THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 9, Issue of March 25, pp. 4239-4246, 1986 Printed in U.S.A.

Purification and Partial Sequence Analysis of a 37-kDa Protein That Inhibits Phospholipase A2 Activity from Rat Peritoneal Exudates*

(Received for publication, October 17, 1985)

R. Blake Pepinsky, Lesley K. Sinclair, Jeffrey L. Browning, Robert J. Mattaliano, John E. Smart, E. Pingchang Chow, Tanya Falbel, Ann Ribolini, Jeffrey L. Garwin, and Barbara P. Wallner From the Biogen Research Corporation, Cambridge, Massachusetts 02142

We have purified from rat peritoneal exudates a 37- kDa protein that inhibits phospholipase At activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide vari- ety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody de- veloped against a 6-kDa phospholipase inhibitor pro- tein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Proper- ties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.

Prostaglandins and leukotrienes, which are produced in response to an injury, are potent mediators of inflammation. Both families of molecules are derived from a common fatty acid precursor, arachidonic acid, which can be released from cell membranes by phospholipase A2 or through a combination of actions by phospholipase C and diacylglycerol lipase. Al- though the inflammatory response involves numerous steps, compounds that inhibit prostaglandin and leukotriene pro- duction reduce inflammation. Recently, a family of proteins that blocks inflammation has been identified (1-3). These proteins also inhibit phospholipase A2 activity and thus pre- sumably stop inflammation at this early stage. Regulation of the expression and activity of these inhibitory proteins by glucocorticoids has been postulated as the mechanism through which steroids act as anti-inflammatory agents (for reviews see Refs. 4 and 5 ) .

Phospholipase A2 inhibitory proteins have been detected in a number of systems, including rat macrophages (6) , rabbit neutrophils (7), rat renal medullary cells (8), and murine and bovine thymus preparations (9). The predominant form of

* This work was supported by Biogen Research Corp. and Yaman- ouchi Pharmaceutical Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

these inhibitors is a 40-kDa protein, although other species with apparent masses of 15, 30, 55, and 70 kDa have been detected. These additional forms all are immunologically re- lated to the 40-kDa protein (8,lO). Partially purified inhibitor proteins have been shown to be active at three levels. First, they inhibit phospholipase A2 activity in in vitro assays. Second, they block prostaglandin production in cellular as- says. Third, they reduce inflammation in in situ model sys- tems for inflammation. These proteins all are referred to as lipocortin, which replaces three other names (macrocortin, lipomodulin, and renocortin) previously used to describe the same set of molecules.

In this report we describe the purification and partial sequence analysis of the major phospholipase AB inhibitor protein found in rat peritoneal exudates. It has a mass of 37 kDa and is closely related to a human phospholipase inhibitor protein that we recently cloned and expressed (11). The 37- kDa protein was detected in cultured cells of diverse origin, including lines derived from macrophages, fibroblasts, and epithelial cells. Expression levels of the inhibitor in these lines varied from 0 to 0.5% of total protein.

MATERIALS AND METHODS

Source of Inhibitor Protein-Male Wistar rats (200-250 g) were acclimatized to laboratory conditions for 24 h and then injected subcutaneously with 0.1 ml of the glucocorticoid dexamethasone phosphate (Lark Laboratories, 1.25 mg/kg rat) in 0.9% NaCl. One h after injection, the rats were killed with Euthasate and the peritoneal cavities rinsed essentially as described (6) with 10 ml of phosphate- buffered saline (50 mM KHzP04, pH 7.3,150 mM NaCl) containing 2 units/ml heparin and 50 p M phenylmethanesulfonyl fluoride. The lavages were cleared of cells and other particulate matter by centrif- ugation for 30 min at top speed in an International centrifuge. The supernatants were combined and additional protease inhibitors added. These included aprotinin to 20 pg/ml, soybean trypsin inhib- itor to 10 pg/ml, and EGTA’ to 0.5 mM. The exudates were incubated at 37 “C for 1 h in the presence of 0.1 units/ml calf intestinal alkaline phosphatase and subjected to the purification protocol described below.

Phospholipase Az Assay-Samples were tested for phospholipase A2 inhibitory activity by an in vitro assay described previously (8). The substrate for phospholipase Az, autoclaved [3H]oleic acid-labeled Escherichia coli, also was prepared as described (8). For each experi- mental point, 200 yl of sample was combined with 50 pl of a 7x buffer (0.7 M Tris-HC1, pH 8.0, 60 mM CaClz) and with 50 pl of a dilute preparation of porcine pancreatic phospholipase Az (Sigma) that contained 100 ng of enzyme and 125 pg of bovine serum albumin. Samples were mixed and kept on ice for 1 h. 25 yl of the substrate was added, and the reaction was performed at 6 “C for 8 min. The

The abbreviations used are: EGTA, [ethylenebis(oxyethyl- enenitri1o)ltetraacetic acid; HPLC, high pressure liquid chromatog- raphy; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel elec- trophoresis; CNBr, cyanogen bromide. One-letter notation for amino acid sequence is derived from the list adopted by the IUPAC-IUB Commission for Biochemical Nomenclature (CBN).

4239

4240 Rat 37-kDa Phospholipase A2 Inhibitor reaction was stopped by adding 100 p1 of 2 N HCl and 100 p1 of 20 mg/ml delipidated bovine serum albumin (Sigma) to each tube. Tubes were held on ice for 30 min and E. coli pelleted by centrifugation for 5 min at 10,000 X g. 250 pl of each supernatant was mixed with 4 ml of scintillation fluid and the residual phospholipase activity quanti- tated by liquid scintillation counting. In all analyses, samples were assayed in duplicate and adjusted for nonspecific release by subtract- ing a control value in which the preparations were assayed without phospholipase A'. One unit of activity inhibits 15 ng of phospholipase Az.

SDS-Polyacrylamide Gel Electrophoresis-Protein preparations were analyzed by electrophoresis in SDS-polyacrylamide gels (15% acrylamide, 0.18% methylene bisacrylamide) by the procedure of Laemmli (12). Stacking gels contained 7.6% acrylamide and 0.21% methylene bisacrylamide. Before electrophoresis, samples were heated for 10 min at 60 "C in electrophoresis sample buffer (2% SDS, 0.05 M Tris-HC1, pH 6.8, 12.5% glycerol, 1.5% 2-mercaptoethanol). Gel profiles were visualized by staining with Coomassie Brilliant Blue R-250 or by Western blot analysis (13). In instances where protein concentrations were below the detection limits of Coomassie staining, polypeptides were visualized by silver staining (14).

Purification of Phospholipase Inhibitor Protein-Fresh exudate preparations from 24 rats were dialyzed overnight a t 4 "C against 40 volumes of 20 mM Tris-HC1 pH 7.7. The dialysate was subjected to ion-exchange chromatography on a DEAE-cellulose column (What- man, DE52; column dimensions, 1 X 17 cm) previously equilibrated with 25 mM Tris-HC1, pH 7.7. Flow-through fractions were combined and concentrated 20-fold by Amicon ultrafiltration (PM-10 mem- brane). The concentrate was subjected to molecular sieving on a gel filtration column (P-150, Bio-Rad) in 25 mM Tris-HC1, pH 7.7, (column dimensions, 2.5 X 40 cm) and 5-ml fractions collected. Aliquots of fractions from both columns were monitored for protein by absorbance at 280 nm and for phospholipase inhibitory activity by the assay as described above. All protein preparations routinely were stored at 4 "C due to loss of activity upon freezing.

Sequence Analysis-Peak fractions from four sets of purifications (100 pg of inhibitor protein) were combined, quick frozen, and lyoph- ilized. Samples were suspended in 1% SDS, dialyzed against 300 volumes of 25 mM Tris-HC1, pH 6.8, 0.2% SDS, and subjected to preparative SDS-polyacrylamide gel electrophoresis. The region of the gel containing the 37-kDa protein was excised and the protein recovered by electroelution (15) in 10 mM NH,HCO,, 0.1% SDS. This region was identified with a radioactive marker (0.2 pg of iodinated 37-kDa protein), which was added to the sample prior to electropho- resis. The position of the radioactive marker was determined by autoradiography of the wet gel at 4 "C. After electroelution, the sample was lyophilized and resuspended in 200 pl of 0.2 M N - ethylmorpholine acetate, pH 8.6. The protein was reduced with 5 mM dithiothreitol for 2 h at 37 "C, alkylated with 10 mM iodoacetic acid for 30 min at 23 "C in the dark, and precipitated with 20% trichlo- roacetic acid. The precipitate was pelleted by centrifugation for 20 min at 10,000 X g and washed twice with 5 ml of -20 "C acetone; each wash being followed by a centrifugation step. Finally, the pellets were dried under vacuum.

Prior to digestion with trypsin, pellets were suspended in 400 pl of 0.1 M ammonium bicarbonate, pH 8.0,O.l mM CaC12. L-1-Tosylamido- 2-phenyletbyl chloromethyl ketone-trypsin (Worthington, 5 pg total) was added to the 100 pg of rat protein and the digestion performed for 16 h at 37 "C. The trypsin was added in three equal aliquots: the first at time zero, the second after 4 h, and the third after 12 h of incubation. The digest was acidified with formic acid to 20% (v/v). Tryptic fragments were separated by reverse-phase HPLC at 40 "C on a C,, column (SpectraPhysics, column dimensions, 0.46 X 25 cm) equilibrated with 0.1% trifluoroacetic acid. Bound components were eluted with a 95-min gradient (0-75% acetonitrile in 0.1% trifluo- roacetic acid) at a flow rate of 1.4 ml/min. For each analysis, 200 half-min fractions were collected and stored at -70 "C for subsequent sequence analysis.

Samples were subjected to protein micro-sequence analysis by Edman degradation in an Applied Biosystems 470A gas-phase se- quenator in the presence of Polybrene. Prior to loading, 50 pg of SDS was added to the selected fractions, the volume reduced to about 75 p1 in a Speed Vac concentrator, and the samples boiled for 3 min. Phenylthiohydantoins from each cycle were analyzed by reverse- phase HPLC on a 5-pm cyano column (0.46 X 25 cm, IBM) as described (16). The eluate from the cyano column was monitored spectrophotometrically both at 269 nm and at 313 nm. Aliquots from

I fraction number

FIG. 1. DEAE column profile of phospholipase inhibitory activity from rats. Peritoneal lavages from 24 dexamethasone- treated rats were dialyzed overnight at 4 "C against 25 mM Tris-HC1 at pH 7.7 and then subjected to DEAE-cellulose column chromatog- raphy. 14-ml fractions were collected during the flow-through and the wash steps. 7-ml fractions were collected during the salt gradient. Protein in each sample was monitored by absorbance at 280 nm. Activity measurements were based on the in vitro phospholipase A2

assay.

each sample sequenced also were subjected to amino acid analysis on a Beckman System 6300 analyzer.

In instances where tryptic profile peaks contained more than one polypeptide, as indicated by sequence analysis, the corresponding fractions from a second tryptic digest were diluted 1:2 with 0.05 M ammonium acetate at pH 6.46 and re-injected onto a CIS column (SpectraPhysics) equilibrated with the same buffer. Fragments were eluted with a gradient of acetonitrile (0-75% in ammonium acetate) otherwise using the same conditions as described above.

Isolation of Rat Peritoneal Cells-In experiments where resident peritoneal cells were analyzed, rats were acclimatized to laboratory conditions for 4 days. Each peritoneal cavity was washed with 25 ml of 0.34 M sucrose and the exudate cells pelleted by centrifugation for 5 min at 2,500 X g. Cell pellets were suspended in lysis buffer (20 mM Tris-HC1, pH 7.5, 50 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 5 mM N-ethylmaleimide) and disrupted by vortex mix- ing for 30 s. Lysates were clarified by centrifugation for 5 min at 10,000 X g. The supernatants were boiled for 3 min in electrophoresis sample buffer and stored at -20 "C. For oil-elicited peritoneal cells, rats were injected intraperitoneally with 8 ml of Fisher light-weight paraffin oil and the cells isolated after 3 days.

Preparation of Antisera-Antisera against SDS-disrupted rat li- pocortin were developed in rabbits using the lymph node immuniza- tion procedure (17). Rat lipocortin in 0.1% SDS was emulsified with Freund's complete adjuvant (25 pg of protein/l ml of emulsion/ rabbit). 400 pl was injected into two lymph nodes and the remainder injected subcutaneously along the back. After 5 weeks each rabbit was boosted with an intramuscular injection of 25 pg of protein emulsified in Freund's incomplete adjuvant. Production of antibodies specific for the rat protein was assessed by enzyme-linked immuno- sorbent assay and by Western blot analysis. Procedures for the preparation of a rabbit antiserum against a phospholipase inhibitor protein, which we purified from Naja naja Cobra venom (Sigma), are described in detail elsewhere.' The Cobra venom phospholipase in- hibitor protein was purified by a modification of the procedure of Braganca and co-workers (18).

RESULTS

Purification and Properties of a Rat Phospholipase Inhibitor Protein-We have purified a 37"kDa phospholipase Az inhib- itor protein from rat peritoneal exudates through a combina- tion of ion-exchange and molecular sieving chromatographies. To assess the success of these steps, aliquots of each column fraction were monitored for protein and for phospholipase Az inhibitory activity. Fig. 1 shows the results obtained when a crude soluble lavage preparation from 24 rats was fractionated by DEAE chromatography. Approximately 95% of the total

' J. Garwin and A. Ribolini, manuscript in preparation.

Rat 37-kDa Phospholipase A:! Inhibitor 4241

protein was retained by the ion exchanger and subsequently eluted with a gradient of NaCI. Three peaks of inhibitory activity were detected a t 50, 400, and 'io0 mM NaCI, exactly as described previously by Flower and co-workers (6). Using the conditions described, approximately half of the inhibitory activity flowed through the DEAE column. This preparation contains only 3-4741 of the starting protein and has a specific activity of about 200 units/mg protein.

The inhibitor in the DEAE flow-through was further puri- fied by gel filtration. As shown in Fig. 2.4, the inhibitory activity elutes as a single broad peak. Maximal activity was centered at fraction 19. The apparent mass of the inhibitor is about 40 kDa, based on its elution profile relative to standard markers. The inhibitory activity is sensitive to digestion with tr-ypsin and is destroyed by boiling (not shown). I t also is inactivated by heparin (see Table I ) . Most contaminating proteins were larger than 40 kDa and thus are resolved from the inhibitor by the sizing step. Fig. 223 shows a Coomassie- stained profile of proteins from the same column samples after SDS-polyacrylamide gel electrophoresis. The two frac- tions with greatest inhibitory activity, fractions 19 and 20, were found to contain a single major protein a t 37 kDa. The purified inhibitor accounts for 0.2% of the total protein in the lavage preparation and has a specific activity of 6,000-10.000 units/mg. In the in vitro phospholipase assay, the inhibitor blocks phospholipase A, in a dose-dependent manner (see Fig. 3).

12 14 16 18 20 22 24 26 28

fraction number

L" a b c d e f g h i j k l

FIG. 2. Gel f i l t ra t ion chromatography of inhibitor protein. DEAE flow-through preparations desrrihrd in Fig. 1 wrre comhined. concentrated, and suhjected to gel filtration chromatography on a P- 1.50 column. 5-ml fractions were collected. In A, aliquots of each fraction were monitored for protein and for phospholipase A2 inhihi- tory artivity. In R, the same samples were analyzed hy SDS-PACE. Proteins were stained with Coomassie Blue. [-una n, markers (phos- phorylase h, 90 kDa: hovine serum albumin, 68 kDa; ovalhumin, 43 kDa: wrhvmotrypninogen, 26 kDa; &lactoglobulin, 18 kDa; lysozyme, 14 kDa). Lanes b-1 correspond to column fractions 14, 16, 17, 18, 19, 20. 21, 22. 23, 25. and 27, respectivelv.

TARI.E I Effect of rangents on inhihi fop actit'ity

Reagents at the concentrations indicated were incuhated for 1 h a t 4 "C either with the 37-kDa inhibitor protein (PIP) alone or with phospholipase A2 alone. Preparations then were subjected to the DhosDholilnase A2 assav. Values listed renresent Der cent inhihition.

Inhihitor (ronrmtration) - inh ih i tor + inhihitor + inhihitor Al + 1'11' Az + PIP Az

e; P - r E

Phenylmethane sulfonyl fluo- ride ( 3 mM)

TLCK" (10 mM) TPCK* (1 mM) Renzamidine (10 mM)

Heparin (0.1 mg/ml) Soyhean trypsin inhihitor (5

Aprotinin (1 mg/ml) Pepstatin A (1 mg/ml) lodoacetamide ( 5 0 mM) Iodoacetic arid (10 mM) N-Ethylmaleimide (10 mM) p-Chloromercurohenzoate ( 1

ZnCI2 (3 mM) FeCIR ( 3 mM) MnCI, (3 mM) CuCI,! ( 3 mM) CaCI, (3 mM)

EDTA (10 mM)

mg/ml)

mM)

69

57 69 54 54 54 67

54 63 63 63 63 63

67 67 67 67 67

48

52 61 64 48 13 66

76 62 70 64 68 67

50 54 67 65 69

11

1 0

12 2 9 c 1

44 0

28 30 27 24

+8 37 9

+5 11

" TIXK, I-chloro-8-tosvlarnido-7-amino-2-heptanone. TPCK, 1,-l-tosvlamido-2-phenvlethyl chloromethyl ketone.

80

7 0

-

60

* 50

40

x 30

0 k m E

* 2 o v 10

0.0 0.5 1 .o 1.5 2.0

pg OF LIPOCORTIN FIG. 3. Dose-response curve of inhibitor protein. Aliquots of

the purified inhihitor were incubated with 100 ng of porcine pan- creatic phospholipase A,, and the samples were assayed for residual phospholipase activity. The data presented show a typical titration curve generated from such an analysis.

To confirm that the 37-kDa protein is the predominant inhibitor in the DEAE flow-through, the same preparations also were screened by Western blot analysis, using a neutral- izing antibody. This antiserum was developed against a het- erologous inhibitor protein from Cobra venom and blocks the phospholipase inhibitory activity detected in the rat prepa- rations. The antiserum also blocks the activity of the rat protein in a cellular assay in which prostaglandin production was monitored.:' Fig. 4 shows results from a Western analysis in which selected gel filtration fractions were screened with the neutralizing antibody. The 37-kDa protein is a prominent immunoreactive band in the column fractions that were most inhibitory (lane f ) . A second immunoreactive species at 30

~

' R. Flower, unpublished results. ~~~ ~ ~~

4242 Rat 37-kDa Phospholipase AP Inhibitor

a b c d e f g h . . F"-----! , - .

c

i

FIG. 4. Western blot analysis of inhibi tory f ract ions. Gel filtration fractions from a G-75 column were subjected to SDS-PAGE and analyzed either directly hy staining with Coomassie Blue (lanes a-d) or by Western blot analysis (lanes e-h) . For the Western anal- vsis, immunoreactive proteins were detected with a neutralizing an- iihody raised against the cobra venom inhibitor protein. Lanes a and 1, :lY-kDa inhibitory peak; lanes b andg, 15-kDa inhibitory peak; lanes c and / I , 12-kDa region; lanes d and e, markers.

kDa is routinely observed. We have shown, however, by peptide mapping that it is derived from the 37-kDa protein (see below). Both bands were the major immunoreactive spe- cies detected when crude lavage preparations were screened by Western blot analysis and when cell lysates from resident peritoneal cells were analyzed (not shown).

The 30-kDa protein was identified as a fragment of the 37- kDa protein by peptide mapping analysis. Gel slices contain- ing both polypeptides were reacted with CNBr and the cleav- age products separated by SDS-PAGE (19). Cleavage products were visualized directly by silver staining (Fig. 5, lanes a and b) and by protein blotting (Fig. 5, lanes c and d). Immuno- reactive fragments were visualized with an antiserum raised against the 37-kDa rat inhibitor. In both analyses, the cleav- age profiles of the 37- and 30-kDa proteins were nearly identical, differing only in the few positions indicated. The amount of the 30-kDa band detected in the peritoneal exu- dates varied from preparation to preparation. It was enriched after prolonged storage of samples at 4 "C and after mild treatment with trypsin.

Partial Sequence Analysis of the Rat 37-kDa Protein-The purified rat 37-kDa protein was carboxymethylated with io- doacetic acid, digested with trypsin, and cleavage products separated by reverse-phase HPLC on a CIS column. Fig. 6 shows two profiles from such an analysis, one monitored at 280 nm (Fig. 6A) and the other at 214 nm (Fig. 6R). After digestion with trypsin, approximately 40 peaks were generated of which about half had absorbance at 280 nm. All of the major peaks were subjected to sequence analysis.

Sequences from 21 tryptic fragments are shown in Table 11. For most fragments, the entire polypeptide chain was sequenced as evident by lysine or arginine at the end of the sequence. In two instances, for T31 and for T38, the signal was lost before the end of the sequence was reached. The

a b c d .. . .

FIG. 5. Cyanogen bromide mapping of the 37- and 30 -kDa proteins. Gel slices containing the specified proteins were incubated with 7 mg/ml CNRr for 1 h at 23 " C , washed, and cleavage products electrophoresed into an SDS-polyacrylamide gel (15% acrylamide, 0.4% methylene hisacrylamide). Fragments were identified directly by silver staining (lanes a and b) and by Western hlot analysis (lanes c and d). Lanes a and c, 37-kDa protein; h e s b and d, 30-kDa fragment. Arrows denote differences in cleavage fragments.

.O1 t I1

I A

-10 0 IO a 30 40 50 w IO w m IW 110 m I # 140 1 5 0 180 170 180 1 8 0

hac1 ion number

FIG. 6. Trypt ic map of c leavage f ragments der ived f rom the 37-kDa protein. Preparations containing 100 pg of purified protein were carboxymethylated with iodoacetic acid and incubated with trypsin for 16 h at 37 "C. Digests were acidified with formic acid and subjected to reverse-phase HPLC on a CIR column. Fractions were monitored simultaneously at 280 nm ( A ) and at 214 nm (R) . Num- bered peaks signify the polypeptides that have been sequenced.

peptide components of peaks T30 and T32 did not sequence at all. In samples where peaks contained more than one polypeptide, such as for T19 and T22, each peak was rechro- matographed in a subsequent HPLC step at neutral pH. Peptides resolved by the second HPLC step were then se- quenced. These fragments are denoted with a and b in the

Rat 37-kDa Phospholipase Az Inhibitor 4243

TABLE I1 Summary of sequence data for rat polypeptides

Tryptic and cyanogen bromide fragments, each containing 1-2 nm of material, were subjected to amino-terminal protein sequence anal- ysis in a gas-phase sequenator. Designations for tryptic fragments correspond to column peaks described in Fig. 6. CNBr 1 and CNBr 2 are the two small CNBr fragments that were sequenced.

Fragment Sequence

a. Tryptic fragments T3 T6 T9 T13 T15 T17a T17b T18 T19a T19b T22a T22b T23 T24

S Y K G D Y E K V Y R E E L K K Y S Q H D M N K A L Y E A G Q R V F Y Q K L Y E A M K A I M V K D I T S D T S G D F R S E I D M N E I K V F Q N Y R S Y P H L R T P A Q F D A D E L L R

T26 A L D L E L K T29 A A Y L Q E T G K P L D E T L K T30 T31 G G P G S A V S P Y P S F N P S S D V A A T32 T34 G T D V N V F N T I L T T R T35 K G T D V N V F N T I L T T R T37 G V D E A T I I D I L T K T38 G L G T D E E x L I x I

b. Cyanogen bro- mide fragments CNBr 1 M K G A G T R R K T L I CNBr 2 M L K T P A Q F D A D E L L R

table. Three of the tryptic fragments contained additional lysines that were not sensitive to trypsin. Fragment T29 contains the trypsin-resistant sequence Lys-Pro. Fragments T35 and T15 have lysines at their amino terminus. Amino- terminal lysines frequently are generated when sequential cleavage sites for trypsin occur in a protein, because trypsin is not an efficient exopeptidase.

In addition to the analysis of tryptic fragments, prepara- tions of the rat protein also were digested with cyanogen bromide. CNBr fragments were resolved into six peaks by reverse-phase HPLC on a Cs column (not shown). Sequences derived from the two smallest CNBr fragments are given at the bottom of the table. Each of the four large peaks contained a mixture of polypeptides by gel analysis and thus were not subjected to sequencing. The combined information from tryptic and cyanogen bromide fragments identifies 186 amino acids of sequence, which represents over 50% of the rat protein. Attempts to sequence the intact protein directly indicated that the amino terminus is blocked.

Recently, we have cloned and expressed the human ana- logue of rat lipocortin (11). It is structurally and immunolog- ically related to the rat protein and has an apparent mass by SDS-PAGE of about 37 kDa. For each of the rat polypeptides described in Table 11, we have identified the cbrresponding region within the human sequence, based on the high degree of sequence homology between the two proteins. Out of the 186 amino acids that we compared, only 23 residues were different. This information is summarized schematically in Fig. 7. Differences between the two sequences are indicated in the figure and then listed at the bottom of the panel. Each of the observed differences could be generated by a single

abcd e f 0 .. .. .. 1 r l 1 1 1 I !, 100

T3 T31 T19a T37 T29

? ! 101 I rnn

I I 200 T3E T9 T13 T19b

CnBrP

! ? ! " ? P ?! ? !! 201 [ I!, I l l 1 I I I I , !300

T17a T35 T23 T22b T15 T26 T18 CnBrl

301 --- 346

v w

T22a-17b T6

Amino acid differences between rat and human sequences Amino acid Designation Rat Human

12 13 16 17 24 28 41

112 124 211 232 235 239 241 245 251 283 284 289 293 295 310 313

a b

d e f

h g

i

k j

1 m n

P 9 r

t

C

0

S

U

V W

CY5 Leu LYS Gln Ala

Ser Met Leu Gln His LYS Asn -4% Gln Ala TYr Glu Ala Arg Thr

Val Glu

?'yr

Trp Phe Asn Glu Thr Ser T h r Leu -4% Glu Gln

LYS Thr LYS Val His Gln Val His Ala ASP Ala

FIG. 7. Comparison of rat and human sequences. The distri- bution of potential trypsin cleavage sites within human lipocortin is shown schematically in the top panel. These sites are indicated by the hash marks along the base-line. Open boxes denote sequenced rat polypeptides described in Table I1 that were localized within this framework based on their homology to human sequences. Differences between the rat and human sequences are indicated above their appropriate position in the schematic and then summarized at the bottom of the panel.

nucleotide change. From the partial analysis presented, we estimate that the rat and human protein sequences are ap- proximately 90% homologous. Furthermore, we infer that there are no gross changes in the portions of the rat protein that were not sequenced since the rat and the recombinant human proteins both are similar in size by gel analysis and both have similar cleavage profiles based on parallel analyses of tryptic fragments by reverse-phase HPLC.

Cellular Sources of Inhibitor Protein-We have screened by protein blotting a variety of cell lines, tissues, and organs for the presence of the 37-kDa protein. In these analyses, fresh cells or tissue were disrupted in lysis buffer containing 5 mM N-ethylmaleimide and then boiled in SDS sample buffer. Lysates were fractionated by SDS-PAGE and the proteins blotted electrophoretically onto nitrocellulose sheets. The blots were incubated with the specific antiserum against the rat 37-kDa protein and immunoreactive bands visualized with a second antibody conjugated with horseradish peroxidase.

The profiles in lanes g and h of Fig. 8 demonstrate the specificity of the antiserum. Lane g shows immunoreactive

4244 Rat 37-kDa Phospholipase A2 Inhibitor

""_ a b c d e f g h i j k l " - . - TABLE I l l Detection of.77-kDa protein in rot tissues

Fresh tissues from a male Wistar rat (1 g/4 ml huffer) were suspended in lvsis huffer and homogenized with a polvtron unit. Particulate dehris was removed by centrifugation (10 min, 10.000 x R ) and the supernatants boiled for 3 min in sample hufler. Parallel sets of cell lvstates, each containing approximately 10 pg of protein, were suhjected to SDS-polvacwlamide gel electrophoresis and then analyzed either hv Coomassie staining or hv \Vestern analysis. Rela- tive amounts of the 37-kDa Drotein are indicated in the tahle.

Tissue

Spleen Thymus Lung Kidney Smooth muscle Heart Hrain Liver Plasma Red blood cells White hlood cells Peritoneal cells

Resting Activated

U'estem (protein)

++++ +++ +++ ++ ++ + - - - - -

+ ++++

FIG. 8. Immunoreac t ive p ro te ins in rat and mouse cel ls . Cell lysates were disrupted in sample huffer and proteins suhjected to SDS-PAGE. Duplicate samples were analvzed by staining with Coo- massie Blue (lanes a-f) and hv \Vestern blot analysis (lanes p / ) . 1.onr.s a and g. resident rat peritoneal cells; lanes h and h, oil-elicited rat peritoneal cells: lanes c and i, mouse RAW 264.7 monocyte/ macrophage line; lanrs d and j , mouse SP2/0 hvhridoma line: lanes e and k, mouse L929 fihroblast line; lanrs f and I, molecular weight markers.

proteins from resident rat peritoneal cells. Lane h is from oil- elicited rat peritoneal cells. Lanes a and b show Coomassie- stained gel profiles of proteins from the same two prepara- tions. While the pattern of total protein is very similar in the two cases, the cells from the oil-treated rats are enriched for the 37-kDa protein. They contain about 10 times as much of the immunoreactive protein as the resident cells from un- treated rats. Fractionation of the oil-elicited cells on a Percoll gradient into monocyte and granulocyte-rich populations re- vealed that both cell types contained elevated levels of lipo- cortin (not shown). This suggests that the increase in lipocor- tin is due to cell activation and not a result of an alteration in the cell population.

Variations in expression levels of lipocortin also were de- tected when organs from rats were screened by immunoblot- ting analysis. Table I11 summarizes the results from some of these studies. We have determined that lung, spleen, and thymus tissue are the richest sources of the 37-kDa protein. Immunoreactive protein was detected a t lower levels in kidney and smooth muscle. The protein was not detected in brain, liver, or various components of blood.

To evaluate better the types of cells that express the inhib- itor, we have screened for the 37-kDa protein in various cell lines. As shown in Table IV, immunoreactive protein was detected in examples from each of the cell t-ypes that were investigated. This result is illustrated best in the lines derived from macrophages, fibroblasts, and epithelial cells. In the eight monocyte/macrophage lines, dramatic variations in the level of expression of the 37-kDa protein were observed, ranging from no detectable protein in the WEHI-3 line to a high level of expression in the RAW 264.7 line. The maximum level of expression represents about 0.5% of the total protein. The 37-kDa protein was detected in a muscle cell line (G8), a

TABLE IV Cellular specificity of .'U-/:l)a profrin

Cell line Protein

Monocytes/macrophages RAM' 264.7 (mouse) ++++ WR19 34.1 (mouse) +++ J774A.l (mouse) ++ P388 Dl (mouse) + WEHI-3 (mouse) US37 (human) THP-1 (human) + HL-BO (human) +

- +

Fihrohlasts L929 (mouse) HSDMlCl (mouse) Halh/c CL.7 (mouse) NIH 3T3 (mouse) CV-1 COS-; (monkey)

Epithelial cells HT29 (human) CCD-2lsk (human) CCD-118sk (human) CHO (hamster)

++++ ++++ ++++

++ ++

+++ +++ ++ ++

Nerve cells PC-12 (rat pheochromocytoma) - Neuro 2A (mouse) +

Other G-8 (mouse muscle) ++++ MLg 2908 (mouse lung) ++++ CTLL-2 (mouse T-cell) + SP2/0 (mouse hvhridoma) - P815 (mouse mastocytoma) + C6 (rat glioma) ++++ K562 (human erythroleukemia) +++

mastocytoma line (P815), a glioma line (CS), an erythroleu- kemia line (K562), and a neuroblastoma line (Neuro 2A). Lanes i-k of Fig. 8 show Western analysis from three of the cell lines described in Table IV. The SP2/0 mouse hybridoma line demonstrates a cell line that is not producing the inhibitor protein. RAW 264.7 and L929 lines are examples of mouse

Rat 37-kDa Phospholipase AP Inhibitor 4245

macrophage and mouse fibroblast lines that express the pro- tein at high levels.

In cell lysates where the 37-kDa protein was enriched, other minor immunoreactive species at 70, 55, 30, 24, and 15 kDa frequently were observed. The relative amounts of these re- lated species varied from sample to sample. Other groups also have observed a family of immunologically related proteins with similar mass distribution to those we describe (8, 10). While we already have established the structural relationship between the 30- and 37-kDa forms, characterization of these other species will require further analysis. The immunoreac- tive 35-kDa band presumably is an unrelated protein, since we have observed that it is not reactive with an antiserum against recombinant human lipocortin that readily reacts with the other species.

DISCUSSION

We have used a combination of DEAE-cellulose and gel filtration chromatographies to purify the major phospholipase A, inhibitor protein found in rat peritoneal exudates. It is a 37-kDa protein that inhibits phospholipase A2 activity both in an in vitro inhibition assay and in a cellular assay in which prostaglandin production was monitored. The inhibitor is trypsin-sensitive, destroyed by boiling, and inactivated by treatment with heparin. That this particular protein inhibits phospholipase A2 was verified by three independent sets of experiments. First, during its purification, column profiles of the inhibitory activity paralleled elution profiles of the 37- kDa protein. Second, the same protein was recognized specif- ically by a neutralizing antibody. Third, we have cloned and expressed its human analogue. Like the rat protein, the re- combinant human protein is a potent inhibitor of phospholi- pase A2 activity. Properties of the inhibitor suggest that it belongs to the family of phospholipase inhibitory proteins referred to as lipocortin. The 37-kDa protein was detected in a wide variety of cell lines, tissues, and organs with no obvious specificity. Expression levels in these preparations ranged from 0 to 0.5% of the total protein.

By comparing the rat sequences with the predicted sequence of its human analogue, we conclude that the primary structure of the inhibitor is highly conserved. In fact, out of the 186 amino acid residues identified by protein sequence analysis, only 23 were different. Differences in sequence appear to be clustered within the molecule, leaving large stretches of amino acid residues with perfect homology. The most variable re- gions were near the amino terminus and within the carboxyl- terminal third of the protein. Both the rat and human poly- peptides are very polar molecules, with approximately 30% of the residues represented by charged amino acids. In addition both proteins contain consensus sequences for phosphoryla- tion by tyrosine and serine kinases. Phosphorylation has been implicated as an important control mechanism through which the activity of the phospholipase A2 inhibitor proteins is regulated (7, 20). Analogous proteins also were detected in cell lines derived from mouse, monkey, and hamster tissues. However, none of these proteins have been sequenced. The data presented here are the first detailed structural analysis of a mammalian phospholipase inhibitor protein. The se- quence and peptide maps generated for the 37-kDa protein should be diagnostic for comparative analysis with d a t e d proteins.

The rat protein also was immunoreactive with an antiserum raised against a 6-kDa phospholipase inhibitor protein from snake venom. This antiserum presumably recognizes the re- gion of the 37-kDa protein that interacts with phospholipase A2, since it blocks the inhibitory activity. The antigenic sim-

ilarity of the rat and snake venom protein suggests that, like phospholipase A2, the inhibitor protein has highly conserved structural elements. Structural similarities for phospholipase Az molecules have been demonstrated by x-ray crystallo- graphic studies for soluble enzymes from diverse origin, in- cluding the enzymes from porcine pancreas (21) and from snake venom (22).

Phospholipase inhibitory activity, which has been attrib- uted to lipocortin-like proteins, is induced by steroids (2,3, 6, 9). Flower and co-workers (1) have suggested that this re- sponse involves two phases: a rapid release of existing pools of inhibitor from cells occurs within an hour after treatment, followed by a slower phase that requires synthesis and release of additional protein. To determine if the inhibitor we char- acterized also is regulated by steroids, levels of mRNA in untreated cells and in dexamethasone-treated cells were com- pared by Northern blot analysis (11). In resident rat perito- neal cells, we observed about a 6-fold increase in the lipocor- tin-specific mRNA within 2 h after steroid treatment. Similar results were obtained with primary cells grown in culture, confirming that this gene is regulated by steroids. In the same sets of experiments, there was no apparent change in the amount of 37-kDa protein detected by protein blotting. How- ever, we have observed dramatic differences by comparing resident and oil-elicited peritoneal cells (Fig. 8). Because of the complexity of factors that control the inflammatory state of modulatory cells, this particular aspect of regulation awaits a more rigorous analysis. As potential mediators of the anti- inflammatory action of steroids, the lipocortin family of phos- pholipase inhibitors represents an exciting family of modula- tory proteins.

Acknowledgments-We thank Richard A. Flavell and Vicki Sat0 for their support and for critical reading of the manuscript, Roderick Flower for helpful discussions, and Neenyah Ostrom for typing the manuscript.

REFERENCES

1. Blackwell, G., Carnuccio, R., DiRosa, M., Flower, R., Parente, L., and Persico, P. (1980) Nature 287, 147-149

2. Hirata, F., Schiffmann, E., Venkatasubramanian, K., Salomon, D., and Axelrod, J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,

3. Cloix, J. F., Colard, O., Rothhut, B., and Russo-Marie, F. (1983)

4. Flower, R. J., Wood, J. N., Parente, L. (1984) Adu. Inflammation

5. Hirata, F. (1984) Adu. Inflammation Res. 7, 71-78 6. Blackwell, G. J., Carnuccio, R., DiRosa, M., Flower, R. J., Lan-

gham, C. J. S., Parente, L., Persico, P., Russell-Smith, N. C., and Stone, D. (1982) Br. J . Pharmacol. 76, 185-194

2533-2536

Br. J. Pharmucol. 79, 313-321

Res. 7,61-69

7. Hirata, F. (1981) J. Biol. Chem. 256, 7730-7733 8. Rothhut, B., Russo-Marie, F., Wood, J., DiRosa, M., and Flower,

R. J. (1983) Biochem. Biophys. Res. Commun. 117,878-884 9. Gupta, C., Katsumata, M., Goldman, A. S., Herold, R., and

Piddington, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1140- 1143

10. Hirata, F., Notsu, Y., Iwata, M., Parente, L., DiRosa, M., and Flower, R. J. (1982) Biochem. Biophys. Res. Commun. 109, 223-230

11. Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press

12. Laemmli, U. K. (1970) Nature 227, 680-685 13. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad.

14. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981)

15. Hunkapillar, M., Lujan, E., Ostrader, F., and Hood, L. (1983)

Sci. U. S. A. 76, 4350-4354

Anal. Biochem. 118, 197-203

Methods Enzymol. 91, 227-236

4246 Rat 37-kDa Phospholipase A2 Inhibitor 16. Hunkapillar, M., and Hood, L. (1983) Methods Enzymol. 9 1 , 20. Hirata, F., Matsuda, K., Notsu, Y., Hattori, T., and DelCarmine,

17. Sigel, M. B., Sinha, Y. N., andVanderLaan, W. P. (1983)Method.s 21. Dijkstra, B. W., Drenth, J., Kalk, K., and Vandermaelen, P. J.

18. Braganca, B. M., Sambray, Y. M., and Sambray, R. Y. (1970) 22. Keith, C., Feldman, D. S., Deganello, S., Glick, J., Ward, K. B., Eur. J. Biochem. (Tokyo) 13,410-415 Jones, E. O., and Sigler, P. B. (1981) J. Biol. Chem. 256,8602-

19. Pepinsky, R. B. (1983) J. Biol. Chem. 258,11229-11235 8607

486-493 R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,4717-4721

En~ymol. 9 3 , 3-12 (1978) J. MOL. nioz. 124,53-60