Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

Molecular and Biochemical Parasitology, 33 (1989) 289-296 Elsevier

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Sequence and expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei M a r k Carrington 1, R o l a n d Btilow 2,*, Heinz R e i n k e 3,** and Peter Overath 2 IDepartment of Biochemistry, Cambridge University, Cambridge, U.K., 2Max-Planck-lnstitut fiir Biologie, Tiibingen, F.R.G. and 31nstitut far Genetik der Universitiit zu KOln, KOln-Lindenthal, F.R.G. (Received 21 November 1988; accepted 22 November 1988)

Trypanosoma brucei contains a membrane-bound phospholipase C which converts the variant surface glycoprotein (VSG), anchored in the membrane by a C-terminal glycosyl-phosphatidylinositol moiety, into a soluble form and diacylglycerol. The amino acid sequence (358 residues) of this enzyme, derived from the nucleotide sequence of the cDNA and the gene, reveals a polypeptide which lacks an obvious N-terminal signal sequence and stretches of exclusively hydrophobic residues. These properties suggest that the phospholipase is synthesized in the cytoplasm and subsequently associates with or translocates across intracellular membranes. There are much higher levels of glycosyl-phosphatidylinositol specific phospholipase C mRNA in bloodstream form than in procyclic form trypanosomes. The phospholipase gene is probably present in one or two copies per haploid genome, probably not associated with VSG expression sites. Key words: Glycosyl-phosphatidylinositol-specificphospholipase C; Life cycle; cDNA; Gene; Trypanosoma brucei Introduction T h e v a r i a n t s u r f a c e g l y c o p r o t e i n ( V S G ) of the mammalian bloodstream form of Trypanosoma brucei was a m o n g s t t h e first p r o t e i n s to b e rep o r t e d to be a t t a c h e d to the p l a s m a m e m b r a n e by a glycosyl-phosphatidylinositol (GPI) anchor linked to the C-terminal ot-carboxyl group through e t h a n o l a m i n e . S u b s e q u e n t l y an i n c r e a s i n g n u m b e r o f p r o t e i n s h a v e b e e n f o u n d to b e a t t a c h e d in

Present addresses: *Dept. of Medical Microbiology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A. **Institut ftir Biochemie der Universit~it, Universit~ttsstrasse, D4800 Bielefeld, F.R.G. Correspondence address: M. Carrington, Dept. of Biochemistry, Tennis Court Road, Cambridge CB2 1QW, U.K. Note: Nucleotide sequence data reported in this paper have been submitted to the EMBL Data Base with the accession number X13292. Abbreviations: VSG, variant surface glycoprotein; GPI, glycosyl-phosphatidylinositol; GPI-PLC, GPI-specific phospholipase C.

a similar m a n n e r (see refs. 1 a n d 2 for review). T h e s t r u c t u r e o f t h e G P I a n c h o r has b e e n recently d e t e r m i n e d for the V S G of T. brucei [3] a n d for the Thy-1 a n t i g e n f r o m rat b r a i n [4]. Specific p h o s p h o l i p a s e s t h a t c o n v e r t G P I - a n c h o r e d p r o t e i n s f r o m m e m b r a n e a t t a c h e d to soluble forms have been isolated from T. brucei [5-7] a n d rat liver [8]; t h e s e h a v e r e c e i v e d c o n s i d e r a b l e i n t e r e s t b e c a u s e o f t h e i r p o s s i b l e role in r e g u l a t ing t h e r e l e a s e of surface p r o t e i n s . T h e b e s t chara c t e r i z e d is the p h o s p h o l i p a s e C f r o m T. brucei [5-7], which cleaves t h e G P I - a n c h o r o f t h e V S G f o r m i n g d i a c y l g l y c e r o l a n d a 1,2-cyclic p h o s p h a t e on t h e inositol ring [3,9]. This e n z y m e b e h a v e s as a non-glycosylated amphipathic membrane protein of 37-40 k D a a n d is highly specific for the G P I - m o i e t y ; p h o s p h a t i d y l i n o s i t o l is o n l y slowly c l e a v e d a n d o t h e r c o m m o n p h o s p h o l i p i d s d o not serve as s u b s t r a t e s . A s p a r t of an i n v e s t i g a t i o n into t h e p o s s i b l e role o f G P I - s p e c i f i c p h o s p h o l i p a s e C ( G P I - P L C ) in t h e s h e d d i n g of V S G d u r ing the life cycle o r a n t i g e n i c v a r i a t i o n , t h e prim a r y s t r u c t u r e of G P I - P L C d e d u c e d f r o m t h e n u c l e o t i d e s e q u e n c e of the c l o n e d c D N A a n d g e n e is r e p o r t e d h e r e .

0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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Transcription of the VSG and expression site associated genes does not occur in cultured procyclic form trypanosomes (equivalent to the life cycle stage that lives in the insect vector midgut) [10]. GPI-PLC activity was not detectable in procyclic form trypanosomes [11]. It is shown here that there are far higher levels of GPI-PLC m R N A in bloodstream form trypanosomes than in procyclic form trypanosomes. However, the GPI-PLC gene(s) are probably not associated with VSG expression sites. Materials and Methods

Purification of GP1-PLC and determination of the amino acid sequence of tryptic peptides. GPI-PLC was isolated using a monoclonal antibody affinity column [5]. After elution from the affinity column the preparation was passed through a protein A-Sepharose column to remove contaminating antibody. 100 Ixg purified enzyme was treated with 0.9 txg trypsin at 37°C; 0.1 i~g trypsin were added at 1 h intervals for 9 h. After 9 h the enzyme digest was dialyzed against water. Trifluoroacetic acid was added to 0.5% and the peptides applied to a 4.6 x 250 mm Bakerband Wide Pore C~sHPLC column. Peptides were eluted using a trifluoroacetic acid/water/acetonitrile gradient. After removal of solvent the peptides were sequenced by automatic analytic Edman degradation on an Applied Biosystems model 470 A gas phase sequencer. Phenylthiohydantoin derivatives were identified using an Applied Biosystems model 120 A P T H analyser. Isolation of DNA and RNA from trypanosomes. R N A was prepared as previously [12] from T. brucei M I A G 209 bloodstream form and from cultured 227.201 procyclic form trypanosomes (these were derived from T. brucei M I A G 201 bloodstream form trypanosomes); both are members of the I L T a R 1 serodeme [13] and from T. brucei M I A G 519, a member of the M I T a R 1 serodeme [14]. The procyclic form trypanosomes had been in culture for at least 25 cell generations. D N A was prepared from T. brucei M I A G 209 bloodstream form trypanosomes [15].

Construction and screening of cDNA and genomic libraries. A cDNA library was constructed in hNM1149 from polyadenylated R N A extracted from T. brucei M I A G 209 [12]. A genomic library was constructed in hEMBL3 from T. brucei M I A G 209 D N A that had been partially digested with Sau3A [16]. The c D N A library was screened using oligonucleotide probes derived from the N-terminal sequence of tryptic peptides of GPI-PLC (see Results section). Oligonucleotides were synthesized using a Biosearch Cyclone D N A synthesizer. Four groups of redundant oligonucleotides were each used to screen 1 x 105 recombinants [17], hybridization and washing was at 40°C in 4 x SSC (600 mM sodium chloride, 60 mM tri-sodium citrate). One plaque was positive with one of the probes, D N A was isolated and the c D N A insert subcloned into the EcoRI site of pBluescribe (Stratagene) to form plasmid pBS1. This plasmid was used to screen the genomic library [17]. A positive plaque was identified (XBS2) and D N A prepared from it for further analysis. DNA sequencing. D N A was sequenced by first subcloning 400-800 bp fragments produced by sonication into the SmaI site of M13mp18 [18], then by sequencing these M13 subclones by dideoxy chain termination reactions [19]. Each base was sequenced on average 5 times and at least once in each direction. Gel electrophoresis, blotting and restriction enzyme mapping. These were all performed as previously described [16]. Results

The sequence of a cloned cDNA and a cloned gene encoding GP1-PLC. It was not possible to determine any amino acid sequence from the intact GPI-PLC molecule, possibly due the blocking of the N-terminus, so the N-terminal sequences of four tryptic peptides were established (Fig. 1). In peptide number 4 there was a region encoded by 20 bases with 256 possible coding sequences; four groups of oligormcleotides, each 64 fold redundant, were synthesised to cover all of these possibilities (Fig. 1). A cDNA clone isolated using

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A Fig. 1. Amino acid sequences of the N-termini of four tryptic peptides prepared from purified GPI-PLC. Question marks designate unidentified or tentatively identified residues. The sequences of the four oligonucleotides derived from peptide 4 are shown underneath the sequence. Each of the four oligonucleotides had a different base in the position indicated by X.

one of these oligonucleotides was sequenced (Fig. 2). The c D N A encoded a polypeptide of 40.66 kDa in agreement with the estimated molecular mass of 37-40 kDa. The predicted polypeptide contained amino acid sequences exactly matching the four tryptic peptides, with the exception that a dubious serine in peptide 3 is, in fact, a tryptophan. From the c D N A data alone it was not certain that the first A U G (Fig. 2, bases 78-81) is the initiation codon. In order to verify this the sequence of the gene was determined (Fig. 2). A plasmid containing the c D N A (pBS1) was used to isolate a clone of a gene (hBS2). A restriction enzyme map of hBS2 was determined (Fig. 3), and the region containing the gene from this clone was sequenced (Fig. 2). The sequence of the gene was identical to that of the c D N A except for the poly(A) tail corresponding to the apparent 3' end of the m R N A (bases 1282-1302). An in-frame termination codon (bases 16-18) was found 70 bp upstream of the proposed initiation codon. This confirmed that the A U G at bases 78-81 is the initiation codon.

The expression of GPI-PLC is life cycle stage regulated. Plasmid pBS1 was used to probe a Northern blot of polyadenylated R N A from bloodstream and procyclic culture form trypanosomes. The probe detected a 3.4 kb m R N A from bloodstream forms, which could only just be detected in m R N A from procyclic culture forms when a long exposure was used (Fig. 4). In isolates from the I L T a R 1 serodeme it was

possible to detect a second, slightly larger, mRNA. However, in R N A extracted from a member of the M I T a R 1 serodeme only a single size m R N A was apparent. It is unclear whether this second m R N A is a transcriptional variant or derived from a related gene. Comparison of the size of the c D N A encoding the whole of GPI-PLC (1.3 kb, Fig. 2) and the size of the m R N A detected by Northern blotting (3.4 kb, Fig. 4) leaves 2.1 kb of unassigned sequence. The presence of the poly(A) tail in the c D N A would seem to indicate a 5' non-translated region of 2.1 kb. However, it is more probable that most of the unassigned sequence is 3' non-translated, and that the poly(A) tail in the c D N A is the result from internal priming of first strand c D N A synthesis at bases 1282-1299 where, in the gene, 14 out of 17 bases are adenines.

Genomic location of GP1-PLC gene(s). To investigate the genomic location and copy number of the GPI-PLC gene(s) a Southern blot of genomic D N A from T. brucei MIAG209 was probed with pBS1. Fig. 5 shows that pBS1 hybridized to only one genomic restriction fragment produced by 7 out of 9 restriction enzymes and to two fragments when EcoRI or PstI were used. Of the restriction enzymes used only NdeI cuts the c D N A (at base 265 in Fig. 2). The two NdeI fragments in hBS2 that hybridized to pBS1 are indicated in Fig. 3~ In genomic DNA, cut with NdeI and probed with pBS1, a long exposure was needed to observe hybridisation corresponding to fragment N2, presumably because the majority of the probe hy-

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bridised to fragment N1 (data not shown). The results are consistent with the restriction e n z y m e map of hBS2. The copy of the GPI-PLC gene in hBS2 corresponds to the smaller (5.8 kb) EcoRI fragment seen in Fig. 5. From these data it is possible that there are two GPI-PLC genes in tandem with the second gene lying to the right of the restriction enzyme map in Fig. 3, or that there is a single gene and that hBS2 represents one of two allelic variants. Whether there are one or two copies of the gene per haploid g e n o m e will be resolved by isolation of further genomic D N A clones.

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Fig. 4. N o r t h e r n blot analysis of polyadenylated R N A from different life cycle stages and different isolates of T. brucei. BSF, m a m m a l i a n bloodstream form; PCF, cultured procyclic form. T h e probe used to detect tubulin m R N A s was a plasmid which contained a complete copy of T. brucei c~ and 13 tubulin. The probe used to detect GPI-PLC m R N A was pBS1. Each lane contained 1 ixg of R N A , the blot was washed in 0.1 × SSC, 0.1% sodium dodecyl sulfate at 60°C for 2 h.

* - Fig. 2. D N A sequence of the c D N A and gene encoding GPI-PLC. The underlined nucleic acid sequence (bases 685-705) corresponds to the oligonucleotide probe. T h e predicted amino acid sequence is shown above the nucleic acid sequence, the tryptic peptides are underlined. T h e in-frame stop codon to the 5' of the initiating A U G is indicated by *.

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Fig. 5. Southern blot analysis of T. brucei M I A G 209 D N A . The probe used was pBS1, each lane contains 3 p.g of D N A which was digested with the enzyme shown, and the fragments separated on a 0.8% agarose gel prior to transfer. T h e blot was washed in 0.1 × SSC, 0.1% sodium dodecyl sulphate at 60°C for 2 h.

Discussion

A number of properties are apparent from the amino acid sequence of GPI-PLC: (i) There is no easily recognizable N-terminal signal sequence, so the polypeptide is not expected to be translocated across the membrane of the endoplasmic reticulum. (ii) The polypeptide is not particularly hydrophilic or hydrophobic, it contains 69 acidic and basic residues (R,K,D and E) evenly distributed throughout the sequence and 87 hydrophobic amino acids (I,L,W,F and Y) also distributed evenly throughout the sequence. There are no extended runs of hydrophobic amino acids and no regions of high potential for forming amphipathic a-helices. (iii) The regions of ~t-helix, turn or [3-

sheet potential are evenly distributed (determined using the Chou and Fassmann algorithm). (iv) There is no hydrophobic sequence at the Cterminus and so GPI-PLC is unlikely to be modified by the addition of a GPI-anchor. The primary sequence does not contain any obvious features to indicate how the protein is associated with the membrane. However, the experimental observation is that the enzyme is membrane associated, requires detergent for solubilization and can be reconstituted into liposomes [5-7]. Therefore, either some part of the sequence must be folded in such a way that it penetrates the lipid bilayer, or the protein is covalently modified with a hydrophobic component. The absence of a N-terminal signal sequence indicates that GPI-PLC is probably synthesized in the cytoplasm and then associates with, or is translocated across, intracellular membranes by an unknown mechanism [20,21]. On the other hand, VSG is co-translationally translocated across the membrane of the endoplasmic reticulum, the GPI-anchor is rapidly added [22,23] and the protein is subsequently routed to the cell surface in vesicles. Such an arrangement effectively separates enzyme and substrate within the cell. On hypotonic lysis of bloodstream form trypanosomes GPI-PLC rapidly converts membrane-form VSG to soluble VSG [24], possibly due to uncontrolled fusion of membranes. In vivo, due to experimental difficulties, it is not so easy to ascertain the role of GPI-PLC in release of VSG on differentiation to insect procyclic forms [25]. The greatly increased level of GPI-PLC m R N A in bloodstream form trypanosomes indicates a probable role for GPI-PLC in the metabolism of the GPI-anchor of the VSG as the VSG is the only abundant GPI-anchored protein in the bloodstream form. Moreover, in T. cruzi the role of a related GPI-PLC in the shedding of the Ssp4 surface antigen upon differentiation of amastigotes to epimastigotes has been clearly shown [26]. Comparison of the amino acid sequence of GPIPLC with published mammalian phospholipase C sequences [27-30] failed to reveal any homology; this possibly reflects the different substrate specificity and/or the evolutionary distance between trypanosomes and mammals. A screen of protein

295

and nucleic acid data bases did not detect significant homology with any entry. In procyclic form trypanosomes the amount of GPI-PLC mRNA is far lower than that in bloodstream form trypanosomes; it was only detectable using a long exposure of a Northern blot (Fig. 5). This is consistent with the observation that it was not possible to detect GPI-PLC activity in cultured procyclic form trypanosomes . This life cycle stage regulation and the probable involvement of GPI-PLC in the turnover of the VSG raised the possibility that GPI-PLC gene(s) were amongst the expression site associated genes. However, on a genomic Southern blot plasmid pBS1 hybridised with only one or two restriction fragments produced by the enzymes used (Fig. 5), this contrasts with the five to twenty fragments observed when expression site associated gene probes were used [31,10]. This means

that the GPI-PLC gene(s) are not dispersed in the genome and that the life cycle stage specific expression of the GPI-PLC gene is probably not directly linked to VSG gene expression by inclusion in VSG gene expression telomeres.

Acknowledgements We would like to thank Linda Allan for help in preparing bloodstream form trypanosomes, Jerry Wells for the gift of 227.201 procyclic trypanosome stabilates, Fritz J~ihnig for help with structural predictions and Christina Nonneng~isser for technical assistance. The oligonucleotide synthesis facility in the Biochemistry Department in Cambridge is supported by the Wellcome Trust. The work in Tiibingen was supported by the Fond der Chemischen Industrie.

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