The multigene immunophilin family of Dictyostelium discoideum. Characterization of microsomal and mitochondrial cyclophilin isoforms

Biochimie 81 (1999) 943-954 © 1999 Socirt6 franqaise de biochimie et biologie molrculaire / l~ditions scientifiques et mfdicales Elsevier SAS. All rights reserved

The multigene immunophilin family of Dictyostelium discoideum. Characterization of microsomal and mitochondrial cyclophilin isoforms A u r r l i e T a p p a r o a, S y l v i e K i e f f e r b, F r a n q o i s C r e t i n c, M i c h e l Satrea, G r r a r d K l e i n a* "Laboratoire de Biochimie et Biophysique des Systkmes hztdgrds ( UMR 314 CEA-CNRS), Ddpartement de Biologie Moldculaire et Structurale, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble cedex 09, France hLaboratoire de Chimie des Protdines, Ddpartement de Biologie Moldculaire et Structurale, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble cedex 09, France ~Laboratoire d'Immunochimie (U238 INSERM), Ddpartement de Biologie Moldculaire et Structurale, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble cedex 09, France

(Received 21 June 1999; accepted 23 July 1999) A b s t r a c t - The sequences of two cyclophilin (Cyp) isoforms from Dictyostelium discoideum have been determined, cyp2 is expressed as a 197 amino acid protein, which contains a 22 amino acid-long signal sequence, characteristic of endoplasmic reticulum localization signals, and that is cleaved in the mature protein. Mature Cyp2 has a molecular mass of 18 986 Da. The cyp3 gene encodes a 174 amino acid protein with a predicted molecular mass of 19 016 Da. Its sequence reveals no targeting sequence. From the MS analysis of affinity-purified cyclophilins from different subcellular compartments, we localized the previously described Cypl (Barisic K. et al., Dev. Genet. 12 (1991) 50-53) in cytosol, Cyp2 in microsomes and Cyp3 in mitochondria, respectively. The expression of cypl mRNA is constant during differentiation, whereas the mRNA level of both cyp2 and cyp3 is regulated and decreases steadily during development. © 1999 Socirt6 franqaise de biochimie et biologic molrculaire / Editions scientifiques et mrdicales Elsevier SAS cyclophilin / immunophilin / development / Dictyostelium discoideum

1. Introduction

Cyclophilins (Cyps) are members of a ubiquitous, highly conserved protein family, first isolated as the intracellular target of the immunosuppressive drug cyclosporin A (CsA)[1]. Their molecular masses range between 18 and 40 kDa. In eukaryotes, Cyps have been localized in cytosol, nucleus, endoplasmic reticulum, secretory pathway, mitochondrion and chloroplast. Cyps have also been found in prokaryotes with a periplasmic localization. Their wide distribution and the high degree of sequence conservation suggest an important role in the cellular economy (for reviews, see [2-4]). Cyps exhibit a peptidyl-prolyl isomerase activity (PPIase or rotamase), which catalyses the cis-trans isomerisation of the amide bond between a proline residue and the preceding amino acid and, as such, they have been proposed to aid in protein folding [5, 6]. Cyps have also been proposed to act as chaperones, chemotactic agents or stress response proteins. Interestingly, proteins binding the immunosuppressant drug FK506 (FKBP) also possess a peptidyl-prolyl isomerase activity [7, 8]. The Cyp/CsA complex is able to bind to the calciumactivated calcineurin and to inhibit its phosphatase activ* Correspondence and reprints

ity. In T cells, this inhibition of calcineurin prevents their activation by decreasing the nuclear level of transcription factor NF-AT and consequently, the expression of interleukin-2 [9]. Cyclophilin A has been demonstrated to bind to the HIV-1 proteins Gag, Nef, Vif, gpl20env. HIV-1 Gagcyclophilin A interactions seem to be essential in HIV-1 replication and may be a potential target in anti-HIV therapies and vaccines [10-13]. Recently, a nuclease activity has been reported for several Cyps, suggesting a possible nucleolytic role of Cyps in apoptosis [14]. CsA, by binding to Cyp3 localised in the mitochondrial matrix, might inhibit the opening of the permeability transition pore [15, 16] described as a regulator of apoptosis [17, 18]. A cyp gene has previously been reported in Dictyostelium discoideum and Southern blot analysis indicated that there was a single cyp gene in this amoeba [19]. We report

here the CsA-affinity purification of 2 new Cyp isoforms in D. discoideum, their partial N-terminal or internal microsequencing and the subsequent sequencing of their gene. Northern blot analysis of the three cyp genes showed that they are differently expressed during the developmental cycle.

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In all cases, cells were washed twice and resuspended in STBE buffer (0.25 M sucrose, 25 mM Tris-Cl pH 8.0, 5 mM [3-mercaptoethanol, I mM EDTA). After breaking cells in a ball bearing cell cracker, subcellular compartments were obtained by differential centrifugation. The 1000g (10 min) nuclear pellet was discarded and the 10000g (15 min) pellet, the 100000g (60 min) pellet and its supernatant taken as mitochondrial, microsomal and cytosolic fractions, respectively. Washed mitochondria and microsomes suspended in STBE buffer were freeze-thawed to release soluble proteins which were then separated from broken membranes by high-speed centrifugation (180 000 g, 90 min).

performed as described [22]. For the MS analysis, proteins were 'in-gel' digested according to [23], except that the alkylation step was omitted. Briefly, the protein bands of interest were excised from the stained gel, washed with 50% acetonitrile, and dried. The dried gel fragments were swollen in 20 ~tL of 25 mM NH4HCO 3, containing 0.5 ~tg of trypsin (Promega, Sequencing grade), and incubated at 37 °C for 3 h. A 0.4 I.tL volume of the digest was added to the dried matrix (0.4 ~tL of c~-cyano-4-hydroxy-trans cinnamic acid 20 mg/mL and 5 mg/mL nitrocellulose in 1:1 isopropanol:acetone) on the MS sample probe and samples were rinsed with 5/xL of 0.1% trifluoroacetic acid. MALDI-TOF (time of flight) mass spectra of peptide mixtures were obtained using a Bruker Biflex mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) equipped with a SCOUT multiprobe inlet and a gridless delayed extraction ion source as described earlier [24]. Mass spectra were acquired as the sum of ion signals generated by irradiation of the target with 100 laser pulses. They were internally calibrated using ion signals from trypsin autodigestion peptides (MH + = 842.50, 1045.55, and 2211.09). Monoisotopic peptide masses were assigned and used for comparison to computergenerated peptide masses of the different Cyp isoforms from D. discoideum. Comparison parameters allowed a maximal peptide mass error of 0.1 Da and considered one incomplete cleavage per peptide.

2.3. Affinity-purification of Cyp isoforms by binding to CsA-Sepharose

2.5. Preparation of genomic DNA and total RNA

2. Materials and methods

2.1. Strains, growth and developmental conditions D. discoideum axenic strain Ax-2 (ATCC 24397) was grown in liquid medium [20] under constant swirling at 170 rpm at 21 °C. Amoebae were collected in their logarithmic growth phase (5 x 10 6 t o l × l 0 7 cells/mL). They were differentiated at 21 °C on filters soaked in 12 mM Na-K phosphate, pH 6.1 [21]. Escherichia coli JM109 (Promega France, Charbonnirres, France) was used to subclone DNA fragments. 2.2. Preparation of subcellular extracts

D-[(3-amino)8-Ala]cyclosporin A (Novartis, formerly Sandoz Pharma Ltd., Basel, Switzerland) was coupled to NHS-activated Sepharose 4B (Amersham Pharmacia Biotech, Les Ulis, France), following the manufacturer's instructions. Soluble extracts from D. discoideum subcellular fractions (I mg protein) were incubated at 4 °C with 50 ~L of packed CsA-Sepharose beads in a final volume of 1 mL TBE buffer (25 mM Tris-C1, pH 8.0, 5 mM ~-mercaptoethanol, 1 mM EDTA) for 120 min at 4 °C on a rotary mixer (12 rpm). Samples were centrifuged at 12000 rpm for l min to separate the CsA-Sepharose beads from unbound proteins. Beads were washed four times with 1 mL TBE buffer (10 rain on a rotary mixer). Proteins retained on the CsA-Sepharose beads were eluted with 50 IxL SDS-PAGE depolymerisation buffer and the eluate was analyzed on 15% polyacrylamide gels. CsAbinding proteins were analyzed by N-terminal microsequencing or by MS. In a control experiment performed with uncoupled deactivated Sepharose beads and a soluble whole cell extract, no protein interacted with the Sepharose beads as determined by SDS-PAGE.

2.4. Amino acid sequencing and mass spectrometry analysis of proteins N-terminal sequencing of SDS-PAGE resolved proteins and of their 'in-gel' generated internal peptides was

Genomic DNA and total RNA were isolated from D. discoideum amoebae as described [25].

2.6. PCR amplification and 5'/3 'RACE experiments Sequences of the oligonucleotide primers employed are given in table 1. They were purchased from Oligo Express (Paris, France). For both cyp2 and cyp3 genes, N-terminal or internal protein sequence data were used together with a highly conserved sequence among Cyp sequences to design two degenerate primers (Cyp2-1F and -2R, Cyp3-1F and -2R, table I) using the D. discoideum codon usage bias. The resulting PCR products were separated in a 1.5% agarose gel, purified with a QIAEX purification kit (QIAGEN, Courtaboeuf, France) and ligated into the T-tailed pGEM-T v.ector (Promega) for cloning and sequencing. The sequence of the full coding cDNA was obtained after 5'/3'RACE experiments performed according to the manufacturer's instructions (Roche Biochem, Meylan, France) with primers Cyp2-3F, -4R, -5R and -6R or primers Cyp3-3F, -4R, -5R and -6R. The full-length genomic sequence including introns was obtained after genomic PCR with primers Cyp2-7F, -9F, - 1OF, - 11F and -8R or Cyp3-7F, -9F, -8R and -10R. The sequences of cyp2 genomic DNA, cyp2 and cyp3 mRNA were deposited in GenBank under accession numbers AF053068, AF123597 and AF121347, respectively.

Characterization of cyclophilin isoforms in D. discoideum

945

Table I. Oligonucleotides used for the cloning of D. discoideum

this property to affinity-purify D. discoideum Cyps from different subcellular compartments using CsA-Sepharose beads (figure 1). A control experiment with human CypA yielded as expected a 18 kDa-band (lane 1). One major protein was retained on the CsA-affinity column after binding of a D. discoideum cytosolic extract. Its apparent molecular mass, deduced from its migration on a 15% polyacrylamide gel in denaturing and reducing conditions, was 20 kDa (lane 2). A less abundant 40kDa-protein had a blocked N-terminus and could probably represent a high molecular mass cytosolic Cyp as found in mammalian cells [26, 27]. The N-terminal sequence of the 20-kDa protein: TTVKPTSPE, was identical to the amino acid 2-10 sequence of the D. discoideum Cypl isoform [19] and its observed MS peaks corresponded to the tryptic peptides generated by theoretical digestion of Cypl (not shown). These results confirmed its proposed cytosolic localisation and indicated a cleavage of the N-terminal methionine after mRNA translation. Soluble protein extracts prepared from mitochondrial (lane 3) and microsomal (lane 4) fractions also yielded a 20-kDa band after polyacrylamide separation of the CsAretained fraction. N-terminal sequencing of the microsomal 20 kDa-band gave the sequence GKDPKITNK. The N-terminus of the mitochondrial protein was blocked, suggesting that mitochondria indeed contained a Cyp isoform different from the cytosolic and microsomal Cyps already described above. Internal peptides of the mitochondrial isoform were generated with EndoLys-C and one of them gave the sequence KVFFQIK.

cyp2 and Q,p3 genes. Primel~'

5 '-3 'Sequencet"

cyp2 IF 2R 3F 4R 5R 6R 7F 8R 9F 10F llF

GGT AAR GAT CCA AAR ATH AC GTR AAA TCA CCA CCT TGA CAC AT GGT TTA GGT ACC AGT GGT AAA CC GGT GTT CCA GAT TGG GAA CCA ATG G GTG TTT GGA CCA GCG TTA GCC CAC CAC CAG TAC CAT CAC CTC ATG AAA GTT ATT TTC GTA GTT TTA GCC TAA TTC ACC AGA GTT TGA GAT TIT AGC GAG GTG ATG GTA CTG GTG GTG CAC TCC AAA CCA GGT CTT TTA TC AAA GAC CCA AAG ATT ACC AAT

cyp3 1F 2R 3F 4R 5R 6R 7F 8R 9F 10R

AAR GTH TTY TTY CAA ATH AA GTR AAR TCW CCW CCT TG CCA TTA GGT AGA GTT GTA TTT G GAC TCT GTG AAA TGA TGA ACC TCA CCA GTA CAT AAT GCT CTA A CTC TGC TGT CTT TGG TAC GA ATG ACA GGA ATT ATT AGA AAT AAA GTC TTA AAG TTG ACC ACA ATC TI'C AAT GAC GGT ATT GGT GGT GAA TCA ATT TAT GGT ACC ATA AAT TGA TTC ACC ACC AAT ACC

"F stands for forward, R for reverse. bThe following conventions were used to describe degenerate positions: R (A, G); W (A, T); Y (C, T); H (A, C, T).

2.7. Plasmid DNA extraction and sequencing Plasmid DNA was purified from white colonies, selected on isopropyl [3-D-thiogalactoside (IPTG)/5-bromo4-chloroindol-3-yl fS-D-galactopyranoside (Xgal)/ampicillin with a QIAprep spin kit (QIAGEN) and sequenced on both strands with the universal T7 and SP6 primers by Genome Express (Grenoble, France).

2.8. Digoxygenin-labeled DNA and RNA probes Generation of digoxygenin (DIG)-labeled DNA and RNA probes, hybridization and chemiluminescent detection with anti-DIG Fab fragments conjugated to alkaline phosphatase and CDP-StarT M as substrate were performed according to the manufacturer's instructions (Roche Biochem). The cyp2 DNA probe corresponding to the second exon was synthesized by PCR using primers Cyp2-11F and Cyp2-6R. The RNA probes for cypl, -2 and -3 corresponded to their full coding sequence. 3. Results

3.1. Evidence for multiple Cyp isoforms in different subcelhdar fractions fi'om D. discoideum A general characteristic of Cyps is their binding to and inhibition by cyclosporin A (CsA). We took advantage of

3.2. Cloning of the microsomal cyp2 and mitochondrial cyp3 genes from D. discoideum D. discoideum cyp genes were cloned by a first genomic PCR amplification using degenerate primers followed by 3'/5' RACE (figure 2A, B). For the cloning of the cyp2 gene, two degenerate primers (table I) were designed from the N-terminal protein sequence (GKDPKITNK) on the one hand (Cyp2-1F) and from a conserved region among Cyps (MCQGGDFT) on the other hand (Cyp2-2R). The PCR product obtained was a 235 bp-long fragment and its translated amino acid sequence presented a high identity (70%) with Cyps from Arabidopsis thaliana, TJypanosoma cruzi or Caenorhabditis elegans. Its sequence was used to design a non-degenerate primer (Cyp2-3F) for the obtention of the 3'-end of the gene by means of a 3'RACE experiment. The sequence obtained in this experiment was 439 bp long and was used to design three specific reverse primers (Cyp2-4R, -5R, -6R) for 5'RACE experiments. The full consensus cDNA sequence was 685 bp long and contained an open reading frame encoding a 197 amino acid protein. PCR amplification of genomic DNA with different combinations of the following primers: Cyp2-3F, -7F, -9F,

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1

kDa

2

3

4

94.0 ---67.0 43.0

30.0

20.1

-

-

-

-

-

-

14.4 Figure 1. SDS-PAGE analysis of D. discoideum Cyp isoforms. Cyps were purified on CsA-Sepharose beads from cytosol, mitochondria and microsomes as described in Materials and methods. Samples (25 ~tL) were analysed by SDS-PAGE. Lane 1,2.5 ~tg human CypA as control; lane 2, cytosolic Cypl ; lane 3, mitochondrial Cyp3; lane 4, microsomal Cyp2. The position of molecular mass markers (in kDa) is indicated.

-10F, -4R, -5R, -6R and -8R, indicated the presence of four introns (115, 91, 84 and 92 bp-long, respectively) in the cyp2 gene. As usually described for D. discoideum genes, the base composition of the four intronic sequences is skewed towards A/T (90%, 92%, 89% and 88% A + T, respectively) as compared to 65% A + T in the exonic portion. Six putative polyadenylation sites were found after the stop codon. Part of the cyp3 gene was amplified using two degenerate primers designed on the determined KVFFQIK sequence (Cyp3-1F) and the Cyp consensus region QGGDFT (Cyp3-2R). The PCR product was a 212 bp-long fragment and its translated amino acid sequence presented a very high identity (80%) with Cyps from C. elegans, Onchocerca volvulus or Digitalis lanata. Its sequence was used to design four non-degenerate primers to obtain the 3'-end (Cyp3-3F) and the 5'-end (Cyp3-4R, -5R, -6R) by means of 3',5'-RACE experiments. The complete cDNA sequence obtained was 586 bp long and contained an open reading frame encoding a 174 amino acid protein. Genomic cyp3 DNA amplified with the combinations of Cyp3-7F, -9F, -8R and -10R contained no intronic sequence.

3.3. Amino acid sequence analysis of D. discoideum Cyp2 and Cyp3 cyp2 codes for a 197 amino acid protein with a predicted molecular mass of 21 399 Da and a computed isoelectric point of 9.3 (figure 3). Analysis of the translated protein sequence revealed a highly hydrophobic N-terminal sequence, characteristic of ER-directed signal sequences [28] found in CypB isoforms and suggested a cleavage site after A22. This prediction was in full agreement with the N-terminal GKDPKITNK sequence determined above. The protein will be referred to as Cyp2. The molecular mass of the mature protein was 18 986 Da and its isoelectric point 9.28. cyp3 encodes a 174 amino acid protein with a predicted molecular mass of 19 016 Da and a theoretical isoelectric point of 8.7. The sequence of the protein revealed no obvious signal for targeting to mitochondria. Cyp2 and -3 are 65-66% identical to each other or to the previously identified Cypl from D. discoideum [19]. The percentage of identity with Cyps from other organisms was even higher. Cypl, -2 and -3 were 67-78% identical over their whole length to the proteins from the nematodes O. volvulus and C. elegans (table H). All three

Characterization of cyclophilin isoforms in D. discoideum

947

A N-terminal peptide from D. discoideum cyclophilin 2

~> 1F

Consensus sequence in cyclophilins 2R 235 pb GenomicPCR

I

I 3F

3'RACE

439 pb 6R

5'RACE

5R

4R

I

I 7F 11F ATG

cDNA sequence

361 pb

9F

10F

8R TAA

k

I.

u i

L L

','.

,

P--

685 pb

-...-.'_... TAA

ATG ~

I

Genomic DNA sequence

I 1067 pb 100 pb

B Internal peptide from D. discoideum cyclophilin 3

~> 1F

Consensus sequence in cyclophilins

.....

•

..............

2R Genomic PCR

I

• 44

3F 3' RACE 5' RACE

I

Genomic DNA sequence

212 pb

I

~"

~ I

7F

cDNA sequence

•

5R 6R 4R

ATG --(

ATG I

502 pb 141 pb

9F, 1OR

8R TAA ~--

586 pb

TAA I

525 pb

Figure 2. A, B. Cloning strategy of D. discoideum cyp2 and cyp3 genes. A. The N-terminal sequence data (hatched rectangle) and a consensus sequence conserved in cyclophilins (black rectangle) were used to design two degenerate primers Cyp2-1F and -2R. PCR amplification of D. discoideum genomic DNA yielded a fragment allowing the design of the non-degenerate Cyp2-3F primer for 3' RACE. Three primers Cyp2-4R, -5R and -6R were then designed to complete the sequence by 5' RACE. The consensus cDNA was determined with the Auto Assembler software (ABI Perkin-Elmer). A full genomic DNA clone finishing at the penultimate codon was obtained with the primers Cyp2-7F and -8R. It contained four introns (grey rectangles) and their sequence was confirmed by PCR on genomic DNA using all combinations of primers Cyp2-7F, -9F, -10F, -1 IF, -4R, -5R, -6R and -8R (see table 1). B. cyp3 cDNA and genomic DNA were sequenced following a strategy similar to that described above. The first genomic PCR used primers Cyp3-1F and -2R. The full cDNA sequence was obtained with primers Cyp3-3F, -4R, -5R and -6R. The coding genomic DNA sequence was established using the four primers Cyp3-7F, -9F, -8R and -10R. No introns were present in cyp3.

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Y Cyp2

1

Cypl

1

Cyp3

1

MKVIFVVLAIVLVTLWAMPSEAGKDPKITNKVFFDIEIDNKPAGRIVFGLYGKTVPKTV MTTVKPTSPENPR

59

..... T-GGVE..KV-ME..AN

..... A

41

..... Q.KQG.T.L--V--E.-ND

..... A

36

I - K . . . . . ~. - G - S . • - V o T o • . C . . ~ - • o M - N . . . . . . . . . N . - A - . I I I , K . . . . . ,Io .G, S • • - V - K - • -V- -~. • , H . T - I . . . . . . . R T . . . .

MTGI-R #

7 aa ~ s e ~

Cyp2

60

I I * . ** * ** ENFRALCTGEKGLGTSGKPL~KDSKFHRIIPNFMIQG~DFTRGDGTGGESIYGKKFNDE

i19

Cypl

42

............

i01

Cyp3

37

............

Wa~er A Box Cyp2

120

Cypl

102

Cyp3

97

PPlase Box

NFKIKHSKPGLLSMANAGPNTNGSQFFITTVVTSWLDGRHTVFGEVIEGMDIVKLLESIG ...L.-FGQ.T

....... A .........

CVAP.D

-.LV .... I-C-.. .... K ...........

96

A 83

.... K-V-.-F-T

AE.PH-N.G

.... V-.KM-AA,

....... V..F .... KV-NAE

179 161 155

A 130

Cyp2

180

S-QSGTPSKIAKISNSGEL

Cypl

162

..... KTT- PVV.A- C.Q.

179

Cyp3

156

TDR. DR-KAACV.

174

E D C . Q.

197

Figure 3. Multiple amino acid sequence alignment of D. discoideum Cypl, -2 and -3. Alignments were performed with Clustal W [55] using the D. discoideum cyclophilin sequences for Cypl (PRF 1713247A), Cyp2 (Genbank AF053068, this work) and Cyp3 (Genbank AFI 21347, this work). Periods (.) represent residues of Cypl or Cyp3 which are identical to Cyp2. Gaps introduced for best alignment are indicated by dashes (-). The cleavage site of the signal peptide in Cyp2 is pointed by a black arrow head. The 7 amino acid insert found in all three isoforms is indicated. PPIase (Prosite PDOC00154) and Walker A (Prosite PDOC00017) signatures are boxed. Residues involved in the binding of CsA are indicated by a star (*). The two amino acid substitutions in Cyp3, W130H and T83I, are pointed by white arrow heads.

isoforms have the 18 amino acid PROSITE [29] consensus sequence for PPIases (figure 3). The 15 amino acids involved in the interaction with CsA [30, 31], and especially the critical tryptophan residue [32] were conserved in Cypl and -2. The mitochondrial isoform is remarkable for two amino acid substitutions, the substitutions of tryptophan 130 into histidine (WI30H) and of tyrosine 83 into isoleucine (T83I). The three D. discoideum Cyps possess a central 7 aa insert located just before the PPIase consensus box. It is of interest to note that for Cypl and -3, this insert contains a consensus ATP/GTP binding motif (Walker A). Similar inserts have been found in Cyps from plants, nematodes and other diverse eukaryotes [33, 34]. Their function has not been established. The three D. discoideum Cyp isoforms possess the putative N-glycosylation sequence NGSQ in a highly conserved region. This site is indeed glycosylated in vitro in Plasmodiumfalciparum Cyp, but little or no N-glycosylation was suggested in vivo [35]. Mature Cyps from D. discoideum have an apparent molecular mass determined by SDS-PAGE of 19-20 kDa, very close to the predicted mass for the products of cyp genes, suggesting that D. discoideum Cyps are probably not glycosylated. Cypl and

-3 isoforms possess the carboxy-terminal CGQL sequence, a signal for geranylgeranylation [36] also found in some plant Cyps [37]. No evidence exists for a membrane association of D. discoideum Cyps.

3.4. Biochemical characterization of D. discoideum Cyp3

Characterized mitochondrial Cyp isoforms generally contain a hydrophobic N-terminal extension that is cleaved in the mature protein [38]. The amino acid sequence of Cyp3 described above lacks any mitochondrial targeting signal. This is also the case for the adenine nucleotide cartier located in the inner mitochondrial membrane [39]. To confirm the mitochondrial localization of Cyp3, the protein purified on CsA-Sepharose beads from a soluble extract of mitochondria, was 'in-gel' digested by trypsin and analyzed by mass spectrometry, without alkylation. All major peaks, except one, corresponded in mass to the theoretical tryptic peptides of Cyp3 (figure 4).

Characterization of cyclophilin isoforms in D. discoideum Table II. Amino acid sequence identities between Cyps from D.

discoideum and other origins. The table summarizes the percentage of sequence identity between D. discoideum Cyps (Cypl, -2, -3 Dicdi) and selected Cyps from human (CypH Homsa, Swiss-Prot P05092), A. thaliana (Cyp5 Arath, Genbank AF020433), O. volvulus (Cyp2 Oncvo, Genbank U47812) and C. elegans (Cyp7 Caeel, Swiss-Prot P52015). Sequences were compared with Clustal W [55]. Underlined values represent the sequences with greatest identity score for each of the D. discoideum genes. For D. discoideum Cyp2, the sequence of the mature protein was used for comparisons.

CypH Homsa Cyp5 Arath Cyp2 Oncvo Cyp7 Caeel Cyp3 Dicdi Cyp2 Dicdi

Cypl Dicdi

Cyp2 Dicdi

Cyp3 Dicdi

Cyp7 Caeel

Cyp2 Oncvo

Cyp5 Arath

68.9

63.4

60.4

70.7

71.9

64.0

64.8

72.0

62.1

65.5

72.5

77.8

68.4

69.6

81.9

70.2

70.2

67.2

64.9

64.9

65.7

949

3.5. Estimation of the number of cyp genes and other immunophilins To estimate the total number of D. discoideum genes coding for Cyps, genomic D N A digested with several restriction enzymes was probed in a Southern blot with a DIG-labeled D N A probe derived from the second exonic sequence of the cyp2 gene and including the PPIase consensus sequence. No restriction site for the endonucleases used was present in the probe sequence. In high stringency conditions, only one band was detected in each lane corresponding to the different digestions, suggesting that Cyp2 is encoded by a single gene in amoeba (figure 5A). Under less stringent conditions, at least four restriction fragments hybridized with the probe (figure 5B). This result can be taken as an indication that besides Cyp 1, -2 and -3, there are still other isoforms to be characterized. Large sequencing programs are currently underway on D. discoideum c D N A [40] and genomic DNA. We searched the actual expressed sequence tag (EST) bank for Cyp sequences. Several clones containing the full-length coding sequence of C y p l (SSK846, SSK590, SSJ878,

4

1- MTGIIR + GSSFHR 2- TAENFR 3- VFFQIK 4- QGNTPLGR 5- SGKPLHYK 6- KVENAETDR 7- TFNDENFLVK 8- VVFELYNDIVPK

2

10 000

690.33 + 690.40l 737.36 781.46 842.45 929.52 1061.52 1226.60 1435.78

.,,

7

5 000

'!r 3

I

T 800

LL-I 1000

1200

1400

m/z Figure 4. Mass spectrometry analysis of mitochondrial Cyp3. Cyp3 was purified on a CsA-affinity column from a soluble extract of washed mitochondria and was analyzed by SDS-PAGE. The band was extracted from the gel, digested with trypsin and the generated peptides analyzed by MS. Numbered peptides correspond to Cyp3 and their sequence is given as an insert. Two minor peptides from contaminant Cypl are indicated by white arrow heads. An unknown peak (MH + = 1368.7), possibly a CsA derivative released from the affinity column, is indicated by the black arrow head.

950

Tapparo et al. Table III. Cyclophilin isoforms from D. discoideum present in the cDNA and genomic DNA databases.

lsoform 21.2-- ' 9.4-6.5-4.3--

...........

.

~ ' q :?"ii!: ':" " " : " ;. ....

Cypl ..

A

+

~

~'"

~:~:!

B

Figure 5. A, B. Southern-blot hybridization of D. discoideum cyp2. Genomic DNA was digested with EcoRI (EI), EcoRV (EV), HindIII (HIII) and XbaI (XI) or double-digested with EcoRIIEcoRV (EI/EV) or XbaIIHindlII (XI/HIII). The digested DNA was electrophoresed on a 0.8% agarose gel and transferred to a nylon membrane. The 256 bp DIG-labeled DNA probe corresponded to the second exonic region (nucleotides 221-476). Positions of DIG-labeled DNA size markers II (Roche) are indicated on the left side of panel A. Panel A and B correspond to high and low stringency conditions, respectively.

SSD343) and Cyp2 (SSD482) are present in the cDNA database (table III). Clones containing even partial sequences for the mitochondrial Cyp3 sequence are still absent. The genomic DNA database was also searched for cyp genes. Clone JAX4a37h02 (538 bp) contains the 3'-end of the cypl gene, and includes a 109 bp intron. Comparison with our results obtained in this work shows that cypl gene likely possesses a second --- 100 bp-long intron on its 5'-side. A representative clone for cyp2 is JAX4a177fll covering a 490 bp region (457-957) from the beginning of intron 2 to 30 bases upstream of TAA. Clone JC2a127c06 contains the majority of the cyp3 gene, lacking only about 40 bp on the 3'-end. Its sequence confirms the absence of introns in this gene. The genomic DNA database shows evidence for the presence of four additional cyclophilin isoforms (table III). Only partial sequences were found for genes cyp4, -5, -6 and -7 (figure 6) and currently, they are not represented in the cDNA databank.

3.6. Estimation of the number of other immunophilins Interestingly, other amoebal immunophilin sequences are present in the databases (figure 7). A first gene coding for a D. discoideum 250-amino acid FKBP homolog (FKBP1) has been described (Dr. M. Clarke, personal communication, Genbank AF122823). It is 30.2% identical in a 96 amino acid overlap to yeast FKBR The complete genes for two low molecular mass FKBPs are present in the EST bank. FC-AY11 encodes a 19 kDaprotein (FKBP2), with a N-terminal hydrophobic sequence of 22-amino acids characteristic of ER-directed signals [28]. FC-BS02 codes for a possibly cytoplasmic

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"Absent from the data base. 11 kDa-protein (FKBP3). FKPB2 is 39.4% identical in a 132 amino acid overlap to FKBP15-1 from A. thaliana and FKBP3 is 56.6% identical in a 106 amino acid overlap to the 12 kDa-FKBP from Drosophila melanogaster. Partial sequences of two other FKBP genes are also present in the EST database. SLB410 (FKBP4) and SLK849 (FKBP5) are the 3'-gene ends whose translation products are highly homologous to the C-terminus of high molecular mass-FKBPs from Saccharomyces cerevisiae and Triticum aestivum, respectively. All four FKBPs 1, 2, 3 and 4 possess the two characteristic FKBP-PPIase signatures 1 and 2.

3.7. Developmental regulation of cyp genes The gene coding for the cytosolic Cypl isoform was not developmentally regulated as shown in figure 8A and as previously reported [19]. In fact, a slight decrease of expression of the 0.7 kb mRNA at 12 h of development was apparent and could also be detected in previous work [19]. In contrast, the expression of cyp2 was strongly regulated during development. A single 0.7-0.8 kb transcript, consistent with the size of Cyp2 preprotein, was observed in vegetative cells and its expression decreased during the early stages of development. After 12 h of differentiation, the cyp2 mRNA was only slightly expressed, as compared to the vegetative situation (figure 8B). The expression pattern of Cyp3 was similar to that of Cyp2, with 'a strong expression in vegetative cells, and a steady decrease during the differentiation cycle (figure 8C). Two transcripts were observed, a short one, 0.7 kb in size like cypl mRNA, and a longer form at 1.5 kb, about ten times less abundant, but regulated in a very similar way.

4. Discussion We report here that, besides the previously described Cypl [19], that we localized in the cytosol, other Cyp

Characterization of cyclophilin isoforms in D. discoideum

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Figure 6. Multiple amino acid sequence alignment of D. discoideum Cyp isoforms. Alignments were performed with Clustal W [55] using the D. discoideum cyclophilin sequence for Cypl (PRF 1713247A) and the D. discoideum partial sequences for Cyp4 (JAX4b20b 11), Cyp5 (JC2a80a 11+JC2d 15a02), Cyp6 (JC2e08d01 ) and Cyp7 (JAX4a219h01). Amino acid identities between the D. discoideum Cyp sequences are boxed.

isoforms exist in D. discoideum, Cyp2 located in the endoplasmic reticulum and Cyp3 in the mitochondrion. The sequence of a gene coding for a FKBP homolog (Genbank accession number AF122823) has been described (Dr. M. Clarke, personal communication). Recent

additions to the database of D. discoideum ESTs and genomic DNA indicate the presence of four additional cyclophilin isoforms (Cyp4-7) and four homologs of FKBPs (FKBP2-5). D. discoideum has thus an immunophilin equipment very similar to mammals, worm, yeast

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of CsA, whereas phenylalanine does not share this property. The three D. discoideum Cypl, -2 and -3 contain a seven amino acid insertion in a region between an a-helical and a p-sheet secondary structure on the basis of the structure of human Cypl [43,44]. Cyp I and -3 contain a Walker AATP/GTP binding site at the start of this insert, as do Cyp2, -3 and -7 from C. elegans [34]. Such a conserved addition suggests a possible function for this region. D. discoideum Cyp isoforms differ in terms of intron positions. qpl genomic sequence was about 750 bp-long and contained thus a total of 200 bp as intronic sequences (not shown), qlp2 has four 100 bp-long introns and q,p3 none. In the various Cyp isoforms from A. thaliolza, the number of introns varies between zero and six and their positions is not conserved [33]. Both Cyps and FKBPs inhibit the protein phosphatase 2B calcineurin, upon binding the immunosuppressive molecules CsA, FK506 or rapamycin [45]. D. discoideum cells harbor a calcineurin gene 1461 and both CsA and FK506 strongly inhibit stalk cell rormation in wild type strains and spore formation in a sporogenous mutant [47]. In view of the now increased number of immunophilins in D. discoideum, the exact target of CsA and FK506 that inhibits calcineurin and affects cell differentiation remains to be determined. A D. discoideum mutant generated by shotgun transformation with antisense cypl cDNA grows and develops almost normally, except for the production of tall fruiting bodies [48]. The function of immunophilins was addressed in yeast by a genetic approach. Yeast mutants lacking all 12 immunophilins are viable, an indication that Cyps and FKBPs are not essential [41]. Calcium and CsA have been shown to play an important role in the control of the mitochondrial transition pore [49, 501. Mitochondria through the two proteins, Cyp3 and the adenine nucleotide carrier that may specifically interact [51], have been proposed to play a central execution role in apoptosis 117, 52, 531, by controlling the release of cytochrome C which in turn can activate caspases and trigger the effector portion of the cell death program [54]. With the cloning of D. discoideum Cyp3 as described in this work and of the adenine nucleotide carrier [39], we haye now the tools to study the implication of mitochondna during differentiation. leucine9

24

I

,

Figure 8. A, B, C, D. Developmental expression analysis of D. discoideum mRNAs for q,pI. -2 and -3. Total RNA was isolated from vegetative amoebae or cells that had been filter-developed for 4, 8, 12, 16, 20 or 24 h. 6 pg (12 pg in condition C) of total RNA for each condition of the time-course were separated electrophoretically, transferred to a nylon membrane and hybridized with a specific digoxygenin-labeled RNA probe. Hybridization was performed in DIG-Easy buffer (Roche) at 50 “C. mRNAs were detected after binding anti-DIG antibodies coupled to alkaline phosphatase with CDP-StarTMas substrate (Roche). A, B, C. mRNAs for c?,pl, -2 and -3. The size of the transcripts (kb) is indicated. Equivalent loading on the gel and transfer to

the membrane was controlled with ethidium bromide stained ribosomal 28s and 18s RNA (D).

and plants, where five to eleven Cyps and three to six FKBPs have been found [33, 34, 411. Cypl and Cyp2 possess the highly conserved CsAbinding domain [31], but the mitochondrial Cyp3 carries a W130H substitution in that domain. The Cyp3 isoform was fully inhibited by 1 FM CsA (not shown), an indication that W or H at the active site lead to CsA sensitivity. This is in good agreement with the results observed for the CsA-sensitive Cyp from Halobacterium cutirubrum where the same W to H substitution is observed [42]. A W to F substitution in E. coli has been shown to reduce the CsA sensitivity of the bacterial Cyp by a factor of 300 [32]. In fact, histidine, like tryptophan, has a hydrogen donor group within a certain pH range and has the capability of hydrogen-bonding with methyl-

Acknowledgments The authors would like to thank Novartis (formerly Sandoz) for the generous gift of D-[(3-amino)8-Ala]cyclosporin A. We thank Dr. M. Clarke for providing unpublished sequence data, Dr. M.-J. Moutin for stimulating discussionsand Drs. L. Aubry and J. Garin for critically reading our manuscript. This work was supported by funds from the Universitd Joseph Fourier, the

C h a r a c t e r i z a t i o n o f c y c l o p h i l i n i s o f o r m s in D. d i s c o i d e u m Centre National de la Recherche Scientifique and the C o m m i s sariat ~ I'Energie Atomique. c D N A sequences were obtained from the Dictyostelium cDNA database (Dic~_cDB, http://www.csm.biol.tsukuba.ac.jp/cDNAprojectlhtml). Genomic sequence data for D. discoideum was obtained from the G e n o m e Sequencing Centre Jena website at http://genome.imbjena.de/dictyostelium/. The German part o f the D. discoideum G e n o m e Project is carried out by the Institute of Biochemistry I, Cologne and The G e n o m e Sequencing Centre Jena with support by the Deutsche Forschungsgemeinschaft (N ° 113/10-1 and

10-2).

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T a p p a r o et al. [45] Liu J., Farmer J.D.J.R., Lane W.S., Friedman J., Weissman I., Schreiber S.L., Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes, Cell 66 (1991) 807-815. [46] Dammann H., Hellstern S., Husain Q., Mutzel R., Primary structure, expression and developmental regulation of a DicO,ostelium calcineurin A homologue, Eur. J. Biochem. 238 (1996) 391-399. [47] Horn E, Gross J., A role for calcineurin in Dictyostelium discoideum development, Differentiation 60 (1996) 269-275. [48] Spann T.P., Brock D.A., Lindsey D.E, Wood S.A., Gomer R.H., Mutagenesis and gene identification in Dictyostelium by shotgun antisense, Proc. Natl. Acad. Sci. USA 93 (1996) 5003-5007. [49] Bernardi P., The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death, Biochim. Biophys. Acta 1275 (1996) 5-9. [501 Connern C.P., Halestrap A.P., Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to Ca 2+, Biochemistry 35 (1996) 8172-8180. [511 Woodfield K., Riick A., Brdiczka D., Halestrap A.P., Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition, Biochem. J. 336 (1998) 287-290. [521 Kroemer G., Zamzami N., Susin S.A., Mitochondrial control of apoptosis, Immunol. Today 18 (1997) 44-51. [531 Marchetti P., Castedo M., Susin S.A., Zamzami N., Hirsch T., Macho A., Haeffner A., Hirsch E, Geuskens M., Kroemer G., Mitochondrial permeability transition is a central coordinating event of apoptosis, J. Exp. Med. 184 (1996) 1155-1160. [54] Cai J., Yang J., Jones D.P., Mitochondrial control of apoptosis: the role of cytochrome C, Biochim. Biophys. Acta 1366 (1998) 139-149. 1551 Thompson J.D., Higgins D.G., Gibson T.J., CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673-4680.