Rapid purification of two lipocortin-like proteins from mice lung

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 153, No. 3, 1988

Pages 1271-1275

June 30, 1988

RAPID PURIFICATION OF TWO LIPOCORTIN-LIKE PROTEINS FROM MICE LUNG Mourad Errasfa* and Fran@oiseRusso-Marie Unitd de Pharmacologie cellulaire, Unitd associde INSERM n ° 285 / Institut Pasteur 25-28, rue du Dr. Roux 75015 Paris France Received May 17, 1988

S u m m a r v : We have purified two proteins ( 40 kDa and 36 kDa) from mice lung by the method of calcium-precipitation / EGTA solubilization and then a separation on a high anion exchanger column (Mono Q HR 5/5. Pharmacia) with a gradient of NaC1. The two proteins were strong inhibitors of t~hospholipase A2 as assessed in vitro with porcine pancreatic phospholipase A2 andI3H~-oleic acid labeled E. Coli membranes as substrate. The 40 kDa protein had a pI of 5.8 and was found to be immunologieally related to human recombinant lipoeortin I. The 36 kDa protein had a pI of 4.7 and cross-reacted with a polyclonal antibody raised against a 32 kDa human lipocortin-like protein described in human blood mononuelear cells. We report here a rapid purification of two distinct lipocortin-like proteins from mice lung. © 1988 Academic Press, "me.

Lipocortins (LCs) are phospholipase A2 (PLA2)-inhibitory proteins, they have been described as corticosteroids (CS)-inducible proteins in a number of tissues including lung (I), neutrophils (2), renomcdullary interstitial cells (3), fibroblasts (4), rat inflammatory exudate (5). LCs are thought to mediate the anti-inflammatory effect of CS. Human L C was cloned and expressed in E.Coli (6), and this allowed to associate L C with other proteins, since a family of calcium / phospholipid / actin-binding proteins (7-10) identifiedas substratc of protein tyrosine kinases and as cytoskelctalproteins were shown to have sequence homologies and immunological cross reactivitieswith human L C (6). All these described proteins are inhibitorsof PLA2, and the mechanism of PLA2 inhibitionwas shown to be due to the binding of L C to the phospholipid substrate in a calcium-dependent manner rather .thanto a direct interactionof L C with PLA2 (11,12). In thispaper, we report a rapid purificationof two PLA2 inhibitoryproteins from mice lung. Immunological studies show that the mice 40 kDa is related to the human recombinant L C I, and the mice 36 kDa * To whom all correspondence should be addressed. Abbreviations: lipocortin (s): LC(s). Corticosteroids: CS. phospholipase A2: PLA2. Hepes: 4- (2-hydroxyethyl)- 1 -piperazineethanesulfonic acid. EDTA: ethylenediamine-tetraacedc acid. PMSF: phenylmethanesulfonyl fluoride. EGTA: ethyleneglycol -bis (beta-aminoethylether) - N , N'-tetracetic acid. FPLC: fast protein liquid chromatography. SDS: Sodium dodecyl sulfate. PAGE: polyacrylamide gel electrophoresis. IEF: isoelectric focusing. BSA-FAF:bovine serum albumin-fatty acid-free. TCA: trichloroacetic acid. PBS: phosphate buffered saline. 0006291X/88 $1.50 1271

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 153, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

related to a 32 kDa LC purified from human blood mononuclear cells (13). In the accompanying paper, we report that these two proteins inhibit cellular PLA2 activity of whole cells. Materials and Methods

Protein extraction and nurification Mice lungs were removed, placed in buffer I (10mM Hepes, 0.15 M NaC1, 5 mM EDTA, 0.2 mM PMSF, 200 U/ml aprotinin, pH 7.4), homogenized (VIRTIS, 4°C, 5 x 15 sec at 5000 rpm), then sonicated (SONIMASSE $20, 4 °C, 3x15 sec, 200 W) and centrifuged 30 rain at 20,000 g at 4 °C (Sorvall RC-5B centrifuge, Du Pont Instruments). Into the resulting supernatant (S 1), CaC12was added to a final concentration of 3 raM. S 1 was kept at 4 °C under shald~g for 3 hours then centrifuged for 30 rain at 40,000 g at 4 °C. The pellet was resuspended in buffer 1I ( 10 mM Hepes, 0.15 M NaCI, 5 mM CaCI2, 0.2 mM PMSF, 200 U/ml aprotinin, pH 7.4) and centrifuged for 30 min at 40,000 g at 4 °C. The pellet was then resuspended in 10 mi of buffer HI (10 mM Hepes, 10 mM EGTA, pH7.4) and centrifuged for 40 rain at 100,000 g at 4 °C (TGA-65 Ultracentrifuge, Kontron). The supernatant $2 was kept at 4 °C for further analysis in FPLC system and SDS-PAGE. The supernatant $2 was mixed with buffer A (20 mM ethanolamine, 0.05 mM PMSF, 200 U/ml, aprotinin, pH 9) and subjected to a Mono Q HR 5/5 column previously equilibrated with the same buffer. Then a NaCI gradient was generated from 0 to 0.35 M over 35 rain with buffer B (buffer A supplemented with 1 M NaC1). The FPLC fractions were tested for their ability m inhibit porcine pancreatic PLA2 in ~tro. The samples which possessed an inhibitory activity on PLA2 were analysed in SDS-PAGE and in native IEF. Gradient SDS gels (10-15% acrylamide) and IEF (1% agarose containing Pharmalyte 4-6.5) were performed using the Phmnacla Phastsystem according to the manufacturer's instructions. Proteins were visualized by silver staining using the Pharmacia developer system. Proteins were measured by the method described by Lowry et al.(14). The FPLC fractions were concentrated and desalted by mean of a microconcentrator system (centricon 10) before testing their activities in vitro. Buffer mixtures eluting as each protein, desalted and concentrated in the same way as the protein FPLC fractions were always used as vehicle of the protein. Determinationof anti-PLA2 activity The anti-PLA2 activity of each fraction obtained after the FPLC separation was performed as previously described (4). Briefly, 50 gl of a protein fraction, or 50 gl of its corresponding vehicle were preincubated for 10 rain with 60 ng of porcine pancreatic PLA2 in a final volume of 3~5Oglin 100 mM Tris-HCl, pH 8 at 4 °C. Reactions were inititiated by addition of 50 gl oflaHJ-olcic acid labeled E.Coli membranes (125 000 cpm) as the susbtrate. T~ae reactions were terminated 10 min later by the addition of 50 gl of 2 M HCI. The released[~HJ -oleic acid was trapped in the supernatant by addition of 100 gl of BSA-FAF (100 mg/ml). The samples were centrifuged at 10,000 g for 5 rain to remove particulate materials, an aliquot of the supematant was counted by liquid scintillation spectromeFy to determine radioactivity. Results were expressed as the percent inhibition of released[3H]-oleic acid in the presence or absence of the protein to be tested minus blanks where PLA2 was omitted. Western blot analvsis The proteins were subjected to elcctrophoresis using the Pharmacia Phastsystem. The gels were detached carefully from the backing sheet and applied on a nitrocellulose sheet as described in (15). The proteins were transfered onto the nitrocellulose sheet in an EMBL transfer-biot apparatus for one hour at 4°C in 20 mM Tris, 150 mM glycine and 20% methanol buffer according to the procedure described by Towbin et ai.(16). The nitrocellulose sheet was then incubated for 30 rain in 3% TCA, 0.2% Ponceau S to visualize the transfered proteins. The ni~oceilulose sheet was washed with distilled water, and then incubated for one hour at 37°C in phosphate buffered saline (PBS) calcium and magnesium free, containing 5% powdered skim milk (R6gilalt, France) to saturate the non-specific sites. The nitrocellulose sheet was then washed five times with PBS and then incubated for one 1272

Vol. 153, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hour at 37 °C with an appropriate dilution of the required antiserum in PBS-5% powdered skim milk (PBS-PSM). The nitrocellulose sheet was washed again 5 times with PBS and incubated for one hour at 37 °C with the anti-rabbit Ig, biotinylated species-specific F(ab') fragment (from donkey) diluted 1:500 in PBS-PSM. The sheet was then washed with PBS and incubated for one hour at 37 °C with the Streptavidin-biotinylated horseradish peroxidase complex diluted 1:400 in PBS-PSM. The sheet was washed again with PBS and incubated with the detection reagent ( 0.5 mg/ml diaminobenzidine in PBS containing 0.02% H202 ) for 5 rain. ]~esults and Discussion The purification on a Mono Q column of the protein extract obtained from mice lung has given two main peaks eluting at 0.20 M (peak a) and 0.25 M (peak b) of NaC1 (Fig. 1A). Both these proteins strongly inhibit porcine pancreatic PLA2 in a dose dependent m~n_ner o n [3H]-oleie acid labeled E. Coli membranes (Fig 1B). When analysed in

SDS-PAGE, the two peaks have an apparent molecular weight of 40 kDa (peak a) and 36 kDa (peak b) respectively (Fig.2A, lanes 1,2) and were corresponding to two major proteins of the total protein extract of mice lung (Fig 2A, lane 3 ). As shown in Figure 2]3, the pls of the 40 kDa (lane 1) and the 36 kDa (lane 2) proteins are of 5.8 and 4.7 respectively. The protein extract of mice lung and the purified proteins on the Mono Q column were screened with an antisenam raised against human recombinant LC I (kindly given by Dr Jeff Browning, Biogen, Cambridge, Ma). Figure 3A shows that this antiserum recognizes the purified 40 kDa (Fig. 3A, lane 1) whereas it does not recognize the purified 36 kDa (Fig.3, lane 2). Nevertheless, this anti-LC I antibody cross reacts with two proteins in the total protein extract migrating at the apparent Mrs of 40 kDa and 36 kDa (Fig 3A, lane 3) demonstrating that another species of LC I-like protein with another Mr (36kDa) is present in

e

A

0.2.

_~

80

,.-. 1

I

60'

E t-

,:

O GO

~0.1.

a

i I I I

40

b

0 0 c a

Z

20 ¸

0

=no o

1o

go

0,1

80

Elution volume (ml)

1 PROTEIN ( pg )

Fit,ure I A- Chromatographic profile of the purified proteins 40 kDa (peak a) and 36kDa (peak b) fxommice lung on a Mono Q column. The supematant $2 was applied onto the column and the proteins eluting with a gradient of NaCI were detecuxlby absorbanceat 280 nm. B- Dose-responsecurve of mice purifiedproteins on PLA2 activity. Different .a~unts of the purified proteins were incubated with 60 ng of porcine pancreatic PLA2 usins[ H]-oleicacid labeled E. Coli membranesas substrate.Results are expressedin % iahibitioffof PLA2 activity. ( • ) : 40 kDa protcin, ([]) : 36k Da protein. 1273

10

Vol. 153, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

1

2

B

3

1

2

6.5

• ~..~ :.

..::...."

94 67 43

•

~.-~

.

•. . . .

:

: t.::.::

30

.~,~

~ .--,,~

~

:-!:..

• : :':i~:,i

20 ~ . 14

:

. :

:i

i •

...

5.8

5.2

::!;

.

/!'")~,'~ .

•

[ ~ !

:••

•L;••

•

• • i :::.i .

•

i• :

•.

4.5

•

....•

4.1

FimLre2 : SDS-PAGE a~A IEF of the purified proteins. Samples eluting from the Mono Q column separation were analyzed by SDS-PAGE (10-15 % gradient gels) and IEF (pH gradient 4-6.5) and visualized by silver staining. A- SDS-PAGE : On the left are indicated the moleettlar weight markers in kDa: phosphorylase b, 94; bovine serum albumin, 67; ovalbumin, 43; carbonic anhydrase, 30; trypsin inhibitor, 20.1; alpha-lactalbumin, 14.4. Lane 1, 2, 3 represent the analysis of the peaks a and b (eluted on the Mono Q colunm) and the total protein extract of mice lung respectively. B- IEF : On the right are indicated the pI markers: human carbonic anhydrase, 6.55; bovine carbonic anhydrase, 5.85; beta-laclalb, min A, 5.2, soybean trypsin inhibitor, 4.55; glucose oxidase, 4.15. Lanes 1 and 2 represent the pI analysis of peaks a and b eluted on the MonoQ column respectively.

A

1

94 67

-* ---

43

---

30

---

2 0 -~ 14 ..~

2

3

4

5

:~ :. • ::

:.~.!:/..~ii.:;.~/i ~ / / . :i::!:;..~. . :

.. .

.:~:-.; ../.". . : ;.:".!....

.... •

4"

•[••::

:

••••

•• •••:••

:,,,-;:.:~!~il :

!.

:L

B

1

2

3

4

5

" :

i•• ••

•

I i..:

•, ,,,~ i,/,,:.: ,:,, ,,,:! ,,, ~,,:,:),~iii::: ;.:

;-

i... •

::. ' . -i :

:

•

:•••

~iii..

.::,.

" "

." •

:

••

• •

•

• ~ " •

•

" "': i

;:':!"i ql;

•

i...

[:

i

Figure 3 : Western blot analysis. Western blot analysis of the proteins using an antiserum (1: 3000) raised against human recombinant LC I (panel A) or an antiserum (1: 500) raised against the 32 kDa LC protein purified from human blood mononuclear cells (panel B). Lane 1: purified mice 40 kDa, lane 2: purified mice 36 kDa, lane 3: the total protein extract of mice lung, lane 4: human recombinant LC I, lane 5: the human 32 kDa LC. The arrows on the left represent the migration of the molecular weight markers. 1274

Vol. 153, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the total protein extract. This protein was present in the flow through of the Mono Q column as assessed by Western blot analysis (data not shown). Thus it appears that in mice lungs there exists at least two protein species immunologically related to human recombinant LC I possessing different Mr and different charges since they do not eoelute on a strong anionic exchange resin. Using an antibody directed against LC II (kindly donated by Dr J. Browning), neither of these proteins (the purified 40 kDa and 36 kDa) were recognized (data not shown). The same proteins were screened with an antiserum raised against a 32 kDa LC from human blood mononuclear cells (13). This antiserum recognizes only the 36 kDa protein either purified (Fig 3B, lane 2) or present in the total protein extract (Fig 3B, lane 3). Neither the mice 40 kDa nor the human recombinant LC I cross react with the 32 kDa antibody (Fig 3B, lanes 1 and 4). In this paper, we report a rapid purification and a semi-characterization of two distinct PLA2 inhibitory proteins from mice lung. The 40 kDa and the 36 kDa proteins are immtmologically related to human recombinant LC I (6) and to the human mononuclear ceils 32 kDa LC (13) respectively. Both of these proteins have a strong inhibitory effect on soluble porcine pancreatic PLA2 in an in vitro assay and are also able to inhibit cellular PLA2 of isolated guinea-pig alveolar macrophages in vitro as it is reported in the accompanying paper.

References °

2. 3. 4. 5. .

,

8.

9. 10. 11. 12. 13. 14. 15. 16.

Blackwell, G., Carnuccio, R., DiRosa, M., Flower, R., Parente, L., and Persico,P. (1980) Nature 287, 147-149 Hirata, F., Schiffmann, E., Venkatasubramanian, K., Salomon, D., and Axelrod,J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2533-2536 Cloix, J.F., Colard, O., Rothhut, B., and Russo-Marie, F. (1983) Br. J. Pharmacol, 79, 312-321 Errasfa, M., Rothhut, B., Fradin, A., BiUardon, C., Junien, LL., Bure, J., and Russo-Marie,F., (1985) Biochim. Biophys. Acta. 847, 247-254 Pepinsky, R.B., Sinclair, L.K., Browning, J.L., Mattaliano, R.J., Smart, J.E., Chow, E.P., Falbel, T., Ribolini, A., Garwin, J.L., and Wallner, B.P., (1986) J. Biol. Chem. 261, 4246-4249 WaUner, 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. 321, 81-84 Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645 De, B.K., Misono, K.S., Lukas, T.J., Mroezkowski, B., and Cohen, S., (1986) J. Biol. Chem. 261, 13784-13792 Gerke, V., and Weber, K. (1984) EMBO J. 3, 227-233 Glenney, L (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4258-4262 Davidson, F.F., Dennis, E.A., PoweU, M., and Glenney, J.R. (1987) J. Biol. Chem, 262, 1698-1705 Aarsman, A. J., Mynbeek, G., Van Den Bosch, H., Rothhut, B., Prieur, B., Comera, C., Jordan, L., and Russo-Marie, F. (1987) FEBS lett, ~ 176-180 Rothhut, B., Comera, C., Prieur, B., Errasfa, M., Minassian, G., and Russo-Marie, F. (1987) FEBS Lett, 219, 169-175 Lowry, O.H., Rosebrough, N.J., and Randall,R.J. (1951) J. Biol, Chem. 193, 265-275 Prieur, B. and Russo-Marie, F. (1988) Anal. Biochem. (in press) Towbin, H., Staechelin, T., and Gordon, J. (1979) Proc. Natl. Aead. Sci. 76, 4350-4354 1275