fk-506 binding protein from tolypocladium inflatum: resistance of fkbpfk-506 complex against...

Vol. 182, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 14, 1992 Pages 1282-1287

FX-506 BINDING PROTEIN FROM TOLYPOCLADIUN INFLATUM: RESISTANCE OF FKBP/FK-506 COMPLEX AGAINST PROTEOLYSIS

Chan Lee, Kai Hoffmann and Rainer Zocher*

Institut fur Biochemie und Molekulare Biologie, Technische.Universit&t Berlin,

Franklinstrasse 29 D-1000 Berlin 10, FRG

Received December 31, 1991

A 12-kDa peptidyl-prolyl-cis/trans-isomerase was purified 22% fold to homogeniety from the cyclosporin producing fungus TOLYPOCLADIUM INE'LATUM. The enzyme is highly sensitive to the immunosuppressant FK-506 but not to cyclosporin and thus belongs to the class of FK-506 binding proteins (FKBP). Interestingly the FKBP/FK-506 complex is resistant against proteolytic digestion by the endoproteases GluC and Lysc, in contrast to the free FKBP, which is readily cleaved by these proteases. This protection may play a role in the effects of FK-506 in the living cell. B 1992 Academic Press, Inc.

FK-506 is a immunosuppressive compound with macrolide

structure. It binds to a cytosolic protein called FK-506-

binding protein (FKBP) (1). The presence of this protein has

been demonstrated in different procaryotic and eucaryotic

organisms (2). FKBPs exhibit, like the earlier described

cyclophilins (3), peptidyl-prolyl-cis/trans-isomerase (PPIase)

activity and are discussed to play a role in protein folding

events (4). Cyclophilins are inhibited in a highly specific

manner by the fungal metabolite cyclosporin (5,6) and FKBPs by

FK-506 (7). Detailed studies revealed that the

immunosuppressive and also some antifungal effects are rather

*To whom correspondence should be addressed.

ooO6-291x/92 $1.50 Copyright 0 1992 by Academic Press, he. All rights of reproduction in any form reserved. 1282


caused by the complex of these immunophilins and their

inhibitors than by PPIase-inhibition (2). We recently

demonstrated, that the cyclophilin of the cyclosporin

producing fungus T. INFLATUM is also sensitive to cyclosporin A

(8). In this report we describe the purification and

characterisation of FKBP from the same fungus.

MATERIALS AND METHODS

Growth of Orsanism This was done by a modified procedure as described (9). The cyclosporin producing fungus Tolypocladium inflatum DSM 915 was maintained on agar slants (2 % maltextract, 0.4 % yeastextract, 2 % agar). Precultures (200 ml in 500 ml Erlenmeyer flasks) were inoculateg by addition of 200 ~1 of a sporesuspension of T.inflatum (10 /ml) and incubated for 48 h in MCP75 medium (7.5 % maltose, 2.5 % tryptone, 0.1 % KH2P04, 0.25 % KCl) in a rotary shaker (160 rpm, 26OC). 10 ml were transfered to 200 ml mainculture (7.5 % maltose, 2.5 % peptone, 0.1 % KH P04,

t+ 0.25 % KCl, pH 5.5) in 500 ml

Erlenmeyer flasks. he culture was was maintained under the same conditions and harvested after 6 days by suction filtration. The mycelium was washed with distilled water and lyophilized.

Measurement of PPIase activity The activity of PPIase was determined as described (6). The peptide succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (Bachem) was used as test substrate. The assay mixture contained 100 mM Tris-HCl buffer (pH 8), 10 ~1 testpeptide (2.3 mg/ml DMSO) and FKBP in a final volume of 400 ~1. The reaction was initiated by the addition of 50 1.11 of A-Chymotrypsin (2.5 mg/ml) and followed photometrically at 390 nm. The inhibition studies were performed by addition of FK-506 (in EtOH) in a concentration range between 6 and 60 nM.

Protein determinations Protein concentrations were determined by a modified Bradford procedure as described (10) with bovine serum albumin as a standard.

SDS-oolvacrvlamide se1 electroohoresis Slab gel electrophoresis was done as described (11). The relative molecular mass of FKBP was determined from its mobility related to those of the standard proteins.

Enzvme ourification All operations were carried out at 4°C. Buffer A (100 mM Tris- HCl, pH 8) was used for all operations. The lyophilized mycelial cake was suspended in 2 ml/g buffer A, containing 300 mM NaCl, and disrupted by using a French Press (16000 psi). After centrifugation (20 min at 10000 g) the supernatant was applied to a Q-Sepharose column (Pharmacia, 12 x 2.5 cm). To the flow through, containing the activity, ammonium sulfate was added to give 80 % saturation. The precipitated protein

1283


was dissolved in 5 ml of buffer A and separated by gel filtration on Ultrogel AcA 54 (1.5 x 120 cm). Active fractions were pooled and passed again through a Q-Sepharose column (1 x 5 cm). The flow through protein was precipitated by adding solid ammonium sulfate (80 % saturation). After centrifugation the pellet was dissolved in 1 ml of buffer A and separated by FPLC using a Superdex 75 16/60 column (Pharmacia) previously equlibriated with buffer A (flow rate 0.5 ml/minute).

Isoelectric focussinq The isoelectric point of FKBP was determined by using Servalyt Precotes (Serva, Heidelberg, Germany) according to the instructions of the manufacturer.

Protease disestion of FKBP/FK-506 complex The enzymatic cleavage of the FKBP/FK-506 complex was performed using the endoproteases GluC (V8) from STAPNYLOCOCCUS AUFIEUS and LysC from Acromobacter. FK-506 and ascomycin (in 4~1 EtOH) were added to the FKBP-solution to give a final concentration of 200 nM for FK-506 and 1 PM for ascomycin. After preincubation for 5 minutes at 37OC the cleavage was started by addition of 0.1 l.(g of protease and incubated for 30 minutes at 37OC. Buffer for V8 digestion was 50 mM ammonium hydrogen carbonate (pH 4) and 100 mM Tris-HCl (pH 8) for V8.

RESULTS

We have purified a FKBP from the cyclosporin producing fungus

T. INFLATUM. The purification steps are given in table 1. As can

be seen, the most effective step of this purification

procedure was the gel filtration on Ultrogel AcA54, leading to

Table 1. Purification of the FKBP

@sspharose 80 awe 25.aci 312 73 4

lblmwlAcA54 36 I,15 lW3 a275 33 104

Q-sepharose 45 OS80 9.750 12.167 30 137

superdex75 10 020 4.mo 2amm 13 225

8 g of lyophilized mycelium was used.

1284


0 1

Fig. 1. SDS-Gel of FKBP. Lane 1, 16/60 (2 ccg) -

FKBP b

f3 w 12

PI - 9.45 - 8.3 -7.7 I g.8

electrophoresis of the last purificationsteps Ultrogel AcA 54 (13 pg); Lane 2, Superdex 75

Fig. 2. Isoelectric focussing of FKBP. Lane 1, FKBP (Qbg); Lane 2, standard proteins.

a thirty-fold increase of the specific activity. An overall

yield of 13 % of the total activity was obtained. Figure 1

shows the SDS-PAGE of the last two purification steps. The

enzyme migrated as a single band after the Superdex

gelfiltration step and was obviously homogeneous.

Like other FKBPs from a variety of organisms the molecular

mass of this enzyme was found to lie in the range of 12 kDa.

Furthermore the enzyme exhibited peptidyl-prolyl-cis/trans

isomerase activity which could be inhibited by the

immunosuppressant FK-506 at a nanomolar level (figure 2) and

by ascomycin (not shown). Another similarity to other FKBPs

(12) lies in the preference of the enzyme for the isomerase

test-peptide succinyl-Ala-Leu-Pro-Phe-p-nitroanilide.

Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide which is a good

substrate for most cyclophilins gave only slow reaction rates

(about l/12 of that of the Leu-peptide; not shown).

Analytical isoelectrical focussing on polyacrylamid gel

1285


100

60 -

45kDa Lc

24 kDa )

IPkDa +

0 I / I I 1 I

1 23456799

Fig. 3. Inhibition of PPIase activity by FK-506. Fig. 4. Protease digestion of the FKBP/FK-506 complex. Lane 1, purified FKBP; Lane 2, GluC digestion of FKBP; Lane 3 GluC digestion of FKBP/FK-506 complex; Lane 4, GluC digestion of FKBP in the presence of 4 ~1 EtOH; Lane 5, LysC digestion of FKBP; Lane 6, LysC digestion of FKBP/FK-506 complex; Lane 7, GluC digestion of FKBP in the presence of EtOH; Lane 8, GluC digestion of FKBP/ascomycin complex; Lane 9, LysC digestion of FKBPjascomycin complex.

yielded PI-value of this enzyme of about 6.8 (figure 3).

During a set of experiments on the limited proteolysis of this

FKBP we found, that the complex between the enzyme and its

inhibitor (FK-506 or ascomycin) exhibits a considerable

protection against proteolytic digestion by the endoproteases

Glut (V8) and LysC. This is shown in figure 4. Lane 3 and lane

6 show the protection of the immunophilin-inhibitor complex

against proteolytic attack, caused by V8 and LYSC,

respectively. Controls are given in lanes 4 and 7, in which

only the solvent EtOH was used without inhibitor. The same

protective effect was observed in the case of the FE-506

homologues compound ascomycin, which has similar

immunosuppressive properties.

To our knowledge this is the first time that the protection of

a immunophilin-inhibitor-complex against proteolytic attack

1286

>ve

-LysC -FKBP


has been described. One may speculate that this protection is

caused by the dramatic conformational change of the FKBP

induced by FK-506.

It cannot be excluded, that this protection may play a

fundamental role in the effects of FK-506.

Work is in progress to study this protective effect in detail

with a variety of FKBPs and also cyclophilins.

ACKNOWLEDGMENTS

This work was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 9, Teilprojekt C3. We thank Ullrich Keller for valuable discussions.

REFERENCES

1)

2) 3)

4)

5)

6)

7)

8)

9)

10) 11) 12)

Sawada, S., Suzuki, G., Kawase, Y. and Takaku, F. (1987) J. Immunol. 139, 1797-2001. Schreiber, S.L. (1991) Science 251, 283-287. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J. (1984) Science 226, 544-547. Tropschug, M., Wachter, E., Mayer, S., SchLinbrunner, E.R. and Schmid, F.X. (1990) Nature 346, 674-677. Takahashi, N., Iiayano, T. and Suzuki, N. (1989) Nature 337, 473-475. Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. and Schmid, F.X. (1989) Nature 337, 473-475.

Siekierka, J.J., Staruch, M.J., Hung, S.H.Y. and Siga1,N.H. (1989) J. Immunol. 143, 1580-1583.

Zocher, R., Keller, U., Lee, C. and Hoffmann, K. (1992) J. Antibiot. (in press).

Billich, A. and Zocher, R. (1987) J. Biol. Chem. 262, 17258-17259. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Laemmli, U.K. (1970) Nature 227, 680-685. Harrison, R.K. and Stein, R.L. (1990) Biochemistry 29, 3813-3816.

1287

fk-506 binding protein from tolypocladium inflatum: resistance of fkbpfk-506 complex against...

Documents