85

Biochimica et Biophysica Acta. 10[.2(1991) 85-93 ,3 1991 ElsevierScience PublishersB.V. 0005-2760/91/$03.50 ADONIS 000527609100108Q

BBALIP 53593

Porcine pancreatic phospholipase A; isoforms: differential regulation by heparin Mitchell B. Diccianni t, Larry R. McLean -" William D. Stuart t Meenakshi J. Mistry ~, Clefts M. Gil t and Judith A.K. Harmony t I Dcpa, imem of ~harmacoloyO'and Cell Biophysics College of Medicine. lJ~m.ersityof Cincinnati. ('in( innati. OH (U.S.A.) and : Merrell Dow Research InMttute. CincinnatL OH (U.S.A.)

(Received 10 October 1990)

Key words: PhospholipaseA,; Heparin; Enzymeisoform;(Porcine pancreas) iso[orms of porcine pancreatic phospholipase A z (PLAz) can be differentially regulated by beparin. The nlajor ,soform of PLA z can bind to beparin-Affigel and its catalytic activity can be inhibited by beparin. The interaction between this PLA., isoform and heparin does not require calcium ion or a functional active site. The sensitivity to beparln inhibition depends on the pH, with optimum sensitivity at pH 5 - 7 and greatly diminished sensitivity as the pH is increased from 7 to 10. A minor isoform of porcine pancreatic PLA z cannot hind to beparin and is resistant to beparin inhibition. The resistant isoform a!~-~"s to be iso.pig PLA2. Heparin affinity chromatography therefore offers a convenient route to the isolation of structurally and functionally distinct c l a ~ s of PLA2 enzymes. The existence of classes of p L s . , that can be differentially regulated by heparin may have important physiological c'onseqnences.

Introduction Phospholipase A z (PLAz; EC 3.1.1.4) is a calcium ion-dependent enzyme that catalyzes the hydrolysis of diaeyl phospholipids to fatty acids and monoacyl phospholipids. Enzyme activity is present in virtually every ceil and tissue type studied [i]. The primary sequences of more than 50 PLA2s, isolated from diverse species such as mammals and the venoms of snakes and insects, are known. These phospholipases show a high degree of sequence homology, particularly in the conservation of the catalytic triad (His-48, Asp-99, HzO) [2]. A comparison of the sequences of 32 PLAzs obtained from mammalian pancreas and snake venom suggests that the genes encoding these enzymes developed from a

Abbreviations: R-PLA2, PLA2 that binds to heparin-Affigel; BPB, p-bromophenacylbrondde; CD. circular dichroism; HPLC, high-performance liquid chromatography; GAG, glycosaminog.lycan;IEF. is~lectric focusing; IRS. interracial recognition site; PAGE, polyaczTlamidegel electrophoresis;PC, phosphatidyIcholine;PLA2, phospholipase A2, PMSF, pbenyl methane sulfonyl fluoride; RP, reversephase; SDS, sodium dodecyl sulfate; UB-PLA2, PLA2 that does not bind to heparin-Affigel. Correspondence: J.A.K. Harmony, Pharmacology and Cell Bio-l'hysics, Universityof Cincinnati. 231 B¢thesdaAveliue. Cincinnati, OH 45267-0575, U.S.A.

common gene through a divergent evolutionary process 13,4]. In addition to homologous PLAzs from different species and from different tissues within a specie, multiple PLA,_ isoforras within a single specie or tissue have been identified. The isoforms have been distinguished by differences in substrate specificity [5-9], by the pH optimum of catalytic activity [6,10,11], by differences in electrophoretic mobility [12-15] and, in the case of the A g k i s t r o d o n piscivorus piscivorus venom PLA 2, by differences in toxicity ]16,17]. The physiological relevance of isoforms of PLA z is not known. A complete understanding of the biological functions of isoforms of PLA_, is contingent on the development of isoform-specific inhibitors and rapid methods to separate isoforms. Here we report that sensitivity to regulation by heparin distinguishes isoforms of P L A , isolated from porcine pancreas. The predominant isoform of porcine pancreatic PLA 2 hinds to heparin and its catalytic activity is consequently inhibited. A minor pancreatic isoform does not bind to heparin and its catalytic activity is not influenced by this glycosaminoglycan (GAG). Comparative functional and physical characterizar.ions of the two PLA,_ isoforms indicates that the hepafin-sensitive enzyme is the prototype pancreatic PLA z, whereas the heparin-resistant isoform is iso-pig PLA 2 [13,18]. The difference in affinities of the two

86 isoforms of PLA z for heparin provides an opportunity to investigate di:;tinct isoforms of the enzyme which may have different physiological roles.

were mixed, the heparin-Affigel pelleted and aliquots removed for activity or protein determination as appropriate.

Materials and Methods

Inactivation of PLA.,

Materials Porcine pancre:~t.i¢ PLA 2 (EC, 3.1.!.4. lot 26F-0366) and heparin from porcine intestinal mucosa (lot 34F0742) were purchased from Sigma. Phosphatidylcholine (dimyristoyl pho~phatidylcholine; PC), 1-myristoyl monoacyl phosphatidylcholine, (monoacyl-PC) and other chemicals were also obtained from Sigma. Electrophoresis supplies and equipment and Affigel-10 were purchased from Bio-Rad. Radiolabeled phosphatidylcholine ( L-a- 1-stearoyl-2-arachidonyl[ 3H]phosphatidyl choline; [3H]PC, 91 Ci/mmol) was obtained from New England Nuclear.

PLA, assay The activity of PLA 2 was determined by the release of all-labeled fatty acid from radiolabeled PC, as previously described [19]). A typical reaction proceeded for 1 h at 37°C and contained (final concentration): 80 /Lg/ml of PLA2, 2.5 mM dimyristoyl phosphatidylcholine, 0.870 Nonidet P-40, 10 mM CaCI 2 and 0.02 /LCi of [3H]PC. Reaction times were established to yield initial rates of hydroly:;is (10-1570 hydrolysis). Reactions were quenched by the addition of 1 ml of Dole's reagent (isopropyl alcohol/heptane/0.5 M H2SO4 (40: 10: 1, v/v)) and heating at 60°C for 20 min. Upon cooling to room temperature, released 3H-labeled fatty acid was extracted with 0.5 ml of heptane and 0.5 ml of distilled water (dH20) and vortexed. A 0.55-ml aliquot of the upper, organic phase was transferred to a separate test tube containing. 0.5 ml of heptane and approx. 20 mg of silicic acid. The ~ubes were again mixed, the silicic acid allowed to settle and 0.9 ml of the heptane solution (containing the rele:~sed 3H-labeled fatty acid) was transferred to 5 ml of Budget Solve (Research Products International) for radioactivity determination. All hydrolysis values are corrected for background (uncatalyzed hydrolysis).

Chromatography of PLA 2 on heparin-Affigel To separate heparin-sensitive and -resistant forms of PLA2, the enzyme was dialyzed into 50 mM Tris-HCl (pH 7.5), containing 10 mM CaCI2 and fractionated on heparin-Affigel. Unbound protein eluted in the application buffer; bound protein was eluted in 50 mM TrisHC1 (pH 7.5), containing 10 mM CaCI 2 and 0.5 M NaCI. Column fractions were dialyzed and analyzed for PLA 2 activity, using the standard assay. To assess heparin binding, 50 ttg of PLA 2 were added to 0.25 ml of heparin-Affigel in 400 ttl of either 1 mM CaCI 2 or 1-5 mM EGTA in 50 mM Hepe~ (pH 7.0). The samples

PLA2 was inactivated with p-blomophenacyl bromide (BPB). The enzyme was suspended at a concentration of 500 /tg/ml in 50 mM Hepes (pH 7.0), containing 100/tM EGTA. At t = 0 rain, BPB (1 m g / m l in acetonitrile; final concentration of 19.6 /tg/ml) or acetonitrile was added and the enzyme was incubated at room temperature until it had lost > 90~ of its original activity. For reduction and alkylation, PLA2 was dialyzed into 160 mM Tris-HCI (pH 8.5), containing 6 M guanidine-HCl. Dithiothreitol was added to the protein solution at a final concentration of 2.9 m g / m l and the enzyme was incubated at room temperalure overnight in a nitrogen atmosphere. The reduced protein was alkylated with iodoacetic acid (20 mg/ml final concentration) for 2.5 h, dialyzed and lyophilized.

Binding of PLA: isoforms to micelles of monoacyl.PC The interaction of PLA2 with phospholipid was determined in 50 mM glycine (pH 10), by monitoring changes in PLA 2 fluorescence intensity or wavelength maximum, using a stirred thermostated (37°C) 1-cm path length quartz cuvette. The excitation and emission wavelengths were 295 and 350 nm, respectively. Excitation and emission slit widths were 8 nm.

Peptide mapping PLA2 (192 /tg) in 50 mM NH4HCO3 was reduced with 20 mM dithiothreitol fer 1 h at 55°C. The protein was then alkylated with a 5 M excess of iodoacetic acid at 4°C, recovered by centrifugation after acetone (4:1, v/v) precipitation at - 2 0 ° C and digested for 18 h at 37°C with trypsin (1:50, protein/protein) in 200 ltl of buffer (2 M urea, 100 mM NH4HCO 3, 1 mM CaCi2). The reaction was terminated by the addition of PMSF (1 raM) and the protein was precipitated with acetone. The peptides were resuspended in 0.1~ TFA (solvent A), applied to a (:;4 reverse phase-high performance liquid chromatography (RP-HPLC) column (Vydac, 4.6 x 25 mm) and resolved with an increasing gradient of solvent B (9570 acetonitrile, 0.1fo TFA): 0-2 rain of 070 B, 2-5 min of 15~ B, 5-50 min of 60~ B and 50-55 rain of 100~ B. The flow rate was 1 mi/min: elnted protein was monitored at 220 and 280 nm.

Gel electrophoresis One-dimensional polyacrylamide gel electrophoresis (PAGE) was performed in sodium dodecyi sulfate (SDS) by the Laemmll [20] procedure, using either 3-2070 acrylamide gradient gels or 13~o acrylamide gels. For two dimensional gel electrophoresis, isoelectric focusing (IEF) gels were cast in glass tubes ( 3 × 11 cm) as

87 described by O'Farrell [21] and modified by Anderson and Anderson [22]. Circular dichroism (CD) Spectra were obtained at room temperature in the absence or presence of 50 # g / m l of heparin on a Jasco J-500A spectropolarimeter at a PLA 2 concentration of 0.1-0.2 m g / m l in 50 m M Hepes (pH 7), c o n t a i n i n g 1 m M CaCI 2. Spectra are the average of nine scans with buffer blanks subtracted from each scan. Secondary structure calculations from the C D spectra were determined according to Bolotina et al. [23] and Morrisett et at. [24]. Secondary structure predictions from the amino-terminal sequence,~ were calculated according to C h o u and F a s m a n [25]. A m i n o acid composition and sequence analysis A m i n o acid compositions were determined with a W a t e r s Pico-tag system. The a m i n o terminal and peptide sequences were d e t e r m i n e d with a n Applied Biosystems model 470-A gas phase sequencer. For sequence analysis, proteins were further purified by R P - H P L C , using a C18 c o l u m n (Bio-Rad, 4.6 x 25 ram). Lyophilized protein was solubilized in 0.1% T F A , applied to the c o l u m n a n d eluted with a n increasing gradient of solvent B (90% acetonitrile, 0.1% TFA): 0 - 2 rain of 0% B, 5 min of 28% B, 26 rain of 39% B a n d 28 rain of 100% B. The flow rate was 1 r a l / m i n . Results

Heparin inhibits the catalytic activity of porcine pancreatic P L A 2 toward nonionic micellar substrates [19]. Such substrates were chosen to minimize possible ionic interactions between heparin a n d the substrate interface, thus revealing potential interactions with the enzyme. The m a x i m u m extent of inhibition varies from 50-85%, d e p e n d i n g o n the enzyme preparation, suggesting that PLA2 consists of heparin-sensitive a n d heparin-resistant forms. T o test the possibility that some b u t not all of the PLA2 can interact with heparin, PLA2 was c h r o m a t o g r a p h e d on heparin-Affigel. T w o protein fractions were o b t a i n e d (Fig. 1A). Approx. 10-30% of the total pro*.ein did not b i n d to heparin-Affigel and was designated U B - P L A 2. The majority of the protein b o u n d to heparin-Affigel and was designated B-PLA 2. B-PLA2 was eluted from heparin-Affigel with 0.5 M NaCI. The catalytic activity of b o t h PLA2 isoforms was similar to that of the unfractionated enzyme (Table l). However, B-PLA 2 was inhibited by heparin, whereas U B - P L A 2 was not succeptible to t-c.parin inhibition. Analysis of unfractionated po:~;ine pancreatic P L A 2 under non-reducing conditions b y S D S - P A G E revealed three b a n d s at 16 (major), 17 and 26 k D a (Fig. IB). U B - P L A 2 was the 17 k D a protein; B-PLA z consisted of the 16 k D a protein and the 26 k D a protein. In different

04, ~

~°~

12 A -10 -o8 ~

02,

i06

too

0

i

Froctton num0er 214-

B 111-68-- • ,t5--

D,,~

"¢-

---mw

15Stds, PLA2 UB B62 B77

B84

Fig. I. Heparin-Affigel chromatography of PLA 2 ~parates PLA 2 isoforms. PLA z (5 mg). dialyzed in 100 ram Tris-HCI (pH 7.5) containing 10 mM CaCI z. was applied to a heparin-Affigel column. Unbound protein was elated in 20 mM Tris-HCI (pH 7,5), 10 mM CaCI 2 The heparin-bindin 8 traction was eluted with a NaCI gradient in the same buffer. (A) Heparin-Affigel elution profile of PLA 2, (B) PLAz isoforms (30 ~tg), separated by heparin-Affigel chromatography, evaluated by gel electrophoresis. PLA2 is non-fractionaled PLAz; UB is the unbound fraction of PLA2; 1~2, B-n and B~ are bound fractions 62 77 and 84, respectively.

experiments, the a m o u n t of the 26 k D a protein detected by S D S - P A G E was highly variable, suggesting that it is a d i m e r of the 16 k D a protein. The sequences of the amino-terminal ten residues of the 17 kDa, the 16 k D a

TABLE I Activity and heparin sensitiviO. of PLA: fractionated on heparin-Affi~,4 PLA 2 was fractionated on heparin-Affigel as described in the Materials and Methods. For this assay, 80/tg/ml of PLA2was present in the standard assay in 50 mM Hepes with 2.5 mM PC. Enzyme

Heparin (p.g/ml) 0

PLA 2

20

125 + 18 a

100 69+4

(-45~) h

UB-PLA2

134+12

B-PLA2

116+ 3

137+10

(+2~)

55+6 ( - 53st)

The nmol PC hydrolyzed/h. h Percent change relative to control.

32+4 (-75~)

131+15

(+1~)

11+1 ( - 90~)

88 TABLE It

10[,

~'.PL4 2 binding to heparin-Affigel is calcium ion-independent, but confimmatwn -dependent

B-PL&z was incubated with heparin-Affigel as described in the Materials and Methods. After removzl of heparin-Affigei, the solution was assayed for protein and PLA 2 activity. BPB is BPB-inactivated B-PLA 2, which retained less than 10% of its original activity; RDA is reduced, denatured and alkylated B-PLA 2, which retained less than 2% of its original activity. Protein was determined by the Bio-Rad (Pierce) assay.

5 mM CaCI 2 1 mM EGTA 1 mM CaCI2/BPB 1 ,-aM CaCI2/RDA

% Activity recovered after heparinAffigel ~

% Protein recovered after heparinAffigel ~.h

% Protein recovered after BSAAffigel ~'~

4 8 n.a. ~ n.a. ~

n.d. d n.d. n.d. TOO+16

96:i:4 101 +6 97+7 60:1:6

The 9~ of total recovered in the supernatant after removal of Affigel by centrifugation. b Data are averages of n = 2. n.a., n~t applicable. d n.d., not detectable.

a n d the 26 k D a p r o t e i n s were identical to t h a t r e p o r t e d for p o r c i n e P L A 2 [26]. T o evaluate e n v i r o n m e n t a l a n d s t r u c t u r a l requirem e n t s for b i n d i n g of B - P L A 2 to h e p a r i n , direct b i n d i n g studies with heparin-Affigel were p e r f o r m e d a n d the results are p r e s e n t e d in T a b l e II. A l m o s t all of the B - P L A 2 b o u n d to heparin-Affigel w h e n r e c h r o m a t o g r a p h e d . This result indicates the c o n s e r v a t i o n o f h e p a r i n - b i n d i n g c a p a c i t y a n d suggests t h a t B - P L A 2 is not a c o m p o n e n t o f a n e q u i l i b i r i u m m i x t u r e o f B- a n d U B - P L A 2. H e p a r i n b i n d i n g did not require c a l c i u m ion since B - P L A 2 b o u n d to h e p a r i n - A f f i g e l w h e n the E G T A c o n c e n t r a t i o n was 1 m M . In fact, i n c r e a s i n g the E G T A c o n c e n t r a t i o n to 5 m M did not alter P L A 2 ' s ability to b i n d to heparin-Affigel (not shown). M o r e o v e r , inactive B - P L A 2 treated with p - b r o m o p h e n a c y l b r o m i d e (BPB) retained its heparin-Affigel b i n d i n g c a p a c i t y . BPB alkylates the active site His-48 residue of P L A e to prevent substrate binding and hydrolysis without inhibiting micellar b i n d i n g [27]. As a control, B - P L A 2 did n o t b i n d to BSA-Affigel u n d e r a n y of the c o n d i t i o n s tested. A l t h o u g h b o t h active a n d inactive B - P L A 2 b o u n d to heparin-Affigel, e n z y m e c o n f o r m a t i o n a p p e a r e d to be i m p o r t a n t in the interaction. T h e evidence for this is the fact that B - P L A 2 d i d not b i n d to h e p a r i n - A f f i g e l a f t e r it h a d been r e d u c e d , d e n a t u r e d a n d a l k l a t e d with iodoacetic acid to prevent refolding (Table I1). T h e c o n f o r m a t i o n s of U B - P L A 2 a n d B - P L A 2 were e x a m i n e d b y circular d i c h r o i s m ( C D ) s p e c t r o s c o p y (Fig. 2). U B - P L A 2 c o n t a i n e d less a-helix t h a n B - P L A 2 (Table Ill) consistera with literature values for P L A a a n d iso-pig P L A 2 [3,29,30] a n d with the values p r e d i c t e d

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-~o -15

-2o

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200

220

240

260

Wavelength, nm Fig. 2. PLA 2 isoforms have different secondary structures. CD spectra of PLA2 isoforms (0.2 mg/ml) in 50 mM Hepes (pH 7.0) and 1 m,~.~. CaC! 2 were obtained on a Jasco J-500A spectropolarimeter. (A) UB-PLA2; (B) B-PLA 2.

[25] f r o m their a m i n o acid sequences. H e p a r i n h a d n o effect o n the s e c o n d a r y s t r u c t u r e o f either P L A 2 isof o r m (not shown). A n a l y s i s o f the P L A 2 f o r m s b y t w o d i m e n s i o n a l gel electrophoresis (Fig. 3) revealed t h a t the t w o i s o f o r m s also differed in c h a r g e . B - P L A 2 h a d a p r e d o m i n a n t p l o f 6.3 c o m p a r e d to U B - P L A 2 with a p r e d o m i n a n t p l o f 6.0, c o n s i s t e n t with the values d e t e r m i n e d for P L A 2 a n d iso-pig P L A 2 [13]. P L A 2 t h a t h a d n o t b e e n c h r o m a t o grapher on heparin-Affigel consisted of major proteins with b o t h p l values (Fig. 3A). B - P L A 2 a n d U B - P L A 2 were purified f u r t h e r b y R P H P L C (Fig. 4), a n d the s e q u e n c e s of the a m ~ n o - t e r m i n a l 30 residues were d e t e r m i n e d (Table IV). T h e a m i n o t e r m i n u s of B - P L A 2 w a s identical to t h a t r e p o r t e d for the p r e d o m i n a n t f o r m o f p o r c i n e p a n c r e a t i c P L A 2 [26]. U B - P L A 2 differed f r o m the p r o t o t y p e e n z y m e at three

"rABLE Ill Secondary structure of P LA 2 isoforms from circular dichroism spectra

Structure predicted a-helix i 0222,deg.cm2/dmol a222h u-helix. 70 ~ fl-shcet, 70 fl-turn, 70

Secondary structure UB-PLA 2 31 - 13500 27

B-PLA 2 32 - 16200 34

35 + l

39 + I

16+ l 12:t: I

16+ 1 8:t: I

a Calculated [25] from amino acid sequences. h Calculated [24l from 0222. c Secondary structure was calculated from the circular dichroism spectrum by the method of Bolotina et al. 123].

89 pH N kDa

[4.71s.215.816.316.716.8I

A

68-43--

Q

-,-.. O v a l

26-18-14--

----

68--

"-

43 --

*",-

- ~

UF-PLA 2

B Oval

26--

18--

• w.=qp.,,---,-..

14--

68 . . . . .

B-PLA 2

C

43--

,qp

'*-"Oval

26--

18-14--

UB-PLA 2

P

Fig. 3. PLAz isoformshave different pl values. PLAz isoforms(20 p.g) and unfractionated PLA2 (20 lag) plus internal standard ovalburain (20 /~g)were analyzed by two-dimensionalgel electrnphoresis. Proteins are aligned relative to ovalbumin.(A) Unfractionated PLA: (UF-PLA2); (B) B-PLAz" (C) UB-PLAz.

positions: an Ala--* Thr substitution at position 12, a His ---, Asp substitution at position 17 and a Met -- Leu substitution at position 20. in addition, peptide mapping and sequencing of three of the resolved peptides (Table 5), w e r e consistent with separation of iso-pig PLA z from its predominant form [12,13,18,31] by heparin affinity chromatography. Peaks 16 and 23 of UB-PLA 2 corresponded to the sequences reported [26] for iso-pig PLA 2. PLA z and iso-pig PLA z can also be distinguished by their pH-rate profiles and by their dependence on calcium ion for activity at alkaline pH.

In the absence of heparin, both isoforms had a pH optimum of 7 (Fig. 5"~ and, at this pH, their specific activities were comparable. However, the catalytic activity of UB-PLA 2 was relatively insensitive to pH, with high catalytic activity maintained over the pH range 5-10, as predicted for iso-pig PLA z [18]. The pH-dependent behavior of B-PLA 2 contrasted markedly with that of UB-PLA z. The rate of hydrolysis catalyzed by B-PLA 2 decreased significantly between pH 7-10, as expected [32] for the prototype pancreatic enzyme [3235]. UB-PLA 2 was not inhibited by heparin at any pH tested. B-PLA z was very sensitive to heparin inhibition in the pH range of 5-7, but became increasingly less sensitive as the pH was raised from 7 to 10. Reduced catalytic activity of the prototype porcine pancreatic PLA 2 at alkaline pH is due to a low-affinity calcium ien binding site important in micellar binding of the enzyme at alkaline pH [32-35]. This calcium ion binding site, distinct from the high affinity site necessary for catalytic activity, is comprised of Glu-71 [32,36-38]. Since iso-pig PLA, lacks Glu-71, its activity toward micellar substrates at pH 10 should be relatively insensitive to calcium ion concentration, in contrast to that of the prototype enzyme. The initial rate of UBPLA z catalyzed hydrolysis at alkaline pH was minimally increased, by 35%, by increasing the calcium ion concentration from 10 to 50 raM. The ,came increase in calcium ion concentration at pH 10 increased B-PLA 2 activity by 300%. To confirm that UB-PLA2 but not B-PLA2 maintained an IRS at alkaline pH, the binding of both isoforms to micelles of monoacyl phosphatidylcholine (monoacyI-PC), a non-hydrolyzable substrate analog [39], was investigated at pH 10. Binding was monitored by fluorescence spectroscopy. When PLA z binds to micelles, the amino-terminus of PLA z, containing the unique Trp at position 3, undergoes a conformationai change, The change is reflected in a change in intrinsic fluorescence, and can be used to monitor the PLA zmi,:elle interaction [33,40,41]. The binding of UB-PLA 2 to monoacyI-PC resulted in a 30% increase in fluores-

TABLE IV Amino.terminal sequences o f B - P L A : and UB-PLA ,

Amino-terminalsequencesof B-PLA2 (identical to porcinepancreatic PLA2) and UB-PLA2weredetermined by gas-phasesequenceanalysis,using an Applied Biosystems470A gas phase sequencer. Aminoacid differences between the three anfino-terminiare underlined. B-PLA 2

1 15 ALA-LEU-TRP-GLN-PHE-ARG-SER-RET-ZLE-LYS-CYS- AL..AA-ZLE-PRO-GLY16 30 SER-H%S-PRO-LEU-RET-ASP-PHE-ASN-ASN-TYR-GLY-CYS-TYR-CYS-GLY-

1 15 UB-PLAa ALA-LEU-TRP-GLN-PHE-ARG-SER-RET-ZLE-LYS-CYS-THR-ILE-PRO-GLY16 30 $ER- ASP-PRO-LEU-LEU-ASP-PHE-ASN-ASN-TYR-GLY-CYS-TYR-CYS-GLY-

90

15

UB-PLA 2 3

5

10 78 9

2

23

/112,=

11 13 l

220

m

Q) o

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L

t

I

i

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10

20

1

15 4

I

. B-PLA2

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280nm

19

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e~

~'- 280r;m I

10

I

I

20

30

Retention Time, min Fig. 4. HPLC separation 0[ tsypic peptides of PLA 2 isoforms. RP-HPLC purified UB-PLA 2 or B-PLA2 was digested with trypsin and the resulting peptides (38 #g of each digest) were applied to a Vydac C4 RP-HPLC column. The peptides were separated with an increasing gradient of 0.l'~ TFA. 95% acetonitrile as described in the Materials and Methods. Peaks are numbered based on retention time. TABLE V

Amino acid sequences of selected t@ptic peptides Peptides generated [rom the tryptic digest of peak 15 of B-PLA 2 and UB-PLA 2 and peaks 16 and 23 of UB-PLA 2 were sequenced. For UB-PLA2 peaks 16 and 23. amino acid differences from the porcine pancreatic enzyme are underlined; the corresponding residue of the porcine pancreatic enzyme is shown in parenthesis. The peaks correspond to residues 1-6 of iso-pig and pig PLA 2 (peak 15). (,3-83 of iso-pig PLA 2 (peak 16) and 11-43 of iso-pig PLA 2 (peak 23). Peak

Sequence

15

ALA-LEU-TRP-GLN-PHE-ARG

1

16

23

6

63 (GLU) 75 PHE-LEU-VAL-ASP-ASN-PRO-TYR-THR-ASN-SER-TYR-SER-TYR76 83 SER-CYS-SER-ASN-THR-GLU-ILE-THR 11(ALA) (HIS) (HET) 23 CYS-THR-ILE-PRO-GLY-SER-ASP-PRO-LEU-LEU-ASP-PHE-ASN24 36 ASN-TYR-GLY-CYS-TYR-CYS-GLY-LEU-GLY-GLY-SER-GLY-THR-

37

43

PRO-VAL-ASP-GLU-LEU-ASP-AR6

91

precipitated, preventing a full investigation of the effect of calcium ion on PLA2-micelle interaction under these conditions. Discussion o

E 40 c -6 20

B

~100 60 -

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I

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I

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10 pH Fig. 5, PLA 2 isoforras are differentially iafluenced by pH. UB-PLA: (A) or B-PLA 2 (B) (80/tg/ml,~ wa~ incubeted for I h at 37°C with 2.5 mM PC in 50 mM sodium acetate (pH 5), 50 mM Hepes (pH 7-8) or 50 mM glycine (pH 10). The heparin concentrations were (o, q)) 0 .ag/mh (O, III) t0 Fg/mh (&, A) 25 Fg/ml. 5

6

7

8

9

cence intensity accompanied by a blue wavelength shift of 7 nm (Fig. 6A). In contrast, B-PLA 2 fluorescence intensity increased by only 10%; no wavelength maximum shift was observed. Increasing the calcium ion concentration from 1 to 30 mM increased the fluorescence intensity of B-PLA z by more than 5-fold and induced a blue wavelength shift of 8 nm (Fig. 6B), suggesting that the B-PLA 2 micelle interaction at pH 10 was dependent on calcium ion. Above 30 mM calcium ion for B-PLA2 and 5 mM for UB-PLA2, the enzyme

360

AI

E 3551 = 350

~33 44510

-

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41-m41-m-i-m-m-nlZ~ll -

~

335

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3

4

5

6

7

8

Monoacy= PC. rnM

__i

i

Ca2" m M

Fig. 6. UB-PLA 2. but not B-PLA2, binds micelles of monoacyI-PC at pH 10. PLA 2 isoforms (75/~g/ml) in 50 mM glycine (pH ]0). 0.l mM CaCI 2 were titrated with monoacyI-PC, then CaCI 2. Protein was

excited at 295 nm and relative fluorescenceintensity(bottom panels) was recorded at the appropriate wavelength maximum(top panels). (A) Fluorescencevs. monoacybPCconcentration.(B) Fluorescencevs. calcium ion concentration.(O) UB-PLA2; (11)B-PLA2.

This is the first report that isoforms of PLA 2 can be distinguished by a regulatory parameter, the inhibition of catalytic activity by heparin. Commercial preparations of porcine pancreatic PLA2 consisted of two isoforms which could be separated by heparin affinity chromatography. The predominant isoform, the prototype enzyme designated here as B-PLA 2, bound to heparin-Affigel and its catalytic activity towara_ nonionic micellar substrates was inhibited by heparin. The minor isoform, designated here as UB-PLA,, did not bind to h,:parin and its activity could not be inhibited by this GAG. UB-PLA_, was shown to be identical to iso-pig PLA 2, first described by van Wezel and de Haas [13]. Evidence for identity was provided by partial amino acid sequence, isoelectric point, the relative insensitivity of the catalytic activity to pH and the absence of calcium ion dependence for micelle interaction at alkaline pH. With respect to heparin regulation, UB-PLA 2 (iso-pig PLA2) was insensitive to heparin at pH values between 5 and 10. In contrast, B-PLA 2 was considerably mnre sensitive to heparin inhibition between pH 5-7 than it was at alkaline pH. The structures of B- and UB-PLA 2 were compared to deduce the nature of B-PLA2-heparin interaction. A direct physical interaction between B-PLA 2 and heparin was indicated by the binding of B-PLA 2 to heparin-Affigel. Heparin-binding typically involves electrostatic interactions of positively charged basic amino acids, linearly arranged in the protein sequence, with negatively charged heparin [42]. Analysis of the primary structure of PLA 2 [43] revealed no linear consensus sequence that might participate in heparin binding. This suggests that the heparin-binding domain in B-PLA 2 is conformationally determined, as has been proposed [44] for antithrombin IlL Although calcium ion can alter the conformation of PLA z [45], it was not required for heparin binding by B-PLAz On the other hand, the alteration of the conformation of B-PLA2 by reduction and alkylation abolished heparin binding, consistent with the importance of conformation. The conformations of B- and UB-PLA 2 in solution, determined by CD, were not dramatically different. Thus, if the heparin-binding property of B-PLA 2 depends on its conformation, the binding domain must reflect localized rather than global conformational requirements. The results reported here suggest that the conformation of the amino-terminus of B-PLA 2 is important in heparin binding. We propose that the amino-terminus may interact directly with heparin. Located in this region of the enzyme were three of the

92

Thr Ala

Phe

Me ~ ~ / - ' N J / Q Gly/~C// Gin ~ Cys

le \~".,-L_\ '~Leu

~ A l e

rg

Pro ~ His/Asp Ser ~ Lys Pro Trp Fig. 7. Helical wheel diagram of the amino-terminal 18 residues of PLA 2. B-PLA 2 has Ala-12 and His-17; U B - P L A 2, Thr-12 and Asp-17.

four amino acid substitutions which distinguished Band UB-PLA2-residues at positions 12, 17 and 20. The amino-terminus is known to play a critical role in the binding of PLA 2 to lipid micelles [32-35,41,46,47]. AIthot,gh the overall conformations of the two isoforms were similar, the conformations of the amino-terminal 30 residues of UB-PLA 2 and B-PLA2, subjected to Chou-Fasman [25] secondary structural analysis, were predicted to be different. UB-PLA 2 was calculated to have about 20% less a-helix (33% predicted) in the amino-terminus compared to B-PLA 2 (53% predicted). In general, the high content of a-helix predicted for the amino-terminus is conserved among PLA2s [3]. The amino-terminal sequences of the isoforms are displayed in a helical wheel diagram, a method introduced by Schiller and Edmundson [48], in Fig. 7. Arg-6, His-17 and Lys-10 project on the same face of an a-helix in the amino-terminus of B-PLA2, creating a region of positive charge for heparin binding. The substitution of a negative charge, Asp-17, within this region of UB-PLA 2 may be sufficient to ablate heparin binding. In support of a role for the amino-terminus in heparin binding are results which eliminate other functional domains of the enzyme. The ability of BPB-inactivated B-PLA 2 to bind to hepatin-Affigel suggests that a functional active site is not important. The lack of a calcium ion requirement for binding mitigates against involvement both of the active site and the low-affinity calcium ion binding site comprised of Glu-71, which is Asn-71 .¢ " in UB-PLA 2. Confirmation of the precise role of the ~P amino-terminal region ir heDarm binding awaits examination of amino-terminal enzyme modification and analysis of heparin interactions with amino-terminal pcptides. In spite of having been recognized for many years, the physiological significance of two PLA 2 isoforms in porcine pancreas remains obscure. There appears to be some difference in substrate specificity between the two isoforms [18]. In the in vitro assays employed here, the major distinctive features of the minor iso-pig isoform are: its general insensitivity to pH and to calcium ion at

alkaline pH and its insensitivity to heparin inhibition. The iso-pig isoform is, in fact. remarkable in its resistance to catalytic modulation by pH. by calcium ion and by heparin. The physiological significance of heparin regulation of PLA 2 is uncertain. Our data with the porcine enzyme suggest that PLA 2 exists in two classes, heparin-sensitive and heparin-resistant. Bosner and colleagues [49] have proposed that heparin-like cell surface molecules sequester and concentrate digestive hydrolytic enzymes, speci~'ically chr,!esterol esterase, at the luminal endothelit~m of the intestine. We suggest that PLA 2 can be simil, rl'.' anchored. The fact that the GAG-bound PLA 2 is likely to be inactive can be important in protecting the luminal cell membrane phospholipids from hydrolysis. The existence of nonbinding isoforms, as is documented here for PLA 2, may serve to ensure a constant level of PLA 2 activity. In tissues, heparin-resistant isoforms of PLA 2 may be important when the inhibitor heparin is abundant, such as released from mast cells at sites of degranulation. Acknowledgements

The authors appreciate the expert assistance of Ms. Terri Beming, who prepared the manuscript and Mr. Gene Fellows, who contributed the figures. This project was supported by grant HL27333 from the National Institutes of Health. M.B.D. was a predoctoral trainee sponsored by Training Grant HL07382 from the National Institutes of Health. Protein sequences were determined in the Protein Chemistry Core Laboratory in the Department of Pharmacology and Cell Biophysics. References

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