1986 Journal Biol Chem by Tanya Falbel

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

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Q 1986 by The American Society of Biological Chemists, Inc.

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, JohnE. 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 exudates37a these inhibitors is a 40-kDa protein, although other species kDa protein that inhibits phospholipase At activity. It with apparent masses of 15, 30, 55, and 70 kDa have been is the predominant phospholipase inhibitor protein in detected. These additional forms all are immunologically rethese preparations andalso is detected in a wide vari- lated to the40-kDa protein (8,lO).Partially purified inhibitor ety of cell lines. Levels of expression range from0 to proteins have been shown to be active at three levels. First, 0.5%of total protein. In the peritoneal preparations, theyinhibit phospholipase A2 activity in in vitro assays. the inhibitor is partially proteolyzed into a series of Second, they block prostaglandin production in cellular aslower mass forms, includingspecies at 30, 24, and 15 says. Third, they reduce inflammation in in situ model syskDa. These fragmentsall are immunoreactive with an tems for inflammation. These proteins all are referred to as antibody raised against the 37-kDa protein. The rat lipocortin, which replaces three other names (macrocortin, protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor pro- lipomodulin, and renocortin) previously used to describe the same set of molecules. tein fromsnake venom. The primary structure of more Inthis report we describe the purification andpartial thanhalfoftheratinhibitorhasbeendeducedby protein microsequence analysis. These sequences are sequence analysis of the major phospholipase AB inhibitor closely related to sequences fromits human analogue, protein found in rat peritoneal exudates. It has a mass of 37 which we recently cloned and expressed (Wallner, B. kDa and is closely related to a humanphospholipase inhibitor P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, protein that we recently cloned and expressed (11). The 37R., Sinclair, L. K., Foeller, C., Chow,E. P., Browning, kDa protein was detected in cultured cells of diverse origin, J. L., Ramachandran, K. L., andPepinsky, R. B. including lines derived from macrophages, fibroblasts, and epithelial cells. Expression levels of the inhibitor in these (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Proper- lines varied from 0 to 0.5% of total protein. ties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins MATERIALS AND METHODS collectively referred to as lipocortin. 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 mlof the glucocorticoid dexamethasone phosphate (Lark Laboratories, 1.25 mg/kg rat) in 0.9% NaCl. One h Prostaglandins and leukotrienes, which are produced in after injection, the ratswere killed with Euthasate and the peritoneal response to an injury, are potent mediators of inflammation. cavities rinsed essentially as described (6) with 10 ml of phosphateBoth families of molecules are derived from a common fatty buffered saline (50 mM KHzP04, pH 7.3,150 mM NaCl) containing 2 acid precursor, arachidonic acid, which can be released from units/ml heparin and 50 p M phenylmethanesulfonyl fluoride. The lavages were cleared of cells and other particulate matter by centrifcell membranes by phospholipase A2or through combination a ugation for 30 min at top speed in an International centrifuge. The of actions by phospholipase C and diacylglycerol lipase. Al- supernatants were combined and additional protease inhibitors though the inflammatory response involves numerous steps, added. These included aprotinin to 20 pg/ml, soybean trypsin inhibcompounds that inhibit prostaglandin and leukotriene pro- itor to10 pg/ml, and EGTA’ to 0.5 mM. The exudates were incubated duction reduce inflammation. Recently, a family of proteins at 37 “C for 1 h in the presence of 0.1 units/ml calf intestinal alkaline that blocks inflammation has been identified (1-3). These phosphatase and subjected to the purification protocol described proteins also inhibit phospholipase A2 activity and thus pre- below. Phospholipase Az Assay-Samples were tested for phospholipase sumably stop inflammation at this early stage. Regulation of A2 inhibitory activity by an in vitro assay described previously (8). the expression and activity of these inhibitory proteins by The substrate for phospholipase Az, autoclaved [3H]oleicacid-labeled glucocorticoidshas been postulated asthe mechanism through Escherichia coli, also was prepared as described (8).For each experiwhich steroids act as anti-inflammatory agents (for reviews 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 see Refs. 4 and 5 ) . dilute preparation of porcine pancreatic phospholipase Az (Sigma) Phospholipase A2inhibitory proteinshave been detected in that contained 100 ng of enzyme and 125 pg of bovine serum albumin. a number of systems, including rat macrophages ( 6 ) , rabbit Samples were mixed and kept on ice for 1 h. 25 yl of the substrate neutrophils (7), rat renal medullary cells (8), and murine and was added, and the reaction was performed at 6 “C for 8 min. The

bovine thymus preparations (9). The predominant form of

* This work was supported by Biogen Research Corp. and Yamanouchi 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 accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

The abbreviations used are: EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid; HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; 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 quantitated by liquid scintillation counting. In all analyses, samples were assayed in duplicate and adjusted for nonspecific release by subtracting 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 belowthe 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 of20mM Tris-HC1 pH 7.7. The dialysate was subjected to ion-exchange chromatography on a DEAE-cellulose column (Whatman, 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 membrane). 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 lyophilized. 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 electrophoresis. The position of the radioactive marker was determined by autoradiography of the wet gel at 4 "C. After electroelution, the 0.2 M N sample was lyophilized and resuspended in 200plof 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% trichloroacetic 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 bya 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-Tosylamido2-phenyletbyl chloromethyl ketone-trypsin (Worthington,5 pg total) was added to the 100 pg of rat protein and the digestion performed for 16 h a t 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% trifluoroacetic acid) at a flow rate of1.4 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 sequenator 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 cyclewere analyzed by reversephase 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 at313 nm. Aliquots from

I fraction number

FIG. 1. DEAE column profile of phospholipase inhibitory activity from rats. Peritoneal lavages from 24 dexamethasonetreated rats were dialyzed overnight at 4 "C against 25 mM Tris-HC1 at pH 7.7 and then subjected to DEAE-cellulose column chromatography. 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 a t 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 theexudate 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 mixing for 30 s. Lysates were clarified by centrifugation for 5 min at 10,000 X g. The supernatants were boiledfor 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 thecells isolated after 3 days. Preparation of Antisera-Antisera against SDS-disrupted rat lipocortin were developed in rabbits using the lymph node immunization procedure (17). Rat lipocortin in 0.1% SDS was emulsified with Freund's complete adjuvant (25 pgof 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 of25pgof protein emulsified in Freund'sincomplete adjuvant. Production of antibodies specific for the rat protein was assessed by enzyme-linked immunosorbent 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 inhibitor 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 combination 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

TARI.EI protein was retained by the ion exchanger and subsequently Effect of rangents on i n h i h i f o pactit'ity eluted with a gradient of NaCI. Three peaks of inhibitory activity were detected a t 50, 400, and 'io0 mM NaCI, exactly Reagents at the concentrationsindicated were incuhated for 1 h a t as described previously by Flower and co-workers (6). Using 4 "C either with the 37-kDa inhibitor protein (PIP) alone or with the conditionsdescribed, approximately half of the inhibitory phospholipase A2 alone. Preparationsthen were subjected tothe DhosDholilnase A2 assav. Values listed renresent Der cent inhihition. activity flowed through the DEAE column. This preparation Az + PIP Az contains only 3-4741 of the starting protein and hasa specific Inhihitor (ronrmtration) -Ali n+h i1'11' hitor inhihitor inhihitor activity of about 200 units/mg protein. Pe; rE The inhibitor in the DEAE flow-through was further puriPhenylmethane sulfonyl fluo69 48 11 fied bygel filtration. As shownin Fig. 2.4, theinhibitory ride ( 3 mM) activity elutes as a single broad peak. Maximal activity was TLCK" (10 mM) 57 52 1 centered at fraction 19. The apparent massof the inhibitor is TPCK* (1 mM) 69 0 61 Renzamidine (10 mM) 54 64 12 about 40 kDa, based on its elution profile relative to standard 54 48 EDTA (10 mM) 2 markers. The inhibitory activityis sensitive todigestion with Heparin (0.1 mg/ml) 54 9 13 tr-ypsin and is destroyed by boiling (not shown). It also is Soyhean trypsininhihitor (5 67 66 1 I). Mostcontaminating inactivated by heparin(seeTable mg/ml) proteins were larger than 40 kDa and thus areresolved from Aprotinin (1 mg/ml) 54 76 44 63 Pepstatin A (1 mg/ml) 62 0 the inhibitor by the sizing step. Fig. 223 shows a Coomassielodoacetamide ( 5 0 mM) 63 70 28 stained profile of proteins from the same column samples Iodoacetic arid (10 mM) 63 30 64 after SDS-polyacrylamide gel electrophoresis. The two fracN-Ethylmaleimide (10 mM) 63 68 27 tions with greatest inhibitory activity, fractions 19 and 20, p-Chloromercurohenzoate ( 1 63 67 24 were found to contain a single major protein a t 37 kDa. The mM) ZnCI2 (3 mM) purified inhibitor accounts for 0.2% of the total proteinin the +8 67 50 54 37 FeCIR( 3 mM) 67 lavage preparation and hasa specific activity of 6,000-10.000 MnCI, (3 mM) 67 67 9 units/mg. In the in vitro phospholipase assay, the inhibitor +5 CuCI,! ( 3 mM) 67 65 blocks phospholipase A, in a dose-dependent manner (seeFig. CaCI, (3 mM) 67 69 11 +

3).

" T I X K , I-chloro-8-tosvlarnido-7-amino-2-heptanone. TPCK, 1,-l-tosvlamido-2-phenvlethyl chloromethyl ketone. 80

x E

2 o v 10

12 14 16 18 20

22 24 26 28

fraction number

0.0

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

0.5 1.o 1.5 pg OF LIPOCORTIN

2.0

FIG. 3. Dose-response curve of inhibitor protein. Aliquots of the purified inhihitor were incubated with 100 ng of porcine pancreatic phospholipase A,, and the samples were assayed for residual phospholipase activity. The data presented show a typical titration curve generated from such an analysis.

FIG. 2. Gel filtration chromatography of inhibitor protein. DEAE flow-through preparations desrrihrd in Fig. 1 wrre comhined. concentrated, and suhjected to gel filtration chromatography on a P1.50 column. 5-ml fractions were collected. In A, aliquots of each fraction were monitored for protein and for phospholipase A2 inhihitory artivity. In R, the same samples were analyzed hy SDS-PACE. Proteins were stained with Coomassie Blue. [-una n, markers (phosphorylase 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.

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 neutralizing antibody. This antiserum was developed against a heterologous inhibitor protein from Cobra venom and blocks the phospholipase inhibitory activity detected in the rat preparations. 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 neutralizingantibody. The 37-kDa protein is a prominent immunoreactive band in the column fractions thatwere most inhibitory (lane f ) . A second immunoreactive species at 30 ~

~~~

' R. Flower, unpublished results.

Rat 37-kDa Phospholipase APInhibitor

4242

a b c d e f g h

a b

F"-----! .

c. d .

- .

c i

FIG. 4. Western blot analysis of inhibitory fractions. 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 analvsis, immunoreactive proteins were detected with a neutralizing aniihody raised against the cobravenom inhibitor protein. Lanes a and 1,:lY-kDa inhibitory peak; lanes b andg, 15-kDa inhibitorypeak; lanes c and / I , 12-kDa region; lanes d and e, markers.

kDa is routinelyobserved.We haveshown, however, by peptide mapping that it is derived from the 37-kDa protein (see below). Both bands were the major immunoreactive species 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 proteinwas identified as a fragment of the 37kDa protein by peptide mapping analysis.Gel slices containing both polypeptides were reacted with CNBr and the cleavage products separatedby 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). Immunoreactive fragments were visualized with an antiserum raised against the 37-kDa rat inhibitor. In both analyses, the cleavage profiles of the 37- and30-kDaproteins were nearly identical, differing only in the few positions indicated. The amount of the 30-kDa band detected in the peritoneal exudates varied from preparation to preparation. Itwas enriched after prolonged storage of samples at 4"C and after mild treatment with trypsin. Partial Sequence Analysis of the Rat 37-kDaProtein-The purified rat 37-kDa protein was carboxymethylated with iodoacetic 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 withtrypsin, approximately40 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,theentire 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

FIG. 5. Cyanogen bromide mapping of the 37- a n d 3 0 - k D a proteins. Gel slices containing thespecified 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

-10

w IO

110

m I#

140 1 5 0

180 170 180 1 8 0

hac1ion number

FIG.6. Tryptic map of cleavage fragments derived from 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 CIRcolumn. Fractions were monitored simultaneously at 280 nm ( A ) and at 214 nm ( R ) .Numbered peaks signify the polypeptides that have been sequenced.

peptide components of peaks T30 and T32did not sequence at all. Insamples where peakscontained more than one polypeptide, such as for T19 and T22,each peak was rechromatographed in a subsequentHPLCstepatneutral pH. Peptides resolved by the second HPLC step were then sequenced. These fragments are denoted with a and b in the

Rat 37-kDa Phospholipase Az Inhibitor TABLE I1 Summary of sequence datafor rat polypeptides Tryptic and cyanogen bromide fragments, each containing 1-2 nm of material, were subjected to amino-terminal protein sequence analysis in a gas-phase sequenator. Designations for tryptic fragments correspond to column peaks described in Fig. 6. CNBr 1and CNBr 2 are the two small CNBr fragmentsthat were sequenced. Fragment

a. Tryptic fragments T3 T6 T9 T13 T15 T17a T17b T18 T19a T19b T22a T22b T23 T24 T26 T29 T30 T31 T32 T34 T35 T37 T38

.. ..

GTDVNVFNTILTTR KGTDVNVFNTILTTR GVDEATIIDILTK GLGTDEExLIxI

b. Cyanogen bromide fragments MKGAGTRRKTLI CNBr 1 MLKTPAQFDADELLR CNBr 2

table. Three of the tryptic fragments contained additional lysines that were not sensitive to trypsin. FragmentT29 contains the trypsin-resistant sequence Lys-Pro. Fragments T35 and T15 have lysines at their amino terminus. Aminoterminal lysines frequentlyare 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, preparations 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 andcyanogen bromide fragments identifies 186 amino acids of sequence, which represents over 50% of the rat protein.Attempts to sequence theintact protein directly indicated that theamino terminus isblocked. Recently, we have cloned and expressed the human analogue of rat lipocortin (11).It is structurally and immunologically related to the ratprotein and has an apparentmass by SDS-PAGE of about 37 kDa. For each of the ratpolypeptides 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 thefigure and thenlisted at thebottom of the panel. Each of the observed differences could be generated by a single

T31

101

T37

T29

r n n

T19a

T3E

T9 T13

100

200

T19b

CnBrP

201

GGPGSAVSPYPSFNPSSDVAA

Sequence

SYK GDYEK VYR EELK KYSQHDMNK ALYEAGQR VFYQK LYEAMK AIMVK DITSDTSGDFR SEIDMNEIK VFQNYR SYPHLR TPAQFDADELLR ALDLELK AAYLQETGKPLDETLK

abcd

4243

[

I!,

T17a

I T35

?!"? l

T23 T22b T15

T26

? !!

I ,

!300

T18 CnBrl

- 301 - -

346 T22a-17b

Amino acid differences between rat andhuman sequences Amino acid

12 13 16 17 24 28 41 112

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

Designation

Rat

Human

a b

CY5 Leu LYS Gln Ala ?'yr Ser Met Leu Gln His LYS Asn -4% Gln Ala TYr Glu Ala Arg Thr Glu Val

Trp Phe Asn Glu Thr Ser Thr Leu -4% Glu Gln

d e f g h i j

1 m n 0

P 9 r S

t U

V W

LYS Thr LYS Val His Gln Val His Ala ASP Ala

FIG. 7. Comparison of rat and human sequences. The distribution 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 approximately 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 fragmentsby reverse-phase HPLC. Cellular Sources of Inhibitor Protein-We have screened by protein blotting avariety 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 5mM N-ethylmaleimide and then boiled inSDS 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

Rat 37-kDa Phospholipase A2 Inhibitor

4244

_" a

TABLE Ill Detection of.77-kDa protein in rot tissues Freshtissuesfrom a maleWistarrat(1g/4 ml huffer) were suspendedinlvsishufferandhomogenizedwith a polvtronunit. Particulate dehris was removed by centrifugation (10min, 10.000 x R ) and the supernatants boiled for 3 min in sample hufler. Parallel sets of cell lvstates, each containing approximately10 pg of protein, were suhjected to SDS-polvacwlamide gel electrophoresis and then analyzed either hv Coomassie staining or hv \Vestern analysis. Relative 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. I m m u n o r e a c t i v e p r o t e i n s i rat n and mouse cells. Cell lysates were disrupted in sample huffer and proteins suhjected to

SDS-PAGE. Duplicate samples were analvzed by staining with CoomassieBlue (lanes a-f) and hv \Vesternblotanalysis (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, mouseRAW 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. (mouse)

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

Cell line

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

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

proteins from resident rat peritonealcells. Lane h is from oilelicited rat peritoneal cells. Lanes a and b show Coomassie(human) stained gel profiles of proteins from the same two preparations. While the patternof total protein isvery similar in the Fihrohlasts two cases, the cells from the oil-treated rats are enriched for ++++ L929 (mouse) the 37-kDa protein. They contain about10 times as much of ++++ HSDMlCl (mouse) the immunoreactive protein as the resident cells from un++++ Halh/c CL.7 (mouse) treated rats. Fractionationof the oil-elicited cells on a Percoll ++ NIH 3T3 (mouse) gradient into monocyte and granulocyte-rich populationsre++ CV-1 COS-; (monkey) vealed that both cell types contained elevated levels of lipoEpithelial cells cortin (not shown). This suggests that the increase in lipocor+++ HT29 (human) tin is due to cell activation and not a result of an alteration +++ CCD-2lsk (human) in the cell population. ++ CCD-118sk (human) Variations in expression levels of lipocortin also were de++ CHO (hamster) tected when organs from rats were screened by immunoblotNerve cells ting analysis. Table I11 summarizes the results from some of pheochromocytoma) (rat PC-12 thesestudies. We have determined that lung, spleen, and Neuro 2A (mouse) + thymus tissue are the richest sourcesof the 37-kDa protein. Immunoreactive proteinwas detected a t lower levels in kidney Other and smooth muscle. The protein was not detected in brain, muscle) G-8 (mouse ++++ MLg 2908lung) (mouse ++++ liver, or various components of blood. CTLL-2 + T o evaluate better the types of cells that express the inhib- T-cell)(mouse SP2/0 hvhridoma) (mouse itor, we have screened for the 37-kDa protein in various cell mastocytoma) (mouse P815 + lines. As shownin Table IV, immunoreactiveprotein glioma) was (rat C6 ++++ detected in examples from each of the cell t-ypes that were K562 (human erythroleukemia) +++ investigated. This result is illustrated best in the lines derived from macrophages,fibroblasts, andepithelial cells. In the eight monocyte/macrophage lines, dramatic variations in the mastocytoma line (P815), a glioma line (CS), an erythroleulevel of expression of the37-kDaprotein were observed, kemialine (K562),and a neuroblastomaline (Neuro 2A). ranging from no detectable protein in the WEHI-3 line to a Lanes i-k of Fig. 8 show Western analysis from three of the high level of expression in the RAW 264.7 line. The maximum cell lines described in Table IV. The SP2/0 mouse hybridoma level of expression represents about 0.5% of the total protein. line demonstratesa cell line that is not producing the inhibitor The 37-kDa protein was detected in a muscle cell line (G8),a protein. RAW 264.7 and L929 lines are examples of mouse

Rat 37-kDaPhospholipase APInhibitor macrophage and mouse fibroblast lines that express the protein 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 related 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 structuralrelationship between the 30- and 37-kDa forms, characterization of these other species will require further analysis. The immunoreactive 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 otherspecies. 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 anin 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 37kDa protein. Second, the same protein was recognized specifically by a neutralizing antibody. Third, we have cloned and expressed its human analogue. Like the rat protein, the recombinant human protein is a potent inhibitor of phospholipase 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 inthesepreparations ranged from 0 to 0.5% of the total protein. By comparing the ratsequences with the predicted sequence of its humananalogue, we conclude that theprimary 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 regions were near the amino terminus andwithin the carboxylterminal third of the protein. Both the rat and human polypeptides are very polar molecules, with approximately 30% of the residues represented by charged amino acids. In addition both proteins contain consensus sequences for phosphorylation by tyrosine and serinekinases. Phosphorylation has been implicated as an important controlmechanism through which the activity of the phospholipase A2 inhibitorproteins 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 sequence and peptide maps generated for the 37-kDa protein should be diagnostic for comparative analysis with d a t e d proteins. The ratprotein also was immunoreactive with an antiserum raised against a 6-kDa phospholipase inhibitor protein from snake venom. This antiserum presumably recognizes the region of the 37-kDa protein that interacts with phospholipase A2, since it blocks the inhibitory activity. The antigenic sim-

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ilarity of the rat andsnake 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 crystallographic studies for soluble enzymes from diverse origin, including the enzymes from porcine pancreas (21) and from snake venom (22). Phospholipase inhibitory activity, which has been attributed to lipocortin-like proteins, is induced by steroids (2,3, 6, 9). Flower and co-workers (1) have suggested that this response involves two phases: a rapid release of existing pools of inhibitor from cells occurs within an hour after treatment, followed bya slower phase that requires synthesis andrelease of additional protein. To determine if the inhibitor we characterized also is regulated by steroids, levels of mRNA in untreated cells and indexamethasone-treated cells were compared by Northern blot analysis (11).In resident rat peritoneal cells, we observed about a 6-fold increase in the lipocortin-specific mRNA within 2 h aftersteroid treatment. Similar results were obtained with primary cells grown in culture, confirming that thisgene is regulated by steroids. In thesame sets of experiments, there was no apparent change in the amount of 37-kDa protein detected by protein blotting. However, 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 antiinflammatory action of steroids, the lipocortin family of phospholipase inhibitors represents an exciting family of modulatory 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, 2533-2536 3. Cloix, J. F., Colard, O., Rothhut, B., and Russo-Marie, F. (1983) Br. J. Pharmucol. 79, 313-321 4. Flower, R. J., Wood, J. N., Parente, L. (1984) Adu. Inflammation Res. 7,61-69 5. Hirata, F. (1984) Adu. Inflammation Res. 7, 71-78 6. Blackwell, G. J., Carnuccio, R., DiRosa, M., Flower, R. J., Langham, C. J. S., Parente, L., Persico, P., Russell-Smith, N. C., and Stone, D. (1982) Br. J. Pharmacol. 76, 185-194 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. 81,11401143 10. Hirata, F., Notsu, Y., Iwata, M., Parente, L., DiRosa, M., and 109, Flower,R. J. (1982) Biochem.Biophys.Res.Commun. 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. Sci. U. S. A. 76, 4350-4354 14. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R.(1981) Anal. Biochem. 118, 197-203 15. Hunkapillar, M., Lujan, E., Ostrader, F., and Hood, L. (1983) Methods Enzymol. 9 1 , 227-236

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16. Hunkapillar, M., and Hood, L. (1983) Methods Enzymol. 9 1 , 486-493 17. Sigel, M. B., Sinha, Y. N., andVanderLaan, W. P. (1983)Method.s En~ymol.9 3 , 3-12 18. Braganca, B. M., Sambray, Y. M., and Sambray, R. Y. (1970) Eur. J. Biochem. (Tokyo) 13,410-415 19. Pepinsky, R. B. (1983) J. Biol. Chem. 258,11229-11235

A2Inhibitor 20. Hirata, F., Matsuda, K., Notsu, Y., Hattori, T., and DelCarmine, R. (1984) Proc. Natl. Acad. Sci. U.S. A. 81,4717-4721 21. Dijkstra, B. W., Drenth, J., Kalk, K., and Vandermaelen, P. J. (1978) J. MOL. nioz. 124,53-60 22. Keith, C., Feldman, D. S., Deganello, S., Glick, J., Ward, K. B., Jones, E. O., and Sigler, P. B. (1981) J.Biol. Chem. 256,86028607