The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas

411

Biochimica et Biophysica Acta, 580 (1979) 411--415 © Elsevier/North-Holland Biomedical Press

BBA Report B B A 31286

THE AMINO ACID SEQUENCE OF THE PHOSPHOLIPASE A: ISOENZYME FROM PORCINE PANCREAS

W.C. PUIJK a, H.M. VERHEIJ a, P. WIETZES b and G.H. de HAAS a

aBiochemical Laboratory, State University of Utrecht, University Center 'De Uithof', Utrecht and bBiochemical Laboratory, State University of Groningen, University Center 'De Paddepoel', Groningen (The Netherlands) (Received May 22nd, 1979)

Key words: Phospholipase A : ; lsoenzyme ; Primary structure; (Porcine pancreas)

Summary The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of porcine isophospholipase differs from the sequence of porcine phospholipase by four substitutions; viz. Ala 12~Thr; His 17÷Asp; Met 2° ~Leu and Glu 71 *Asn.

Phospholipase A2 (EC 3.1.1.4) has been isolated from several sources, including mammalian pancreas, snake and bee venom [1, 2]. The primary structures of all vertebrate phospholipases have been shown to be homologous [ 2, 3 ] although the differences between individual phospholipases can be as big as fifty percent. Both snake venoms and mammalian pancreas were found to contain isoenzymes [2]. Usually the enzymes and isoenzymes which can be isolated from one animal species are characterized by relatively few substitutions. Studies concerning structure-function relationships which make use of enzymes from one animal species would be more straightforward than a comparison of phospholipases from e.g. porcine and equine pancreas. From porcine pancreas about 5% of the phospholipase can be isolated as an isoenzyme; both enzymes are very similar with respect to binding of Ca 2÷, monomeric and micellar substrate, but the isoenzyme shows a lower activity when assayed in the egg-yolk assay [4, 5]. Amino acid analyses showed that the isoenzyme contains one histidine and one methionine residue less than the major occuring enzyme and evidence was obtained that His 17 and Met 2° are lacking from the sequence [4]. The isoenzyme has been used for chemical modification of the single

412 Met residue [5] and the absence of Met 2° and His 17 has made possible an unequivocal assignment of the 1H-NMR resonances of the histidine residues 17, 48 and 115 and methionine residues 8 and 20 found in the major porcine pancreatic enzyme [6]. In this paper we describe the chemical and enzymatic fragmentation of porcine isophospholipase and the isolation of the peptides. The peptides were compared with respect to electrophoretic mobility, RF values and amino acid composition with peptide fragments predictable from the sequence of the major porcine enzyme [ 7]. When deviations were found the peptides were analyzed by Edman degradation in combination with dansylation. Phospholipase A2 isoenzyme from porcine pancreas was obtained by tryptic activation of its zymogen as described before [4]. Cyanogen bromide cleavage was carried out in 70% formic acid. Before enzymatic digestion, the protein was reduced and cysteines were converted into thialaminines or Smethylcysteines (8). Tryptic and c h y m o t r y p t i c digestion was carried out at an enzyme: substrate ratio of 1:200 (mol/mol) for 1 h at 40°C, using TPCKtrypsin (Serva) or chymotrypsin crystallized three times (Fluka). Before use, chymotrypsin was incubated for I h, at 25°C with a 3 mM solution of tosyllysine chloromethyl ketone in 1% (w/v) ammonium bicarbonate solution. Digestion with staphylococcal protease specific for cleavage of glutamoyl bonds was carried out at an enzyme: substrate ratio of 1:100 (mol/mol) for 2 h at 40 ° C. All digestions were done in 1% ammonium bicarbonate solution (pH 7.8) at a substrate concentration of 50--150 nmol/ml. Peptides were separated on Sephadex G50 and G25 (Pharmacia, Uppsala, Sweden), b y high voltage electrophoresis and by paper chromatography as described before [8]. The charges of peptides were estimated from their electrophoretic mobility at pH 6.5 [9]. Small peptides were sequenced according to a manual Edman procedure [ 10] in combination with dansylation [ 11]. A u t o m a t e d Edman degradation was carried out on 150 nmol thialamininated CNBr fragment 9--124 in a Beckman spinning-cup sequenator, model 890C, using a quadrol double cleavage program (Beckman No. 072172 C). Anilinothiazolinone derivatives of amino acids were converted in 1 M HC1, 0.1% (v/v) ethanethiol, for 7--10 min at 80°C under nitrogen. The phenylthiohydantoins were extracted with ethyl acetate and identified by high-performance liquid chromatography as described b y Frank and Stubert [12] on an LC 20 separator coupled to an LC 3 UV detector (Pye Unicam, Cambridge, U.K.). Tryptic and c h y m o t r y p t i c digestion of both thialamininated and Smethylated protein and digestion of thialamininated protein with staphylococcal protease yielded peptides most of which could be fit into the know sequence of porcine phospholipase A2 on the basis of their amino acid composition and their electrophoretic and chromatographic behaviour (Fig. 1). A few peptides which showed different analytical data than could be anticipated from the known sequence could be tentatively fit into the sequence around residue 11--25 and residue 70--73 on the basis of homology. The properties of the deviating tryptic and c h y m o t r y p t i c peptides obtained from thialamininated protein are enlisted in Table I. As was shown before [7], in the thialamininated protein two tyrosine

413

.

C

.

Ala-Leu- rp-Gln-

C

e- rg-Ser-Me -

I

H~s

Z

e- ys-Cys-

r-

!I

~i

,~

10

~c

e- ro-

y-Ser- sp- ro

T1

Ic

30

Leu-Leu-Asp- Phe-Asn-Asn-Tyr'-Gly-Cys-myr-Cys-Gly-Leu-G1y - G l y - S e r - G l y - T h r T 1

SP

SP C C 40 ~ 50 Pro-Val -Asp-G1 u! Leu-As p-Arg-Cys -Cys-Gl u-Thr-Hi s!Asp-As n-Cys-Ty r!Arg-Asp

~C ~SP 60 ,C C ~70 GT.u A1a- Lys-As n- Leu-As p- Ser-Cys-Lys- Phe~-Leu-Val -As p-Ash- Pro-Tyr-Th'r-#~s n- {eer

I!.

--~i

T2

SP 1 80 Tyr-Ser-Tyr-Ser-Cys-Ser-Asn-Thr-Glu-lle~ i

C

SP

Th r- Cys- A sn-Ser- L ys- A sn- A sn

C

90 Ala

C I00

Cys-Glu-Ala-Phe-Ile-Cys-Asn-Cys-Asp-Arg-Asn-Ala-Ala-Ile-Cys-Phe-Ser-Lys

11o

ic

isP

Ic

12o

A1a- Pro-Tyr'-As n- Lys -Gl u-Hi s- Lys-Asn- Leu-Asp-Thr-Lys- Lys -Tyr- Cys

Fig. 1. The primary structure o f p o r c i n e isophospholipase A 2 and t h e sites o f cleavage o n the r e d u c e d and a l k y l a t e d p r o t e i n by c h y m o t r y p s i n (C) and s t a p h y l o c o c c a l protease (SP). The recovered t rypt i c p e p t i d e s are i n d i c a t e d by solid lines u n d e r the sequence. The results obt a i ne d by E d m a n d e g r a d a t i o n axe indicated by arrows (-9. A b o v e t h e residues w h i c h are n o t c o n s e r v e d in p o r c i n e p h o s p h o l i p a s e the residues f o u n d in t h e l a t t e r e n z y m e are s h o w n a b o v e the i s o p h o s p h o l i p a s e s e q u e n c e .

(Tyr 2s and Tyr 73 ) bonds are very sensitive to (pseudo) chymotryptic cleavage during tryptic digestion. This effect can be largely overcome by shortening the incubation time but in studies like the one presented here smaller peptides have an advantage over bigger ones. From the charge and the amino acid composition of peptide C2, it is clear that Glu 71 has been replaced by a neutral amino acid. Two steps of Edman degradation in combination with dansylation confirmed the sequence to be Thr 7° -Asn-Ser-Tyr 73 . Cyanogen bromide cleavage of the thialamininated protein confirmed the conclusion o f van Wezel and de Haas [4] that the single methionine residue is located at position 8. The resulting fragment (residues 9--124) was used for analysis by automated Edman degradation. Unequivocal assignment of the residues could be made except for step 3 (Cys ]~ ) and step 19 (Cys 27 ) since no attempts were made to determine the water-soluble phenylthiohy-

414

TABLE I AMINO ACID COMPOSITION (MOL PER MOL OF P R O T E I N ) OF PORCINE I S O P H O S P H O L I P A S E A2 The c o m p o s i t i o n w a s d e t e r m i n e d by a m i n o acid a n a l y s e s and the values for i s o p h o s p h o l i p a s e and p h o s p h o l i p a s e w e r e t a k e n f r o m the s e q u e n c e ( i n t e g e r s ) . F o r the p e p t i d e s the a m i n o acid c o m p o s i t i o n (tool p e r tool o f p e p t i d e ) and the e l e c t r o p h o r e t i c b e h a v i o u r and charge at p H 6.5 as d e t e r m i n e d by the m e t h o d of O f f o r d [ 9 ] are given. Amino acid

Isophospholipase

Phospholipase

T1

Asx Thr Ser GIx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Trp Lys His Arg

21.6 7.3 9.9 5.6 4.8 5.8 6.9 13.3 2.0 0.9 4.4 7.6 8.1 5.1 1.0 9.1 2.0 4.1

22 6 10 7 5 6 8 14 2 2 5 7 8 5 1 9 3 4

4.1 (4) 1.0 (1) 1.1 (1)

24 7 10 6 5 6 7 14 2 1 5 8 8 5 1 9 2 4

Residue M o b i l i t y R e l a t i v e to Asp Charge at p H 6.5

T2

3.1 (3) 1.0(1) 1.1 (1)

2.0 (2) 2.1 (2)

CI

C2

4.1 (4) 1.2 (1) 1.1 (1)

1.0 (1) 0.9 (1) 1.0(1)

2.1 (2) 1.2 (1)

2.0*(2)

0.9"(1) 1.0 (1)

1.0 2.0 1.7 1.0

(1) (2) (2) (1)

11--28 0 0

0.8 (1) 1.6 (2) 0.8 (1)

63--73 0.15 --1

1.8 2.2 1.0 1.1

(2) (2) (1) (1)

9--25 0 0

0.9 (1)

70--73 0 0

* As thialaminine.

dantoin derivatives of thialaminine. The sequence found: ile.Lys I0 -X-Thr-Ile-Pro-Gly-Ser-Asp-Pro-Leu-Leu

20 - A s p - P h e - A s n - A s n - T y r - G l y - X - T y r

is in perfect agreement with the amino acid analyses of peptides T~ and C~ (Table I) and with their charge. Compared to the porcine phospholipase A2 three substitutions were found to be present Ala 12 ÷Thr; His l~*Asp and Met 2° ÷Leu. A high degree of homology is found for the two porcine enzymes. Although the degree of homology for the bovine and equine enzyme is less pronounced the substitutions found in the porcine isoenzyme are not uncommon in the structures of the equine and bovine enzyme [8, 13]. Two of the four substitutions (His 17*Asp and Glu 71 *Asn) effect the charge of the protein. In addition to the isoenzymes which are subject of this study, multiple enzyme forms from porcine pancreas have been isolated by two groups [14, 1 5 ] . The amino acid analyses shown for these preparations do not fit the analytical data given in Table I and it remains difficult to explain these discrepancies. In our laboratory a constant ratio of isoenzyme and enzyme has been found in about 10 different preparations on different scales during a period of several years and no indication of additional isoenzymes at a level higher than 1% when assayed with the egg-yolk test has been obtained. In this respect it should be mentioned that the possible detection of isoenzymes does depend on the enzyme assay. A similar conclusion was drawn by Tsao

415 et al. [15] using different substrates. While the a m o u n t of isoenzyme in porcine pancreas is a b o u t 5 percent on a weight basis, it comprises only 3 percent of the activity when tested in the egg-yolk lipoprotein; whereas it exceeds 15 percent of the total enzymatic activity when measured with a micellar system containing short-chain lecithin (dioctanoylphosphocholine). Understanding of the relative rates on different aggregated substrates is an intriguing although not yet solved problem. For the bovine pancreas it has been shown that both isoenzymes found in this animal can be isolated from one pancreas [16]. For the porcine isoenzymes this question has not been answered yet since the amount of phospholipase A2 which can be isolated from one pancreas is t o o low to allow for a fair characterization. Because of its different charge isophospholipase can be separated readily from phospholipase b y ion-exchange chromatography and commercial preparations of porcine phospholipase A2 should be free of the isoenzyme. However, preparations of porcine prophospholipase are often contaminated with low amounts of phospholipase A2 and it has been assumed that this is due to a low extent of activation of the zymogen. More recent data has shown that this assumption is not correct and that the phospholipase activity on micelles present in preparations of prophospholipase must be ascribed to contamination with the isophospholipase A2 • Part of this work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organisation for the Advancement of Pure Research (ZWO). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Evenberg, A., Meyer, H., Verheij, H.M. and de Haas, G.H. (1977) Biochim. Biophys. Acta 491, 265--274 Lee, C.Y. (1979) Snake Venoms, Springer, Berlin Heinrikson, R.L., Krueger, E.T. and Keim, P.S. (1977) J. Biol. Chem. 252, 4 9 1 3 - - 4 9 2 1 Van Wezel, F.M. and de Haas, G.H. (1975) Biochim. Biophys. A c t a 410, 299--309 Van Wezel, F.M., Slotboom, A.J. and de Haas, G.H. (1976) Biochim. Biophys. Acta 452, 101--111 Aguiar, A., Slotboom, A.J., Jansen, E.H.J.M. and Williams, R.I.P., Eur. J. Biochem., in t h e p r e s s Puijk, W.C., VerheiJ, H.M. and de Haas, G.H. (1977) Biochim. Biophys. A c t a 492, 254--259 Evenberg, A., Meyer, H., Gaastra, W., Verheij, H.M. and de Haas, G.H. (1977) J. Biol. Chem. 252, 1189--1196 Offord, R.E. (1966) Nature 211, 591--593 Tart, G.E. (1975) Anal. Biochem. 63, 361--370 Gray, W.R. (1972) Methods Enzymol. 25B, 333--3 34 Frank, G. and Strubert, W. (1973) C h r o m a t o g r a p h i a 6, 522--524 Fleer, E.A.M., Verheij, H.M. and de Haas, G.H. (1978) Eur. J. Biochem. 82, 261--269 Yamaguchi, T., Okawa, Y. and Sakaguchi, K. (1973) J. Biochem. (Tokyo) 73, 187--190 Tsao, F.H.C., Cohen, H., Snijder, W.R., KSzdy, F.J. and Law, J.H. (1973) J. Supramol. Struct. 1, 490--497 Dutilh, C.E., van Doren, P.J., Verheul, F.E.A.M. and de Haas, G.H. (1975) Eur. J. Biochem. 53, 91--97