cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequences

Gene. 109 O991) 255-258

© 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50

255

GENE 06219

cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequences* (Recombinant DNA; immunoblotting; immunophilin; immunosuppression; rapamycin)

Patricia A. Nelson, Judith A. Lippke, Mark A. Murcko, Sandra L. Rosborough and Debra A. Peattie Immunology, Molecular Biology and Molecular Modeling. Vertex Pharmaceuticals Incorporated. Cambridge, MA 02139-4211 (U.S.A.)

Received by S.T. Case: 23 July 1991 Accepted: 24 August 1991 Received at publishers: 30 September 1991

suMMARY A F K B P cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence

has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A.

INTRODUCTION

The immunosuppressive compounds Cyclosporin A (CSA) and FK506 (FK) have been shown to be potent immunosuppressive drugs both in vitro and in vivo. They bind to and inhibit the pcptidyl-prolyl cis-trans isomerases cyclophilin (Handschumacher et al., 1984; Harding et al., 1986) and FK506 binding protein (FKBP) (Harding et al., 1989; Siekierka et al., 1989), respectively. CSA and FK are

Correspondence to: Dr. P.A. Nelson, Vertex Pharmaceuticals Incorporated, 40 Ailston St., Cambridge, MA 02139-4211 (U.S.A.) Tel. (617)576-311!; Fax (617)499-2437; E-mail: [email protected]. * On request, authors will supply detailed experimental evidence for conclusions reached in this short communication

Abbreviations: aa, amino acid(s); b, bovine; bp, base pair(s); eDNA, complementary DNA; CSA, immunosuppressant compound Cyclosporin A; h, human; EK, immunosuppressantcompoundFK506; FKBP, FK-bindirr~protein;FKBP,gene (DNA) encodingFKBP; m, muririe;nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open rel:.di::~ fi ame;PVDF, immobiion-Ppolyvinylidenedifluoride; Rap, immunosuppressant compound Rapamycin; TBST, 20raM Tri~.HCI/500mM NaCI/0.3°o Tween20 pH 7.5; UTR, untranslated region.

inhibitory in T lymphocyte mitogenesis assays (Lin et al., 1991). In addition, inhibition of mRNA transcription for IL-2 is a downs,ream activation event known to be inhibited by CSA and FK, presumably after the drugs complex with their respective cytosolic binding proteins. The drugs appear to inhibit completely transcription activated by NF-AT, NF-IL2 A, and NF-IL2 B, and to inhibit partially transcription activated by N F r B (Mattila et al., 1990; Banerji et ai., 1991). These findings suggest that CSA and FK may interfere with the activity of a novel Ca 2 + dependent step that regulates several transcription factors. Rapamycin (Rap) is another immunosuppressant that binds to FKBP, but it does not prevent transcription of IL-2 message, even at concentrations as high as 1/~M (Bierer et ai., 1990; Nelson et al,, 1991). Several other proteins that bind FK and Rap have been described, and their role in immunosuppression is being investigated (Fretz et al., 1991). It has been proposed recently that the FK-FKBP complex inhibits activation of T lymphocytes through specific binding to calcineurin A (Liu et al., 1991), a calmodulin regulated protein phosphatase whose role in T cell activation has yet to be elucidated. These new, compelling results have recently been revic~ved (McKeon, 1991) and

256

(b) mFKBP protein sequence

promise to focus increased attention on the molecular mechanisms of immunosuppression. In the work presented here, the FKBP cDNA encoding murine FKBP has been cloned, and its complete nt sequence has been determined. The cDNA segment encodes an 108 aa protein that differs from the hFKBP by three aa and from the bFKBP by five aa.

The ORF encodes an 108-aa protein that differs from the hFKBP (Maki et al., 1990; Standaert et al., 1990) and bFKB P (Lane et al., 1991) by three and five aa, respectively (mThrS°-hMetS°-bVaP°; mlle76-hThr76-bThr76; mSer 79hPro79-bPr079; mHis9S-hHis95-bAsn95; mVa199.hVa199. bile99; Fig. 1). Since all substitutions are conservative, we predict that mFKBP will share the j~-sheet structure and FK/Rap-binding pocket that has been reported recently for recombinant hFKBP (Michnick et al., 1991; Van Duyne et al., 1991) and native bFKBP (Moore et al., 1991). The structure obtained from computer modeling studies is shown in Fig. 2. None ofthe aa changes are contact residues for FK binding. It also appears that none of the residues point directly at what is a potential binding surface for the putative target protein, calcineurin A, although aa 50, 79 and 95 could be on the outer periphery of a complex that contains FKBP.

EXPERIMENTAL AND DISCUSSION

(a) Cloning and sequencing mFKBP A eDNA encoding the routine FK506-binding protein (mFKBP) has been cloned from a Stratagene mouse thymus eDNA library using a radiolabelled hFKBP eDNA (D.A.P., K. Hsiao and J.A.L., unpublished) as a probe, and the entire nt sequence has been determined (Fig. 1). The full length clone MF6-4-2 contains a 1556-bp segment that includes a 97 nt 5'-UTR, an ORF extending from nt 98-421, a 3'-UTR from nt 422-1527, and a 29-nt poly(A) tail. The nt sequence is largely identical to two hFKBP eDNA sequences (Maki etal., 1990; Standaert et al., 1990), even within the 5'- and 3'-UTRs.

(c) Protein isolation and Western immunoblotting procedure Immunosuppression studies using murine bioassays and FK or Rap indicate there is a munne equivalent of bFKBP

1 CTTCGGCTTCGT~CG~T~CGGCTCCACGCCGCCCGTCGCG~CGCCACCCGCGTCCTTTTCCTCCTCCTCCGCCAA~GC~G~CG~CGCCGC~GCCGcC 98 ATG G G A GTG CAG GTG GAG ACC A T C T C T C C T G G A GAC GGG CGC A C C

l~'Met Gly Val

Gin Val

Glu Thr

lie

Ser

161 ACC TGC GTG GTG CAC TAC A C G G G G A T G

221'Thr Cys Val

Vat

H i s Tyr Thr Gly Mot

CTT G A A GAT GGA AAG A A A

Leu G l u Asp Gly

Pro Phe Lys Pile Thr

Leu Gly

t y s Gin Glu Val

lie

+" 287 GTA GCC C A G A T G A G T G T G GGT C A G A G A

641'Val A l a G i n Met Ser Val

T T T GAT TCC TCT C G G G A C

Lys Lys Phe Asp Ser Set Arg Asp

224 A G A A A C n A G CCT T T T A A G TTT A C A C T A GGC A A G CAG GAG GTG A T C

431'Arg Asn t y s

T T C C C A A A G CGC G G C C A G

Pro Gly Asp Gly Arg Tier Phe Pro Lys Arg Gly G i n

C G A GGC T G G G A G G A A G G G

A r g Gly T r p Glu G l u Gly

t.

G C C A A A CTG A T A A T C T C C T C A G A C T A T GCC T A T G G A

C~ly G i n A r g A l a

tys

Leu l i e

+

lie

Ser Ser Asp Tyr A l a Tyr

Gly

,4.

350 GCC A C C G G G CAC C C A GGC A T C A T C C C A C C A C A T GCC A C T CTT G T T T T T GAT G T G GAG C T T C T A

851'Ala Thr Gly H i s Pro G~y I i o

lie

P r o Pro H i s A l a Thr

Leu Val

Phe Asp Val

G l u Leu Leu

413 A ~ A CTG G A A TGA•AGAAGTGG•CT••TCC•TTAGCTcTGCACGTGGATCTGCCATGGAGGAAT•TGGTACcTC•AGATGGGTGc•••TGAAT

1061~Lys Leu G l u 505 CCATGGGGAG~TCTTCCTG~CGT~AC~AC~CTTTGTATAAACACC~A~GACTGAATG~GTTCCGT~ACT~AG~TTTG~TT¢GG&~AC~C~T 602 GTC•TCTTCCCCC•T•TGTATGTGTGTTGA•cTAAA•TATATGC•ATAAACCTCAAGTTACTTTATTTTGGGGTG•AG•CT•AGTTT•TGTTTC•GA 699 TCTAAGTTTCCAATGAAGTATATTGTCAAGTGTTAACAGCACAAGCGATGGGTTAACTTAGAATAGAAATTGGTGTTGGGGGGGGATTTGCA~GAAT 796 TTTTATTTTATTTTATTTT•ATTTTTTGGATGAAAATTTTATCTATTATATATTAAGA•ATTCTG•TGCTGTGCTGCAAAGcCATAGC•GATT•GAG 893 ATGCTGTTGAGG•TGGATTTTTCT•TAGTGAGGGAGGTCCTGTTAAACTGAAAGCCCTA•CC•AAGTGAGGTGGG••GGA•G•GGGGG&GAG&G••T 990 TGGCTT•TG•CAGTC•TTC•A••TT•TGAAA•T•G•TGCcTTTTAAAGCAGATCCCTT•••TG•T•TGTTGGA••cTAC•GTAT•TGTc•CTGGGTT 1087 GGCAGAGA•TTGAAAGC•TTTG•GACCTGGCTTAATTTGTTTTTCATcCTATGGTTTTT•TAATGGATTTT•TGGA•TTTTGTAATCTTGT•AcTAT 1184 T•A•GCT••••TTT•TAAATTTTAAGA••TTCAGTGGAAAGTTTAAATTGAAGGTG•TGTTTGTAGA•T•A••A••CAACGGA•G•C•AG•C•••&• 1281 cG~AA~TCCTTGAGTGTT~T~TAAG.~ATACGATGCTGGT~ATCACAGCTTCAGCATCTCCTGGGTTTTGATGCTTGGcTCTTTG~TGAT~TTGG~TT 1378 CCTGGCTTTT•CTCCTTCAGTTC•TTCTCACCCTCTGCTGTC•TTGTGTAGTGATTTGGTGAGAGAAAGCTTTTGT•CCCTGCCCTCTGCACTACAT

1475 ~ G e ~ Q T T C ~ T T T r ^ ~ A T T G C ~ T ~ G T G C T ~ T ~ G C T G G C T T ~ C T e ~ ~ Fig, !, The eDNA and predicted aa sequence of murine FKBP. The murine eDNA clone was isolated from a Stratagene (La Jolla, CA) mouse thymns eDNA library (catalog No. 935303) in the 2ZAP' vector, and the eDNA insert was excised as a plasmid via helper phage infection. DNA was purified using Qiagen (Chatsworth, CA) columns, and the complete nt sequence of both strands was determined by the dideoxy method (Sanger et al., 1977) using -~-~S-dATP(Biggin et al., 1983) as the radiqlabd. Synthetic oligos (Promega, Madison, WI) were used as primers in the initial sequencing reactions using the T3 and T7 promoters; oligos for additional sequencing reactions were synthesized as necessary, based on obtained nt sequence, on an Applied Biosystems 380A DNA synthesizer. The mFKBP eDNA sequence has been assigned GenBank accession No. X60203. The predicted aa sequence of mFKBP differs from that predicted for hFKBP (Maki et al., 1990; Standaert et el., 1990) at three positions (e) and the determined bFKBP (Lane et al,,

1991) at five positions ( + ).

257

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~

9 9 mVAL 9 9 hVAL 9 9 bILE

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~

7 6 mILE 7 6 hTHR ~

•

kDa 46

1

2

34

56

=

22 14

m

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2.4

5 0 mTHR 50 hMET 50 bVAL

Fig. 2. Ribbon diagram tracing the alpha carbons of FKBP using the published coordinates for hFKBP (Van Duyne et aI., 1991).The N and C terminal aa are labeled, and for clarity, a portion of the ribbon (aa 24-47) has been removed.The side chains of the five aa residues which vary between mouse,humai~and bovineare pictured. Alsoshown are the nonhydrogen atoms of the FK506compound to delineate the active site region. The figure was generated with Insight 11 (Biosym, San Diego, CA).

and hFKBP (P.A.N., unpublished). In addition, an 11.8-kDa murine protein, a major FK binding protein, was isolated from FK affinity columns (data not shown). To demonstrate the presence of mFKBP, mouse EL4 cells were washed with PBS, the cell pellet was iysed in a hypertonic 1 M Tris. HCI pH 7 buffer, the lysate was diluted to 0.1 M Tris. HCI and sonicated. Protein content was determined using Coomassie protein assay reagent (Pierce, Rockford, IL) which employs the method developed by Bradford (1976). Proteins were separated on a 0.1% SDS-12.5~o PAGE (Laemmli, 1970) and transferred to PVDF membrane (Millipore, Bedford, MA). After the transfer was complete, the nonspecific binding sites on the membrane were blocked for 1 h with a solution of 10% BLOTTO in TBST. The blocked membrane was incubated with primary rabbit anti-bovine FKBP antibody diluted in TBST for 90 rain at room temperature. After washing away unbound FKBP antibody with TBST, the blot was incubated for 1 h in TBST containing horseradish peroxidase-conjugated-goat anti-rabbit IgG. The membrane was again washed with TBST and finally developed using the Enhanced Chemiluminescence (ECL) Western blotting detection system (Amersham, Arlington Heights. IL). The Western-blot analysis (Fig. 3) demonstrates that a murine protein band with migratory properties identical to purified bFKBP is detected with rabbit polyclonal antibody raised against a synthetic peptide of aa 1-13 of the FKBP N terminus. This protein band is detected only weakly with

Fig. 3. Western analysis ofmFKBP and bFKBP with rabbit anti-bFKBP antibodies. Purified bFKBP (0.6 ng; lanes l, 3 and 5) from calf thymus and total protein (20 ~g; lanes 2, 4 and 6) isolated from the murine EL4 lymphoma cell line were resolved on a 0.1% SDS-12.5% polyacrylamide gel (Laemmli, 1970). After transfer to PVDF, lanes ! and 2 were incubated with antibody raised against native bFKBP, lanes 3 and 4 were incubated with antibody to N-terminal residues !-13 of bFKBP, and lanes 5 and 6 (negative control) were incubated with the secondary horseradish peroxidase-conjugate.goatanti-rabbit lgG (Sigma, St. Louis, MO). The blot was developed using the Enhanced Chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL). The approximate Mr (kDa) of marker proteins are shown on the left.

antibody to whole native bFKBP. This result, consistent with the fact that four of the five aa differences between mFKBP and bFKBP sequences are on the external surface of the protein when visualized by computer modeling (Fig. 2), would be explained by dissimilar antigenic determinant(s) on the proteins. The additional bands of higher Mr may be proteins sharing antigenic similarities witll the 11.8-kDa FKBP.

(d) Conclusions Elucidation of the mFKBP sequence should further mechanistic studies of the suppression observed with immunosuppressants such as CSA, FK and Rap in in vitro cellular proliferation assays and should facilitate comparison of in vivo versus in vitro mechanisms of drug action. Analysis of the regulation of this important protein in murine transplant models, with and without the concomitant infusion of drugs, may reveal the normal function and immunosuppressive role of mFKBP and its interaction v~ith other key molecules in T cell activation pathways.

ACKNOWLEDGEMENTS

The authors thank Mark Fleming for oligo synthesis, Holly Hodgdon for assistance with the mFKBP work, Dr. David Pearlman for aid ~ith the computer modeling studies, and Dr. Matthew Harding for valuable discussion.

258 REFERENCES Banerji, S.S., Parsons, J.N. and Tocci, MJ.: The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-ATI. Mol. Cell. Biol. 11 (1991) 4074-4087. Bierer, B.E., Mattila, P.S., Standaert, R.F., Herzenberg, L.A., Burakoff, SJ., Crabtree, G. and Schreiber, S.L.: Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FKS06 or rapamycin. Prec. Natl. Acad. Sci. USA 87 (1990) 9231-9235. Biggin, M.D., Gibson, T.J. and Hong, G.F.: Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Prec. Natl. Acad. Sci. USA 80 (1983) 3963-3965. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976) 248-254. Fretz, H., Albers, M.W., Galat, A., Standaert, R.F., Lane, W.S., Burakoff, S.J., Bierer, B.E. and Schreiber, S,L.: Rapamycin and FKS06 binding proteins (immunophilins). J. Am. Chem. Soc. 113 (1991) 1409-141 I. Handschumacher, R.E., Harding, M.W., Rice, J., Drugge, R.J. and Speicher, D.W.: Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226 (1984) 544-546. Harding, M.W., Handschumacher, R.E. and Speicher, D.W.: Isolation and amino acid sequence of cyclophilin. J. Biol. Chem. 261 (1986) 8547-8555. Harding, M.W., Galat, A., Uehling, D.E. and Schreiber, S,L.: A receptor for the immunosuppressant FKS06 is a cis-trans peptidyl-prolyl isomerase. Nature 341 (1989) 758-760. Laemmli, U.: Cleavage of structural proteins during the ass~mb~, of the head of bacteriophage T4. Nature 227 (1970)680-685. Lane, W.S., Galat, A., Harding, M.W. and Schreiber, S.L.: Complete amino acid sequence of the FKS06 and rapamycin binding protein, FKBP, isolated from calf thymus. J. Prot. Chem. 10 (1991) 151-160. Lin, C,S., Boltz, R.C, Siekierka, JJ. and Sigal, N.H.: FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes. Cell. lmmunol. 133 (1991) 269-284.

Liu, J., Farmer, .I.D., Lane, W.S., Friedman, J., Weissman, I. and Schreiber, S.L.: Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FKS06 complexes. Cell 66 (199 I) 807-8 ! 5. Maki, N., Sekiguchi, F., Nishimaki, J., Miwa, K., Hayano, T., Takahashi, N. and Suzuki, M.: Complementary DNA encoding the human T-cell FKS06-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin. Prec. Natl. Acad. Sci. USA 87 (1990) 5440-5443. Mattila, P.S., Uilman, K.S., Fiering, S., Emmel, E.A., McCutcheon, M., Crabtree, G.R. and Herzenberg, L.A.: The actions of cyclosporin A and FKS06 suggest a novel step in the activation of T lymphocytes. EMBO J. 9 (1990) 4425-4433. McKeon, F.: When worlds collide: immunosuppressants meet protein phosphatases. Cell 66 (1991 ) 823-826. Michnick, S.W., Rosen, M.K., Wandless, TJ., Karplus, M. and Schreiber, S.L.: Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin. Science 252 (1991) 836-839. Moore, J.M., Peattie, D.A., Fitzgibbon, MJ. and Thomson, J.A.: Solution structure of the major binding protein Ibr the immunosuppressant FKS06. Nature 351 (1991) 248-250. Nelson, P.A., Kawamura, A., Akselband, Y., Peattie, D.A., AIdapo, R.A. and Harding, M.W.: Effect of immunosuppressive drugs on cytokine gene transcription studied by message amplification phenotyping (MAPPing) PCR. Trans. Prec. 23 (1991) 2868-2870. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain terminating inhibitors. Prec. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Siekierka, J.J., Hung, S.H.Y., Poe, M., Lin, C.S. and Sigal, N.H.: A cytosolic binding protein f~t immunosuppressant FKS06 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341 (1989) 755-757. Standaert, R.F., Galat, A., Verdine, G.L. and Schreiber, S.L.: Molecular cloning and overexpression of the human FKS06-binding FKBP. Nature 346 (1990) 671-674. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. and Clardy, J.: Atomic structure of FKBP-FKS06, an immunophilinimmunosuppressant complex. Science 252 (1991) 839-842.