 /~1 DMSO were added before incubating another 30 rain at room temperature. The sample was then diluted by the addition of 500 /~1 water, and incubated 3 h at room temperature. Protein fragments were removed from reagents by passage over a Pharmacia PD-10 column equilibrated in water, and the protein-containing pool lyophilized. The lyophilized fragments were subsequently reduced and labeled at cysteines with iodo-1[14C]acetamide as described above, and the labeled peptides recovered in 4 M guanidine-HC1, aac-labeled tryptophan peptides in 4 M guanidine-HC1 were syringed through a 0.6/~m nylon filter before loading onto the same Alltech C18 colunm described above, which was equilibrated in 65% buffer A (0.1% heptafluorobutyric acid in H20 ) and 35% buffer B (0.1% heptafluorobutyric acid in 70% isopropanol/30% acetonitrile (v/v)). Peptides were eluted with the reported gradient. V8 proteinase digests of 14C-labeled phospholipase A e and separation of V8 peptides on HPLC V8 proteinase digestion of phospholipase A 2 was carried out under two different sets of conditions. In the first, 14/~1 of proteinase (20 mg/ml, approx. 60 U/mg) in H20 was added to 100 nmol [14C]carboxyamidomethylated phospholipase A z in 0.1 M sodium phosphate buffer (pH 7.8). The reaction was allowed to proceed 90 rain at 37 °C and stopped by separation on HPLC. In the second case, 133 nmol of [14C]carboxyamidomethylated phospholipase A 2 in 2 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 0.67 M guanidine-HC1 was digested with 45/~1 of V8 proteinase (20 mg/ml, approx. 60 U/ml) for 90 min at 37°C. Again, the reaction was terminated by injection onto HPLC. 14C-labeled phospholipase A 2 peptides from V8 proteinase digestion were separated on the C18-HPLC column described above, using the reported gradients.

*36

V8 peptides were detected by radioactivity and also by absorbance at 230 nm. Unlabeled as well as labeled peptides were thus recovered and sequenced. Peaks were pooled and rechromatographed before being subjected to automated Edman degradation, except where it is indicated that peaks fractions were taken directly for sequence determinations.

Amino acid sequence determinations and compositions Peptides, 0.5-1.0 nmol, were subjected to automated Edman degradation on an Applied Biosystems 470A Gas Phase Sequencer with an on-line P T H amino acid analyzer (Applied Biosystems 120A Analyzer). In this on-line chromatography system, PTH-[14C]carboxyamidomethylated cysteines had almost the same retention time as PTH-glutamic acid. Therefore, aliquots of each step from a sequence run were assayed by scintillation counting to distinguish the two and confirm the locations of cysteines. The entire protein and selected peptides were subjected to amino acid analysis as described elsewhere [3], using norleucine as an internal standard.

A

Alignments of phospholipase A 2 sequences The phospholipase A 2 sequences used were obtained from the National Biomedical Research FoundationProtein Identification Resource Protein Sequence Database, Release 15.0, with the exception of the N. naja naja phospholipase A 2 (reported herein), and the N. naja oxiana phospholipase A 2 [14]. The alignment was generated using the progressive alignment method of Feng and Doolittle [15]. The similarity weighting scale used was a modified version of Dayhoff's Log Odds Matrix which has been reported previously [16]. Results

Circular dichroism The phospholipase A 2 from N. naja naja gives a CD spectrum similar to spectra of other phospholipases A 2 [17], showing extrema at about 194, 204 and 220 nm. As expected by comparison to other venom phospholipases A E, the CD spectra of non-denatured protein are dominated by the a-helical components. Fig. 1 shows spectra of the N. naja naja enzyme taken in the presence and absence of 2.5 M sodium perchlorate a n d / o r 10 m M CaC12 at 20, 50 and 80°C. From panel A, it can be seen that the enzyme is stable in the absence of any buffers for long periods at temperatures up to 60 o C, but shows some flexibility at 50 to 60 ° C and has begun to denature at 80 ° C. Addition of 10 m M CaC12

B

*Ca 2+

+24 I

+12

*12 I..

0

"1=' -'12 ~E

,$E

-24

24

-36 190 200 2113 220 230 240 ~. (nrn)

h 210 ~ 2~0 ~ 230 240 200 * (rim}

-3E9 0.

D

+36~i; +24 "

x o E "D

Cat* 1

+12

~

I

*Ca2 ÷

+3E +24 ÷1~

° ¢

~

Hydropathy plots of phospholipases A 2 Hydropathy plots of three phospholipase A 2 s e q u e n c e s were generated according to the method of Kyte and Doolittle [13].

,36

-CaZ +

-1

-24

-36 L 220 , 230 ~ 240 , 190 2bOO 210 (rim)

12

-24

190 200 2"f0 220 230 240 ^ (nrn)

Fig. 1. Thermal stability of N. naja naja phospholipase A2. Circular dichroism spectra are shown for 2.8-10-6 M phospholipase A 2 in H20 (A and B) or 2.5 M NaC104 (C and D) at 20 (. ), 50 ( - - - - - - ) , 60 (. . . . . . ) and 80 ( . . . . . . ) degrees C. CaCI2, when present (B and D), was 10 raM. The solutions were preincubated > 12 h at each temperature before spectra were recorded.

(panel B) causes an enhancement of the amplitudes of the spectra and makes those taken at 20 ° , 50 ° and 60 ° C almost completely superimposable. Although the enzyme has begun to take on the characteristics of a random coil at 80 ° C, this is less pronounced than in the absence of metal ion (panel A). The observed stabilizing effect of Ca 2÷ on the enzyme's overall tertiary structure is probably an effect of ionic strength, since spectra which were taken in the presence of 10 m M MnC12 or 0.5 M NaC104 at 20 ° were identical (data not shown). Furthermore, at 2.5 M NaC104 (panel C), virtually the same effects are seen as with 10 m M Ca 2+ alone. In this case, however, it is only the spectra taken at 50 and 60 ° C that are superimposable on the three lower-temperature curves of panel B. At 20 ° C, 2.5 M NaC104 enhances the a-helical extrema even further, and at 80 ° C, the enzyme is more denatured in 2.5 M NaC104 than in H 2 0 alone or with 10 m M Ca 2+. Addition of C a 2+ t o the 2.5 M NaC104 sample (panel D) caused only minor changes. The changes induced in the spectra

I0 300

6000 A

DE5

DEI

.•

DE3~4

I

2000

.?

[--

OE6

4500

,

5O

B

CD3c

5"

200

~" 1500 g

r ......... _1

3000

_1 ,~ 0

E e~ DE

-&

/,

20

)

~'J

I 40

---r-" 60

O~

80

60

'~

C

120

4O

15oo

30 ....... /

CD5b

/ /

300

L

&

I

v

1200-

o

8oo-

60 ~"

>

II

'°

20

/

500

240

80

lOOO E

180

5O

CD5a

E 0 10 ~