- Email: [email protected]
Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity
Molecular and Biochemical Parasitology 81 (1996) 53-64
Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity’ Manju Josh?, Gregory P. Pogue’+, Robert C. Duncan”, Nancy S. Lee”, Nishi K. Singh”, Chintamani D. Atreya”, Dennis M. Dwyerb, Hira L. Nakhasi”.* “Laboratory
of Molecular
bCeN Biology Section.
Pharmacology, Division of’ Hemurologic Products, Center jbr Bioiogics Evaluation and Research, Food and Drug Administration, Bethesda MD 208924425, USA Laboratory of’ Parasitic Diseases, NIAID, National Institutes of’ Health. Bethesda MD 208924425. USA
Received 22 February 1996; revised 21 May 1996: accepted 24 May 1996
Abstract Calreticulin has been implicated in multiple cell functions. Recently, we have shown that both human and simian calreticulin are RNA binding proteins and that their binding activity is due to phosphorylation. To demonstrate that the RNA binding property of calreticulin is an intrinsic part of this multi-functional molecule and is evolutionarily conserved. we isolated and characterized the calreticulin gene from the unicellular parasite, Leishmaniu donovani. Amino acid sequence homology between human and Leishmaniu calreticulin (L. d. cal) is limited, but like the human homologue, L. d. cal binds Cat +, can be phosphorylated in vitro and binds certain RNA sequences in a phosphorylation-dependent manner. Unlike human calreticulin, L. d. cal is glycosylated and its binding to endogenous Leishnxmiu RNA is phosphorylation-independent. The binding of L. d. cal to Leishmunia RNA suggests that the RNA binding activity of calreticulin has remained evolutionarily conserved.
Kepmds:
Calreticulin;
RNA binding
protein;
Phosphorylation;
Glycosylation;
Trypanosomatid;
Functional
conser-
vation
L. d. Cal., Leishmania donovani calreticulin; RV, rubella virus. * Corresponding author. Laboratory of Molecular Pharmacology, DHP/OTRR/CBER/FDA, Bldg. 29 Rm 107, 8800 Rockville Pike, Bethesda, MD 20892-0425. Tel: + 301 496 2205; fax: + 301 496 1810; e-mail: [email protected] ’ Note: The nucleotide sequence data reported in this paper is available in the NCBI, GenBank and the accession number is u49191. ’ Present address: Biosource Technologies, Inc., 3333 Vaca Valley Pkwy, Vacaville. CA 95688, USA. Abbreviations:
0166-6851/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PZI S166-6851(96)02676-X
54
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
1. Introduction
Calreticulin is a major Ca+ + binding protein which is localized mainly in endoplasmic reticulum [ER] [l]. The protein initiates with a signal sequence for ER entrance and terminates with a KDEL signal sequence which is the retention signal for ER resident proteins [2]. However, nonER locations for this protein have been also reported: the nucleus of some cells [3], the acrosome of sperm cells [4], cytotoxic granules in T-cells [5] and in the cytoplasm [6,7] suggesting multiple functions outside of the ER. Calreticulin cDNA clones have been isolated from a variety of sources, including human, drosophila and schistosomes [1,8- 131. The phylogenetic diversity of these sources suggest that calreticulin is an ubiquitous protein whose amino acid sequence is conserved among various species. Calreticulin has been implicated in multiple cell functions, based on different functional domains found in its primary structure [14] and its presence in different cell compartments [3-71. For example, the N-terminal domain of calreticulin has been shown to modulate expression of glucocorticoid receptor regulated genes both in vitro and in vivo via protein-protein interaction [6,15]. Specific interaction of the C-terminal domain of calreticulin with coagulation factors (IX, X, prothrombin) suggested that it has a potentially antithrombotic function and indeed the administration of calreticulin was shown to prevent coronary thrombosis [16]. P-domain of calreticulin has a significant similarity with an integral ER membrane protein calnexin which has been shown to have chaperone function [l]. Like calnexin, calreticulin has been shown to be a lectin and participate in molecular chaperone of glycoprotein folding [17,18]. Recently, we have shown that both human and simian calreticulins are RNA binding proteins which interact with the 3’-cis acting element of Rubella virus (RV) RNA in vitro and in RV infected Vero 76 cells [19-231. The 3’-cis acting element of RV consists of an inverted repeat sequence of 12 nucleotides located at the 3’ end of the genomic RNA which is capable of forming stem-loop (SL) structure and is necessary for initiation of (-) strand RNA synthesis of RV RNA
[23]. Further, the RNA binding activity of calreticulin was dependent on its phosphorylation [21,22]. An increase in the binding affinity was observed after RV infection [22], coinciding temporally with the appearance of (-) strand RNA synthesis [23]. Both human and simian calreticulins have phosphoprotein isoforms in vivo and autophosphorylate in vitro [19-231. The in vitro RNA binding and the autophosphorylation activity resides in the N-terminal domain of these molecules [22]. In the present study, we explored the possibility that the newly discovered RNA binding property of calreticulin of higher eukaryotes is conserved in evolutionarily divergent species such as a simple unicellular organism, Leishmania. Leishmania is a trypanosomatid protozoan parasite which has a life cycle characterized by the presence of an extracellular, flagellated promastigote form in sand fly vectors [24] and a nonmotile amastigote stage within phagolysosomes of mammalian macrophages [25]. Little is known about RNAprotein interactions in these trypanosomatid parasites and what role such interactions play in cellular metabolism. However, much progress has been made in identification and elucidation of the functions of many of RNA binding proteins in higher eukaryotes [26]. As a first step towards studying the conservation of calreticulin RNA-binding activity in Leishmania and its potential role in trypanosomatid biology, we have isolated the leishmanial homologue of human calreticulin and characterized its functions. To our knowledge, this is the first detailed study of an RNA-binding protein in Leishmania, characterized both with heterologous and homologous RNAs.
2. Materials and methods 2.1. In vitro culture of parasites isolation
and RNA
A cloned line of L. donovani (WHO designation: MHOM/SD/62/1S-Cl,,) was used in all experiments [27]. Promastigotes and in vitro ‘amastigotes’ were grown as described previously
55
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 5.3-64
[27]. Cells of both phenotypes were harvested and processed as described [27]. Total RNA was isolated and Northern blot analysis was performed according to Joshi et al. [27]. Nick-translated cloned L. d. cal DNA was used to probe RNA blots. 2.2. Cloning of the Leishmania calreticulin (L. d. cul) gene and construction of its chimeric form Two degenerate oligonucleotide primers corresponding to amino acids (aa) 135 - 142 (S-primer, ATGTTCGGCiTCCGjCGACATCTGCGG) and aa 272-279 (3’-primer, CCACTCG/ACCCTTGTACTCCGGGGTT) of human calreticulin that are conserved among known calreticulins [lo] were used to amplify the L. d. cal gene from Leishmania donouani genomic DNA in a PCR reaction. A 485-bp PCR fragment was generated, cloned into the PCRII vector (Invitrogen, Inc). Sequence analysis of this PCR fragment showed significant homology with the human calreticulin sequence. The cloned 485-bp fragment was then used as a probe to screen a Leishmania DNA cosmid library (a gift from Dr. Buddy Ullman, Oregon Health Science University, Portland, OR, USA). A 4.5-kbp fragment containing L. d. cal sequence obtained from the cosmid library was subcloned into a Bluescript vector (Stratagene, PBS), and subjected to nucleotide sequence analysis (Sequenase Kit, USB). Primers corresponding to the part of the putative N-terminal leader sequence (amino acids MLAAVVGV) and the C-terminal end (amino acids SEDKEDL) plus a termination codon (Fig. 1) were used to PCR-amplify the entire L. d. cal open reading frame. PCR products were cloned into the PCRII vector (Invitrogen, Inc.). The entire coding sequence of the mature L. d. cal was PCR-amplified using a 5’-EcoRI site containing primer corresponding to the N-terminus coding sequence (EIFFHEEF) and a 3’ primer corresponding to the C-terminus codons (SEDKEDL) including a BarnHI site. Chimeric L. d. cal protein containing sequences from drosophila calreticulin gene was constructed by using the modification of the 5’ primer described above, where the first 12 amino acids of the mature L. d. cal were replaced by drosophila calreticulin (SAEVY LKENFDN) in frame with codons encoding
MDGW... of the L. d. cdl sequence. The amplified DNA fragments following digestion with EcoRI and BarnHI enzymes were cloned into similarly digested PET 22b( + ) expression vector containing 6-histidine residues at the carboxy terminal end (Novagen). Plasmid DNA transformation and expression of recombinant calreticulin was performed according to the manufactures protocol (Invitrogen, Inc.). 2.3. In vitro translation
of L. d. cal
Approximately 1 ,ug of PTA L. d. cal plasmid DNA was incubated in the T7 RNA polymerase transcription-coupled-translation (T7driven TNT) reaction containing 40 PCi of [35S] methionine (800 Ci mmol _ ‘, specific activity) according to manufacturer’s protocol (Promega, Inc.). In parallel reactions, canine pancreatic microsomal membranes were added and following 2-h incubation, microsomal membranes were pelleted by centrifugation at 12 OOOXg for 10 min and resuspended in 1X phosphate buffer saline (PBS). To test for the importation of the L. d. cal into the lumen of microsomal membranes, equal aliquots of total translation reactions either containing or not containing membranes, were treated with thermolysin protease in the presence or absence of 1% NP40 [28]. To confirm the glycosylation of the L. d. cal, equal aliquots of total translation products synthesized in the presence or absence of microsomes were incubated in a buffer containing 0.05M NaP04 buffer (pH 7.5) supplemented with 1% NP40 [28]. The samples were incubated at 37°C for - 2 h in the presence or absence of N-glycosidase F (PNGase F), New England Biolabs [28]. To each of these samples, SDS-PAGE sample buffer [21] was added to 1X concentration and boiled. All translation reactions were analyzed by 10% SDS-PAGE followed by autoradiography [ 191. 2.4. Expression chimeric .form.
of recombinant
L. d. cal and its
The L. d. cal protein or its chimeric form purified by lysing the cells in buffer A (8M containing O.lM PO4/0.01 M Tris-HCl pH The cell debris was removed by centrifugation
was urea 8.0). and
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 53-64
56
P 7
45
L.d.cal.
MLLSVPLLLGLLGLAV----AEPAVYFKEQFLDGDGDGWT-SRWIESKHKSD II: I I:I I I II III III I I I II MLAAW---GVLVLCVYWQAEIF--FHEEFNTMDGWVQS---E--HTSD
Bu . -Cal.
FGKFV-LSSGKFYGDEEKDKGLQTSQDARFYALS-----ASFETFSNKGQ
89
Hu.-cal.
:/I L.d.cal. Hu. -Cal.
L.d.cal.
Hu. -Cal.
I
II
I
I
II:
II
I I :I I I :I
I
II
40
I
YGK-VALSVGAIHVDAEKEQGLKLMEDAKFYAVSKKLPKAV----SNDGK 5'PCR Primer -> TLWQFTVKHEQNIDCGGGYVKLFPNS-LDQTDMHGDSEYNIMFGPDICG :::j I:11 II : III I:I:I I III I II:/ I :IIIIII SIIVSFSVKNEQKLTCGGTYLKFF--SELDQKDLHGESAYWLMFGPDIIG
85 138 I
133 185
L.d.cal.
PGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTL-IVRPDNTYE--VK I: I I I I I: I :I I I Il:ll: I:1 III I SSTRL-HSRD-YrGTNHLWKKLWRPKTDKATHWTVEIA_PNTYQLYV-
Hu .-Cal.
IDNS--QVESGSLEDDWDFLPPKKIKDP-DASKPEDW-DE~KIDDPTDS
231
L.d.cal.
224
Hu. -Cal.
I I I II I ::I1 IIII I II I II II I: I I I :I : -DGMHIQ-E-GSFEEEWDMLPPKTIPDPTDE-KPADWVDDMMb-DDPSDT ---_ 3'PCR Primer +-
KPEDWDK-PEHIPDPDAKKPEDWDEEMDGEW---E-P--PVIQNPEYK-G
273
II
L.d.cal.
IIIII I II:IIIIII II I I I I KPEHWDDEPATITDSEAVKPVDWD---DGGRRRVEAPKIPKIPNPNYRVG EWKPRQIDNPDYKGTW----IHPEIDN------PEY---SPDPSIYAYDN I I Il:/lI I II I I I I GRHVR-IHNPEYKGEWAARQI-P---NTGLQGGPEPVQGSRRCSTSASMS
I
FGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNE--TWGVTKA-AEKQM ll:lI::I I I:1 II I I I --------WQVEGGTVFDDIIIGDDIT--EVLGVVKSTYG---AMAEK--
I
Hu.-cal. L.d.cal. Eu.-cal. L.d.cal. Hu .-Cal. L.d.cal. Hu.-cal. L.d.cal.
KDKQDEEQR-LKEEEEDKKR--KEEEEA--EDKEDD-EDKDE--DEEDEE I II III/ II II II I I -----E--RDLIQAEE-KKEATKEPAEAAAE-KPNVGEHADHTPDEGDSE DKEEDEEEDVPGQAKDEL ::/ I D----____----_KEDL
1:
179
I
271 310 I
316 357 III
351 399 II
I
I
392 417 397
Fig. 1. Comparison of human (Hu-Cal) and Leishmania calreticulin (L. d. cal) amino acid sequences. Vertical bars represent identical amino acids, the two dots represent conservative changes. The amino acids from which oligonucleotides for PCR primers were derived are shown. SP denotes the signal peptidase cleavage site. The glycosylation sites are indicated by a black diamond. Two putative protein kinase C phosphorylation sites are underlined by thick lines, whereas two putative casein kinase phosphorylation sites are indicated by thin lines. The putative nuclear localization sequence is denoted by a dashed underline.
the supernatant was passed through a Ni-agarose beads column (Qiagen), followed by washing with buffer A at pH 6.3. The recombinant calreticulins bound to Ni-agarose beads were renatured by step wise reduction in urea concentration in buffer A until final wash contained only 0.1 M Sodium phosphate and 1mM Tris-HCl pH 8.0. Calreticulin proteins were eluted from the Ni-agarose beads in buffer containing 250 mM Imidazole, 300 mM
NaCl, 0.1 M Sodium phosphate, 1 mM Tris-HCl (pH 8.0). The eluted fractions were renatured by dialysis against in a buffer (10 mM Tris-HCl pH 7.4, 40 mM KCl, 5% glycerol and 100 PM PMSF) for approximately 12 h using Slide-A-lyzer (Pierce) dialysis membranes. Analysis of proteins isolated in this manner revealed that the induced protein, L. d. cal or chimeric L. d. cal, were > 95% pure in final solution by Coomassie Blue staining.
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 53-64
2.5. Immunoblot analysis of recombinant and its binding to 45Ca + +
L. d. ccl1
A polyclonal rabbit sera raised against recombinant human calreticulin (LAR 086; kind gift of L. A. Rokeach or obtained from Affinity Bioreagents, Inc. Golden, Co) was used to determine immunogenic cross-reactivity between calreticulins from human and Leishmaniu sources. One to five pg of recombinant calreticulin proteins were separated in a 10% SDS-PAGE gel, transferred to nitrocellulose membranes and analyzed by Western blot [29] using calreticulin antibody ( l/l000 dilution). The proteins were visualized by the ECL light reaction system (Amersham, Inc.). The binding assay for 4s Ca + + was performed according to Maruyama et al. [30]. Briefly, proteins on the filters were incubated with Ipci/ml 45Ca in buffer C (60 mM KCl, 5 mM MgCl,, 10 mM imidazole-HCl, pH 7.0) for 10 min. The filters were washed with distilled water. dried and exposed to x-ray film. 2.6. RNA electrophoretic
mobility
shift assay
57
brane in nondenaturing running buffer (25 mM Tris and 200 mM glycine). The membrane was stained with 0.1% Ponceau S and photographed. The membrane was then washed 3 times with TBS-T (20 mM Tris-HCl pH 7.4, 100 mM NaCI, 1 mM EDTA, 0.1% Tween 20) and incubated in 2 ml TBS-T with 1~10~ cpm in vivo labeled total L. donovani RNA at room temperature for 30 min. The membrane was washed 4 times with TBST-T and visualized by autoradiography. Labeled RNA was prepared from 3x10’ promastigotes of cultured L. donovani, first incubated for 1 h in phosphate free RPM1 1640, followed by addition of 2 mCi of “‘P-orthophosphate and incubated for 3 h [32]. RNA was extracted with RNA STAT-60 according to the manufacturer’s protocol (TELTEST ‘B’. Inc.).
3. Results 3.1. Isolation and expression of the Leishmania calreticulin gene and comparison to human calreticulin.
(REMSA)
RNA-protein interactions by REMSA were performed as described before [20]. Radiolabeled [(a-“P) CTP] RV 3’ ( + ) SL RNA was transcribed from a T7 promoter as described [20]. 2.7. In vitro autokinase phosphatase
activity
and alkaline
treatment
In vitro autokinase activity was measured as described earlier using Y-~‘P ATP [19]. The labeled proteins were separated in 10% SDS-PAGE and visualized by autoradiography. The alkaline phosphatase (AP) treatment was done according to Nakhasi, et al. [22]. After the treatment, proteins were used directly in REMSA [22]. 2.8. RNA binding assay for endogenous Leishmania
RNA
Purified recombinant proteins were separated on non-denaturing, discontinuous polyacrylamide gel [31] and transferred to nitrocellulose mem-
We used a PCR based strategy to isolate a calreticulin homologue of the human gene. A 4.5-kb fragment from the Leishmunia cosmid library was cloned and nucleotide sequence of - 1.4 kb was determined. The nucleotide sequence of the 1.4-kb DNA fragment has been deposited in the GenBank with an accession number U49191. The first AUG codon was assigned on the basis of a largest open reading frame (ORF) originating from such an AUG and on the size of the protein coded in an in vitro translation system by this ORF that had a calculated molecular weight similar to known calreticulins. The deduced amino acid sequence of the ORF revealed a putative signal sequence of 18 hydrophobic amino acids at the N-terminus followed by 379 amino acids (Fig. 1). The ORF terminates with KEDL, a putative ER retention signal similar to the KDEL found in human calreticulin [9]. Two putative glycosylation sites were identified at residues 126 and 154 in the mature L. d. cal protein (Fig. 1). In addition, two putative protein kinase C phosphorylation sites at Leishmania
58
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
residues 55-51, 74-76; and two casein kinase sites at residues 170-174, 304-308 were identified (Fig. 1). A putative nuclear localization site at residues 179-186 w’as also identified in the mature L. d. cal protein (Fig. 1). A comparison of the deduced amino acid sequence of L. d. cal with human calreticulin showed an overall homology of 42% (Fig. 1). Amino acid sequences comparison between the three putative domains revealed a 52% homology in the P-domain, 47% in the N-terminal domain, and 29% in the C-terminal domain. Even though there is a limited amino acid sequence homology between the human and Leishmania calreticulins, it was of interest to test structural similarities between the two calreticulins. The ORF of L. d. cal was expressed in E. coli and western blot analysis of the recombinant protein was performed using polyclonal antibodies against human calreticulin (Fig. 2). The L. d. cal showed two Coomassie stained bands (Fig. 2, lane 2) and both forms reacted with the human antibody (Fig. 2, lane 4). Recombinant human calreticulin expressed as a maltose binding fusion protein (H. Cal-MBP) was used as a positive
+
H.cal-Ab
+
kDa
12
34
Fig. 2. SDS-PAGE and western blot analysis of recombinant human and recombinant Leishmania calreticulin. H. Cal.MBP, human calreticulin expressed as fusion protein with maltose binding protein. L. d. Cal.. Leishmania donouani calreticulin expressed as histidine tagged protein. Lanes 1 and 2 represent Coomassie stain of the two proteins on SDS-PAGE. Lanes 3 and 4 represent immuno-reactive bands of the two proteins using polyclonal antibody against human calreticulin.
L. don. RNA Promastigote Amastigote
-1.9 kb-
Fig. 3. Northern blot analysis of total RNA from L. donovani pro- and in vitro grown ‘amastigotes’. Twenty fig of total RNA from amastigotes, lane 1, and from promastigote. lane 2, were separated on 15% agarose gel containing 6% formaldehyde. The approximate size of the L. d. cal mRNA is indicated by an arrow.
control for human calreticulin antibody (Fig. 2, lanes 1 and 3). The specificity of human calreticulin antibody was demonstrated by its inability to react with other proteins such as maltose binding protein, MBP (data not shown). To demonstrate that calreticulin gene was in fact expressed in the parasite, Northern blot analysis of the total RNA isolated from both L. donovani pro- and in vitro grown ‘amastigotes’ was performed and probed with cloned L. d. cal DNA (Fig. 3). Calreticulin mRNA was present in both Leishmania forms (Fig. 3, lanes l-2). Further, the size of the L. d. cal mRNA ( - 1.9 kb) was similar to that reported in other organisms [7-121 and corresponds to the size predicted from the Leishmania genomic clone. 3.2. Leishmaniu calreticulin is a glycoprotein. To test whether putative glycosylation sites in the mature L. d. cal have functional significance, we translated L. d. cal RNA in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomes. L. d. cal RNA translated in rabbit reticulocyte lysates lacking microsomes synthesized two polypeptides of - 60 and 63 kDa
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
59
Microsomal Mem.: k;;
97 -
431
2
3
4
5
6
7
8
9
10
Fig. 4. Glycosylation of in vitro synthesized L. d. cal. SDS-PAGE analysis of proteins synthesized from transcripts of plasmid PTA L.Cal. which contains the entire L.Cal ORF, and were incubated in rabbit reticulocyte lysates alone (lanes l-3, 7-8) or in the presence of canine pancreatic microsomes (lanes 4-6, 9-10). Translation products were treated with either NP40 buffer and thermolysin protease, Prt-ase + NP40 (lanes 3 and 6) or thermolysin protease (Prt-ase) alone (lanes 2 and 5). Translation products were treated with either N-glycanase F, Buffer + N-gly-ase (lanes 8 and IO) or without, Buffer (lanes 7 and 9). The location of molecular weight standards (kDa) are shown at left.
(Fig. 4, lane 1). In contrast, L. d. cal RNA translated in the presence of microsomal membranes yielded two proteins of - 70 and 73 kDa (Fig. 4, lane 4). To establish that the L. d. cal signal sequence led to the import of the L. d. cal protein into the lumen of the microsomes, the translation products were treated with the protease thermolysin. Protease treatment of L. d. cal translated in the absence of microsomes with or without detergent (NP40) led to the complete digestion of the translation product (Fig. 4, lanes 2-3). Following translation of L. d. cal in the presence of microsomes, the microsomes were pelleted by centrifugation and then subjected to various treatments. In the absence of NP40, the microsomes protected the majority of the 70-73 kDa L. d. cal translation product from being digested by the protease (Fig. 4, lane 5). In contrast, addition of NP40 to the latter resulted in the degradation of the - 70-73 kDa proteins to an - 60 kDa species (Fig. 4, lane 6). These results indicate that the L. d. cal contains
a functional signal sequence for import into microsomes. Presumably glycosylation of the L. d. cal protein within such microsomes imparts resistance to its complete digestion with thermolysin. To determine whether the addition of carbohydrates was responsible for higher molecular weight of L. d. cal, we treated L. d. cal translation products with N-glycanase in the presence or absence of microsomes. Treatment of L. d. cal translated in the absence of microsomes with the N-glycanase had no effect on its mobility (Fig. 4, compare lane 8 with lane 7). Incubation of L. d. cal translated in the presence of microsomes with N-glycanase led to a reduction in the size of the labeled proteins from - 70-73 kDa to - 60-63 kDa (Fig. 4, lane 10); the later molecular size being identical to calreticulin observed in translation reactions without microsomes (Fig. 4, lane 1). Thus. confirming that translation of L. d. cal in the presence of microsomes resulted in the glycosylation of the protein.
60
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
3.3. L. Cal is a Ca + + binding protein.
3.4. L. Cal is an autokinase
To determine whether L. d. cal binds Ca+ +, SDS-PAGE gel analysis of a fusion protein consisting of human calreticulin and MBP (H. CalMBP), the Leishmania his-tag calreticulin (L. d. cal-6His) and a his- tag DHFR (DHFR-6His) protein was performed. Proteins were transferred to a nitrocellulose membrane and incubated with 45Ca. Both H. Cal-MBP (Fig. 5, lane 1) and L. d. cal (Fig. 5, lane 2) bound labeled calcium. It was previously shown that MBP alone does not react with 45Ca [19]. No reactivity was observed with the control DHFR-6His protein (Fig. 5, lane 3). On the contrary, it appeared to block background binding of Cat + to membrane.
Recombinant DHFR, L. d. cal, Bovine serum albumin and MBP-human Cal proteins were analyzed on a SDS-PAGE and stained with coomassie blue (Fig. 6, Lanes l-4). Upon incubation with 32Py-ATP in a kinase reaction buffer, only L. d. cal (Fig. 6, lane 6) and H. Cal-MBP (Fig. 6, lane 8) were labeled. Some cleavage of the H. Cal-MBP fusion protein occurs during isolation and the slight radioactivity associated with a lower molecular weight band in Fig. 6, lane 8, corresponds to free calreticulin resolved under these conditions. No significant radioactivity was associated with the DHFR-6His protein (Fig. 6, lane 5). These results demonstrate that L. d. cal, like H. Cal-MBP, can be phosphorylated in vitro in the absence of cellular extract. 3.5. L. d. cal is an RNA binding protein.
45Ca overlay: Mr kDa
97 68 4330 18 -
Fig. 5. Autoradiogram of 45Ca labeled proteins after SDSPAGE and transfer to nitrocellulose membrane. Lane 1, human calreticulin expressed as a fusion protein with maltose binding protein (H. Cal.-MBP); lane 2, Leishmania calreticulin expressed as a His-tag protein (L. d. cal-6His); lane 3, DHFR6His protein. Protein molecular weight markers are shown.
Since the only known RNA ligand for human calreticulin is RV 3’ ( + ) SL RNA [21-231, we first tested the RNA binding activity of the L. d. cal protein with the RV 3’ ( + ) SL RNA in EMSA in the presence or absence of alkaline phosphatase (AP) treatment (Fig. 7A). L. d. cal in the absence of AP treatment bound RV RNA (Fig. 7A, lane 2) and this interaction was abrogated by pretreatment of the protein with AP (Fig. 7A, lane 3). The interaction of L. d. cal with RV RNA was determined to be specific since non-specific RNAs such as poly I-C and poly AGC did not compete with binding to RV RNA (data not shown). These results showed that the recombinant L. d. cal had the capacity to bind RV RNA and that a phosphorylated form of L. d. cal is necessary for such an interaction. In order to determine whether L. d. cal binds to an endogenous leishmanial RNA, we metabolically labeled RNA in cultured Leishmania donouani promastigotes with 32P-orthophosphoric acid. The labeled RNA was tested for binding to calreticulins immobilized on a nitrocellulose membrane. Recombinant L. d. cal, drosophila/L. Cal (D/L. Cal), and MBP-human calreticulin (H. CalMBP) were resolved in non-denaturing polyacrylamide gels and transferred to nitrocellulose
M. Joshi et al. / Molecular and Biochemical Parasito1og.v 81 (1996) 53-64
61
Coomassie Stain: 32P r_ATP: Mr kDa
20097-
+
H.caCMBP
*
L.d.cal-GHis
66-
43-
30-
16-
1
2
3
4
5
6
7
6
Fig. 6. Autokinase activity of recombinant Leishmania calreticulin. DHFR-6His (lanes 1 and 5), L. d. cal-6His (lanes 2 and 6), BSA (lanes 3 and 7), H. cal-MBP (lanes 4 and 8). Proteins were either separated by SDS-PAGE and stained with Coomassie blue, lanes l-4, or incubated with y-‘*ATP alone in a kinase reaction prior to electrophoresis and the dried gel autoradiographed, lanes 5-8. Arrows denote the phosphorylated proteins. Protein molecular weight markers are shown.
membranes. Under these nondenaturing conditions, the recombinant proteins which migrate as a single band in denaturing SDS-PAGE (data not shown), are resolved as multiple bands as seen by Ponceau-S staining of these membranes (Fig. 7B). Labeled promastigote RNA bound only to the slowest migrating form of both L. d. cal and D./L. cal (Fig. 7C, lanes 1 and 3) whereas significantly less binding was observed with H. CalMBP (Fig. 7C, lane 4). Interestingly, pretreatment of L. d. cal with AP resulted in its enhanced binding to Leishmania RNA (Fig. 7C, lane 2). On the contrary, phosphatase treatment of H. CalMBP did not enhance binding to Leishmania RNA (data not shown). Further, other recombinant proteins such as MBP or glutathione-s-transferase (GST) proteins did not bind labeled L. d.
RNA in this assay (data not shown). These results demonstrate that L. d. cal: (1) binds to its cognate RNA in Leishmania, and (2) that phosphorylated isoforms of leishmania calreticulin are not needed for RNA binding activity.
4. Discussion Previously, we showed that both human and simian calreticulin are RNA binding proteins and that they bind the RV 3’ (SL) RNA which is necessary for RV replication [19-231. The RV RNA binding activity of human calreticulin resides in the N-terminal domain [20]. The human calreticulin is devoid of the conserved motifs present in most RNA binding proteins [27], how-
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
62
Protein:
-
L.d.cal
Phosphatase:
-
-
+
A
Phosphatase:
f-
-
4-
-
Complex
-
Fig. 7. (A) REMSA of L. d. cal with a RV 3’ (+ ) SL RNA probe. Lane 1, probe alone. Lane 2 with L. d. Cal protein in the absence of alkaline phosphatase (AP) treatment and lane 3 with L. d. cal protein in the presence of AP. RNA-protein complex is indicated by an arrow. Free probe is also indicated. (B) Ponceau S. staining of the indicated proteins. Roman numerals I-V indicate the position of multiple forms of calreticulin. Lane 1, L. d. cal, without AP treatment; lane 2, the same protein pretreated with AP; lane 3, chimeric drosophila/leishmania calreticulin (D./L. cal) without AP treatment; lane 4, fusion protein of human calreticulin and maltose binding protein (H. Cal-MBP) without AP treatment. (C) The same filter shown in panel B incubated with 1~10~cpm of in vivo “P labeled total Leishmania RNA. The roman numeral-labeled arrows indicate the position of stained bands shown in (B).
ever, its RV RNA binding activity is dependent on the phosphorylation of the protein [19,20]. Thus, from these studies it is evident that RNA binding activity of the phosphorylated form of calreticulin [ 19,201 is an important function of this multifaceted protein for RV replication. Similarities in amino acid sequences among calreticulins from various species has been implicated in the conservation of specific functions [14]. The presence of RNA binding activity of calreticulin may suggest that it has an important role in cellular metabolism. It was of interest, therefore, to ascertain whether this function is conserved in calreticulins from evolutionarily divergent organisms. We choose to study this function in calreticulin from the Leishmania parasite, a unicellular organism, distantly related to multicellular organisms. Since RV RNA is the only RNA ligand known to bind to human calreticulin, we first tested the RNA binding activity of L. d. cal with RV RNA. It was clear from several in vitro experiments that recombinant L. d. cal binding to RV RNA was specific and that such binding was also phosphorylation-dependent, similar to human calreticulin. Further, there is an RNA binding activity in the Leishmania cell extracts that can interact with RV RNA in vitro (data not shown). Even though L. d. cal binds to RV RNA, it is certainly not its physiological ligand. Therefore, it was important to demonstrate whether an endogenous RNA of Leishmania could interact with its own calreticulin. In the present study, we have demonstrated that Leishmania promastigote RNA can be bound by the recombinant L. d. cal and the binding to endogenous RNA does not need phosphorylated isoforms of calreticulin as was observed for its binding with RV RNA [19]. On the contrary, the MBP-human calreticulin fusion protein binds significantly less to Leishmania RNA. Recently, we (Nakhasi, unpublished data) and Cheng et al. [33] have shown similar phosphorylation independent binding of human calreticulin to cellular RNA (hYRNA, a small cytoplasmic RNA of higher eukaryotes). At present, the Leishmaniu RNA ligands for L. d. cal are not known and are the subject of future studies. Thus, it is clear that L. d. cal exhibits dual RNA binding activity depending upon the source of RNA ligand.
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
Little is known about the RNA binding proteins or their ligands in Leishmania or other relevant trypanosomatids. The study of RNA protein interaction is of enormous importance in the trypanosomatids because these parasites employ unique biochemical mechanisms such as trans-splicing of leader RNA and RNA editing in gene expression [34,35]. These, in addition to the routine biochemical processes such as translation, RNA processing, transport of RNA from different organelles and storage of RNAs in RNP-complexes to protect from degradation imply a complex regulation of RNA metabolism by cellular proteins [34,35]. Recently, a cellular protein from Leishmania donovani promastigotes was shown to interact with a non-physiological ligand such as antisense RNA of the 5’ untranslated region of /I-tubulin [36]. However, this protein was also shown to interact with several species of small rRNAs and weakly with tRNA [36]. In neither case was the identification of the protein and the mechanism of interaction evident [36]. Besides having conserved the RNA binding activity, L. d. cal has also retained Ca+ + binding and in vitro phosphorylation functions. This is evident by the significant sequence homology in the P-domain and confirmed by in vitro Ca’ + binding. In addition, we demonstrated. in vitro, that L. d. cal unlike human [9], rabbit [8] or chicken liver [37] calreticulins, can be glycosylated. Recently, however, calreticulin isolated from bovine brain [38] and CHO cell [39] has been shown to be glycosylated. In CHO cells, glycosylation of calreticulin occurs in response to acute heat stress and may play role in the cellular thermotolerance [39]. At present, however, it remains to be seen whether L. d. cal is glycosylated in vivo or undergoes change in glycosylation during temperature shift from promastigote to amastigote form [24]. Further, whether such a change in glycosylation plays a role in parasite differentiation similar to the change in thermotolerance in CHO cells [39] remains to be established. In conclusion, the current study demonstrates that the RNA binding activity of calreticulin, besides other functions such as Ca+ + binding and ability to phosphorylate in vitro. has re-
63
mained evolutionarily conserved from this unicellular protozoan to humans. Acknowledgements
We thank Drs. Tosato and Kaplan for critical reading of the manuscript. We also thank Dr. Rokeach for providing anti-human calreticulin antibody and Dr. Buddy Ullman for the Leishmania DNA cosmid library. We also thank S. Baksh for providing protocol for the Ca+ + binding assay. We would like to thank Ms. Smriti (Sumi) Nakhasi for her excellent editorial assistance. References [I] Michalak, M.. Mimer, R.E., Burns, K. and Opas. M. (1992) Calreticulin. Biochem. J. 285, 681-692. [2] Burns, K. and Michalak, M. (1993) interaction of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes. FEBS lett. 318, 18 I- 185. [3] Opas, M., Dzaik, E., Fliegel, L. and Michalak, M. (1991) Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J. Ceil. Physiol. 149, 160&171. [4] Nakamura. M.. Moriya. M., Baba, T., Michikawa, Y.. Yamanobe, T., Arai, K., Okinaga, S. and Kobayashi, T. (I 993) An endoplasmic reticulum protein, calreticulin is transported into the acrosome of rat sperm. Exp. Cell Res. 205, 101~110. [5] Corad, M.E., Umbreit, J.N., Moore, E.G. and Harper, K.P. (1991) Molbiferrin, a homologue of ROSS-A autoantigen and calreticulin. Blood 78. 89a. [6] Burns, K., Duggan, B., Atkinson, E.A., Famulski. K.S., Nemer, M., Bleackely, R.C. and Michalak, M. (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367. 4677480. [7] Sueyoshi, T.. McMullen, B.A., Marnell, L.L., Clos. T.W.D. and Kisiel, W. (1991) A new procedure for separation of protein Z. prothrombin fragment 1.2 and calreticulin from human plasma. Throm. Res. 63. 569575. [8] Fliegel, L.. Burns, K., McLennan, D.H., Reithmeier. R.A.F. and Michalak, M. (1989) Molecular cloning of the high affinity calcium-binding protein(calreticulin) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264. 21522-21528. [9] McCauliffe, D.P., Lux, F.A., Lieu. T.S.. Sanz. 1.. Hanke. J.. Newkrik, M.M.. Bachinski, L.L., Itoh, Y., Siciliano, M.J., Reichlin, M., Sontheimer, R.D. and Capra, J.D. ( 1990) Molecular cloning, expression, and chromosome 19 localization of a human ROSS-A autoantigen. J. Clin. Invest. 85. 137991391.
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I Molecular and Biochemical Parasitology 81 (1996) 53-64
1101Khahfe, J., Pierce, R.J., Godin, C. and Capron, A. (1993) Cloning and sequencing of the gene encoding Schistosoma mansoni calreticulin. Mol. Biochem. Parasitol. 62. 3133 316. [111 Smith, M.J. (1992) A C. elegans gene encodes a protein homologous to mammalian calreticulin. DNA Seq. 2, 235-240. WI Smith, M.J. (1992) Nucleotide sequence of a Drosophila melanogaster gene encoding a calreticulin homologue. DNA Seq. 3, 247-250. [I31 Chen, F., Hayes, P.M., Mulrooney, D.M. and Pan, A. (1994) Identification and characterization od cDNA clones encoding plant calreticulin in Barely. Plant Cell 6, 835-843. u41 Nash, P.D., Opas, M. and Michalak, M. (1994) Calreticulin: not just another calcium-binding protein. Mol. Cell. Biochem. 135, 71-78. [I51 Dedhar, S., Rennie, P., Shago, M., Hagesteijin, C.-Y.L., Yang, H.. Filmus. J., Hawley, R.G., Bruchovsky, N., Cheng, H., Matusik, R. and Giguere, V. (1994) Inhibition of nuclear hormone receptor activity by calreticulin. Nature 367, 480-483. P61 Kuwabara, K., Pisky, D.J., Schmidt, A.M., Benedict, C., Brett, J., Ogawa, S., Broekman, M.J., Marcus, A., Sciacca. R.S., Michalak, M., Wang, F., Pan, Y.C., Grunfeld, S., Patton, S., Malinski, T., Stem, D.M. and Ryan, J. (1995) Calreticulin, an antithrombotic agent which binds to vitamin K-dependent coagulation factors, stimulates endothelial nitric oxide production, and limits thrombosis in canine coronary arteries. J. Biol. Chem. 270, 81798187. 1171Peterson, J.R., Ora, A., Van, P.N. and Helenius, A. (1995) Transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins. Mol. Biol. Cell 6, 1173-l 184. [I81 Otteken. A. and Moss, B. (1996) Calreticulin interacts with newly synthesized human immunodeficiency virus type I envelope glycoprotein suggesting a cheprone function similar to that of calnexin. J. Biol. Chem. 271, 977103. [I91 Singh, N.K., Atreya, C.D. and Nakhasi, H.L. (1994) Identification of calreticulin as a rubella virus RNA binding protein. Proc. Natl. Acad. Sci. USA 91, 12770-12774. PO1 Atreya CD., Singh, N.K. and Nakhasi, H.L. (1995) The rubella virus RNA binding activity of human calreticulin is localized to the N-terminal domain. J. Viral. 69, 38483851. Pll Atreya, C.D. Pogue, G.P., Singh, N.K. and Nakhasi, H.L. (1996) Role of calreticulin in Rubella virus replication, In: Calreticulin, Molecular Biology Intelligence Unit (Michalak, M., ed.), R.G. Landes, Co., Austin, TX, pp. 89- 115. P21 Nakhasi, H.L., Rouault, T.A.. Haile, D.J., Liu, T-Y. and Klausner, R.D. (1990) Specific high-affinity binding of host cell proteins to the 3’ region of rubella virus RNA. New Biol. 2. 2555264. ~231Nakhasi, H.L., Singh, N.K., Pogue, G.P., Cao, X.Q. and Rouault. T.A. (1994) Identification and characterization
of host factor interactions with cis-acting elements of rubella virus RNA. Arch. Virol. (Suppl) 9, 255-267. 1241Killick-Kendrick, R (1979) In: Biology of Kinetoplastida (W.H.R. Lumsden and D.A. Evans, eds.), Academic Press, New York, Vol. l., pp. 3955449. 1251Chang, K-P and Dwyer, D.M. (1976) Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro. Science 193, 6788680. WI Burd, C.G. and Drefuss, G. (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 6155621. ~271Joshi, M., Dwyer, D.M. and Nakhasi, H.L. (1993). Cloning and characterization of differentially expressed genes from in vitro grown ‘amastigotes’ of Leishmania donovani. Mol. Biochem. Parasitol. 58, 345-354. 1281Markoff, L., Chang, A. and Falgout, B. (1994) Processing of flavivirus structural glycoproteins: stable membrane insertion of premembrane requires the envelope signal peptide. Virology 204, 526-540. 1291Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nat]. Acad. Sci. USA. 76, 4350-4354. [301 Maruyama, K., Mikawa, T. and Ebashi, S. (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J. Biochem. (Tokyo) 95, 51 I-519. [311 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G. and Smith, J.A. (1989) In: Current Protocols in Molecular Biology, Wiley, New York, pp. 10.2.5. 1321Bates, P. and Dwyer, D.M. (1987) Biosynthesis and secretion of acid phosphatase by Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 26, 289-296. 1331Cheng. S.T., Nguyen, T.Q., Wang, Y.S., Capra, J.D. and Sontheimer, R.D. (1996) calreticulin binds hYRNA and the 50 kD polypeptide component of the Ro/SSA-ribonucleoprotein autoantigen. J. Immunol. 156, (in press) [341 Borst, P. (1986) Discontinuous transcription and antigenic variation in trypanosomes. Ann. Rev. Biochem. 55, 701732. [351 Landweber, L.S. and Gilbert, W. (1994) Phylogenetic analysis of RNA editing: a primitive genetic phenomenon. Proc. Natl. Acad. Sci. USA 91, 9188921. [361 Ghosh, A., Ghosh, T., Ghosh, S., Das, S. and Adhya, S. (1994) Interaction of small ribosomal and transfer RNAs with a protein from Leishmania donouani. Nucl. Acids Res. 22, 1663-1669. [371 Lewis, M.J., Mazzarella, R.A. and Green, M. (1985) Structure and assembly of the endoplasmic reticulum. The structure of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes. J. Biol. Chem. 260. 3050%3057. [381 Matsuoka, A, Seta, K., Yamakawa, Y., Okuyama, T., Shinoda, T. and Isobe, T. (1994) Covalent structure of bovine brain calreticulin. Biochem. J. 298, 435-442. [391 Jethmalani, S. and Henle, K.J. (1994) Prompt glycosylation of calreticulin is independent of Ca+ + homeostasis. Biochem. Biophy. Res. Commun. 205, 780-787.
Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity’ Manju Josh?, Gregory P. Pogue’+, Robert C. Duncan”, Nancy S. Lee”, Nishi K. Singh”, Chintamani D. Atreya”, Dennis M. Dwyerb, Hira L. Nakhasi”.* “Laboratory
of Molecular
bCeN Biology Section.
Pharmacology, Division of’ Hemurologic Products, Center jbr Bioiogics Evaluation and Research, Food and Drug Administration, Bethesda MD 208924425, USA Laboratory of’ Parasitic Diseases, NIAID, National Institutes of’ Health. Bethesda MD 208924425. USA
Received 22 February 1996; revised 21 May 1996: accepted 24 May 1996
Abstract Calreticulin has been implicated in multiple cell functions. Recently, we have shown that both human and simian calreticulin are RNA binding proteins and that their binding activity is due to phosphorylation. To demonstrate that the RNA binding property of calreticulin is an intrinsic part of this multi-functional molecule and is evolutionarily conserved. we isolated and characterized the calreticulin gene from the unicellular parasite, Leishmaniu donovani. Amino acid sequence homology between human and Leishmaniu calreticulin (L. d. cal) is limited, but like the human homologue, L. d. cal binds Cat +, can be phosphorylated in vitro and binds certain RNA sequences in a phosphorylation-dependent manner. Unlike human calreticulin, L. d. cal is glycosylated and its binding to endogenous Leishnxmiu RNA is phosphorylation-independent. The binding of L. d. cal to Leishmunia RNA suggests that the RNA binding activity of calreticulin has remained evolutionarily conserved.
Kepmds:
Calreticulin;
RNA binding
protein;
Phosphorylation;
Glycosylation;
Trypanosomatid;
Functional
conser-
vation
L. d. Cal., Leishmania donovani calreticulin; RV, rubella virus. * Corresponding author. Laboratory of Molecular Pharmacology, DHP/OTRR/CBER/FDA, Bldg. 29 Rm 107, 8800 Rockville Pike, Bethesda, MD 20892-0425. Tel: + 301 496 2205; fax: + 301 496 1810; e-mail: [email protected] ’ Note: The nucleotide sequence data reported in this paper is available in the NCBI, GenBank and the accession number is u49191. ’ Present address: Biosource Technologies, Inc., 3333 Vaca Valley Pkwy, Vacaville. CA 95688, USA. Abbreviations:
0166-6851/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PZI S166-6851(96)02676-X
54
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
1. Introduction
Calreticulin is a major Ca+ + binding protein which is localized mainly in endoplasmic reticulum [ER] [l]. The protein initiates with a signal sequence for ER entrance and terminates with a KDEL signal sequence which is the retention signal for ER resident proteins [2]. However, nonER locations for this protein have been also reported: the nucleus of some cells [3], the acrosome of sperm cells [4], cytotoxic granules in T-cells [5] and in the cytoplasm [6,7] suggesting multiple functions outside of the ER. Calreticulin cDNA clones have been isolated from a variety of sources, including human, drosophila and schistosomes [1,8- 131. The phylogenetic diversity of these sources suggest that calreticulin is an ubiquitous protein whose amino acid sequence is conserved among various species. Calreticulin has been implicated in multiple cell functions, based on different functional domains found in its primary structure [14] and its presence in different cell compartments [3-71. For example, the N-terminal domain of calreticulin has been shown to modulate expression of glucocorticoid receptor regulated genes both in vitro and in vivo via protein-protein interaction [6,15]. Specific interaction of the C-terminal domain of calreticulin with coagulation factors (IX, X, prothrombin) suggested that it has a potentially antithrombotic function and indeed the administration of calreticulin was shown to prevent coronary thrombosis [16]. P-domain of calreticulin has a significant similarity with an integral ER membrane protein calnexin which has been shown to have chaperone function [l]. Like calnexin, calreticulin has been shown to be a lectin and participate in molecular chaperone of glycoprotein folding [17,18]. Recently, we have shown that both human and simian calreticulins are RNA binding proteins which interact with the 3’-cis acting element of Rubella virus (RV) RNA in vitro and in RV infected Vero 76 cells [19-231. The 3’-cis acting element of RV consists of an inverted repeat sequence of 12 nucleotides located at the 3’ end of the genomic RNA which is capable of forming stem-loop (SL) structure and is necessary for initiation of (-) strand RNA synthesis of RV RNA
[23]. Further, the RNA binding activity of calreticulin was dependent on its phosphorylation [21,22]. An increase in the binding affinity was observed after RV infection [22], coinciding temporally with the appearance of (-) strand RNA synthesis [23]. Both human and simian calreticulins have phosphoprotein isoforms in vivo and autophosphorylate in vitro [19-231. The in vitro RNA binding and the autophosphorylation activity resides in the N-terminal domain of these molecules [22]. In the present study, we explored the possibility that the newly discovered RNA binding property of calreticulin of higher eukaryotes is conserved in evolutionarily divergent species such as a simple unicellular organism, Leishmania. Leishmania is a trypanosomatid protozoan parasite which has a life cycle characterized by the presence of an extracellular, flagellated promastigote form in sand fly vectors [24] and a nonmotile amastigote stage within phagolysosomes of mammalian macrophages [25]. Little is known about RNAprotein interactions in these trypanosomatid parasites and what role such interactions play in cellular metabolism. However, much progress has been made in identification and elucidation of the functions of many of RNA binding proteins in higher eukaryotes [26]. As a first step towards studying the conservation of calreticulin RNA-binding activity in Leishmania and its potential role in trypanosomatid biology, we have isolated the leishmanial homologue of human calreticulin and characterized its functions. To our knowledge, this is the first detailed study of an RNA-binding protein in Leishmania, characterized both with heterologous and homologous RNAs.
2. Materials and methods 2.1. In vitro culture of parasites isolation
and RNA
A cloned line of L. donovani (WHO designation: MHOM/SD/62/1S-Cl,,) was used in all experiments [27]. Promastigotes and in vitro ‘amastigotes’ were grown as described previously
55
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 5.3-64
[27]. Cells of both phenotypes were harvested and processed as described [27]. Total RNA was isolated and Northern blot analysis was performed according to Joshi et al. [27]. Nick-translated cloned L. d. cal DNA was used to probe RNA blots. 2.2. Cloning of the Leishmania calreticulin (L. d. cul) gene and construction of its chimeric form Two degenerate oligonucleotide primers corresponding to amino acids (aa) 135 - 142 (S-primer, ATGTTCGGCiTCCGjCGACATCTGCGG) and aa 272-279 (3’-primer, CCACTCG/ACCCTTGTACTCCGGGGTT) of human calreticulin that are conserved among known calreticulins [lo] were used to amplify the L. d. cal gene from Leishmania donouani genomic DNA in a PCR reaction. A 485-bp PCR fragment was generated, cloned into the PCRII vector (Invitrogen, Inc). Sequence analysis of this PCR fragment showed significant homology with the human calreticulin sequence. The cloned 485-bp fragment was then used as a probe to screen a Leishmania DNA cosmid library (a gift from Dr. Buddy Ullman, Oregon Health Science University, Portland, OR, USA). A 4.5-kbp fragment containing L. d. cal sequence obtained from the cosmid library was subcloned into a Bluescript vector (Stratagene, PBS), and subjected to nucleotide sequence analysis (Sequenase Kit, USB). Primers corresponding to the part of the putative N-terminal leader sequence (amino acids MLAAVVGV) and the C-terminal end (amino acids SEDKEDL) plus a termination codon (Fig. 1) were used to PCR-amplify the entire L. d. cal open reading frame. PCR products were cloned into the PCRII vector (Invitrogen, Inc.). The entire coding sequence of the mature L. d. cal was PCR-amplified using a 5’-EcoRI site containing primer corresponding to the N-terminus coding sequence (EIFFHEEF) and a 3’ primer corresponding to the C-terminus codons (SEDKEDL) including a BarnHI site. Chimeric L. d. cal protein containing sequences from drosophila calreticulin gene was constructed by using the modification of the 5’ primer described above, where the first 12 amino acids of the mature L. d. cal were replaced by drosophila calreticulin (SAEVY LKENFDN) in frame with codons encoding
MDGW... of the L. d. cdl sequence. The amplified DNA fragments following digestion with EcoRI and BarnHI enzymes were cloned into similarly digested PET 22b( + ) expression vector containing 6-histidine residues at the carboxy terminal end (Novagen). Plasmid DNA transformation and expression of recombinant calreticulin was performed according to the manufactures protocol (Invitrogen, Inc.). 2.3. In vitro translation
of L. d. cal
Approximately 1 ,ug of PTA L. d. cal plasmid DNA was incubated in the T7 RNA polymerase transcription-coupled-translation (T7driven TNT) reaction containing 40 PCi of [35S] methionine (800 Ci mmol _ ‘, specific activity) according to manufacturer’s protocol (Promega, Inc.). In parallel reactions, canine pancreatic microsomal membranes were added and following 2-h incubation, microsomal membranes were pelleted by centrifugation at 12 OOOXg for 10 min and resuspended in 1X phosphate buffer saline (PBS). To test for the importation of the L. d. cal into the lumen of microsomal membranes, equal aliquots of total translation reactions either containing or not containing membranes, were treated with thermolysin protease in the presence or absence of 1% NP40 [28]. To confirm the glycosylation of the L. d. cal, equal aliquots of total translation products synthesized in the presence or absence of microsomes were incubated in a buffer containing 0.05M NaP04 buffer (pH 7.5) supplemented with 1% NP40 [28]. The samples were incubated at 37°C for - 2 h in the presence or absence of N-glycosidase F (PNGase F), New England Biolabs [28]. To each of these samples, SDS-PAGE sample buffer [21] was added to 1X concentration and boiled. All translation reactions were analyzed by 10% SDS-PAGE followed by autoradiography [ 191. 2.4. Expression chimeric .form.
of recombinant
L. d. cal and its
The L. d. cal protein or its chimeric form purified by lysing the cells in buffer A (8M containing O.lM PO4/0.01 M Tris-HCl pH The cell debris was removed by centrifugation
was urea 8.0). and
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 53-64
56
P 7
45
L.d.cal.
MLLSVPLLLGLLGLAV----AEPAVYFKEQFLDGDGDGWT-SRWIESKHKSD II: I I:I I I II III III I I I II MLAAW---GVLVLCVYWQAEIF--FHEEFNTMDGWVQS---E--HTSD
Bu . -Cal.
FGKFV-LSSGKFYGDEEKDKGLQTSQDARFYALS-----ASFETFSNKGQ
89
Hu.-cal.
:/I L.d.cal. Hu. -Cal.
L.d.cal.
Hu. -Cal.
I
II
I
I
II:
II
I I :I I I :I
I
II
40
I
YGK-VALSVGAIHVDAEKEQGLKLMEDAKFYAVSKKLPKAV----SNDGK 5'PCR Primer -> TLWQFTVKHEQNIDCGGGYVKLFPNS-LDQTDMHGDSEYNIMFGPDICG :::j I:11 II : III I:I:I I III I II:/ I :IIIIII SIIVSFSVKNEQKLTCGGTYLKFF--SELDQKDLHGESAYWLMFGPDIIG
85 138 I
133 185
L.d.cal.
PGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTL-IVRPDNTYE--VK I: I I I I I: I :I I I Il:ll: I:1 III I SSTRL-HSRD-YrGTNHLWKKLWRPKTDKATHWTVEIA_PNTYQLYV-
Hu .-Cal.
IDNS--QVESGSLEDDWDFLPPKKIKDP-DASKPEDW-DE~KIDDPTDS
231
L.d.cal.
224
Hu. -Cal.
I I I II I ::I1 IIII I II I II II I: I I I :I : -DGMHIQ-E-GSFEEEWDMLPPKTIPDPTDE-KPADWVDDMMb-DDPSDT ---_ 3'PCR Primer +-
KPEDWDK-PEHIPDPDAKKPEDWDEEMDGEW---E-P--PVIQNPEYK-G
273
II
L.d.cal.
IIIII I II:IIIIII II I I I I KPEHWDDEPATITDSEAVKPVDWD---DGGRRRVEAPKIPKIPNPNYRVG EWKPRQIDNPDYKGTW----IHPEIDN------PEY---SPDPSIYAYDN I I Il:/lI I II I I I I GRHVR-IHNPEYKGEWAARQI-P---NTGLQGGPEPVQGSRRCSTSASMS
I
FGVLGLDLWQVKSGTIFDNFLITNDEAYAEEFGNE--TWGVTKA-AEKQM ll:lI::I I I:1 II I I I --------WQVEGGTVFDDIIIGDDIT--EVLGVVKSTYG---AMAEK--
I
Hu.-cal. L.d.cal. Eu.-cal. L.d.cal. Hu .-Cal. L.d.cal. Hu.-cal. L.d.cal.
KDKQDEEQR-LKEEEEDKKR--KEEEEA--EDKEDD-EDKDE--DEEDEE I II III/ II II II I I -----E--RDLIQAEE-KKEATKEPAEAAAE-KPNVGEHADHTPDEGDSE DKEEDEEEDVPGQAKDEL ::/ I D----____----_KEDL
1:
179
I
271 310 I
316 357 III
351 399 II
I
I
392 417 397
Fig. 1. Comparison of human (Hu-Cal) and Leishmania calreticulin (L. d. cal) amino acid sequences. Vertical bars represent identical amino acids, the two dots represent conservative changes. The amino acids from which oligonucleotides for PCR primers were derived are shown. SP denotes the signal peptidase cleavage site. The glycosylation sites are indicated by a black diamond. Two putative protein kinase C phosphorylation sites are underlined by thick lines, whereas two putative casein kinase phosphorylation sites are indicated by thin lines. The putative nuclear localization sequence is denoted by a dashed underline.
the supernatant was passed through a Ni-agarose beads column (Qiagen), followed by washing with buffer A at pH 6.3. The recombinant calreticulins bound to Ni-agarose beads were renatured by step wise reduction in urea concentration in buffer A until final wash contained only 0.1 M Sodium phosphate and 1mM Tris-HCl pH 8.0. Calreticulin proteins were eluted from the Ni-agarose beads in buffer containing 250 mM Imidazole, 300 mM
NaCl, 0.1 M Sodium phosphate, 1 mM Tris-HCl (pH 8.0). The eluted fractions were renatured by dialysis against in a buffer (10 mM Tris-HCl pH 7.4, 40 mM KCl, 5% glycerol and 100 PM PMSF) for approximately 12 h using Slide-A-lyzer (Pierce) dialysis membranes. Analysis of proteins isolated in this manner revealed that the induced protein, L. d. cal or chimeric L. d. cal, were > 95% pure in final solution by Coomassie Blue staining.
M. Joshi et al. / Molecular and Biochemical Parasitology 81 (1996) 53-64
2.5. Immunoblot analysis of recombinant and its binding to 45Ca + +
L. d. ccl1
A polyclonal rabbit sera raised against recombinant human calreticulin (LAR 086; kind gift of L. A. Rokeach or obtained from Affinity Bioreagents, Inc. Golden, Co) was used to determine immunogenic cross-reactivity between calreticulins from human and Leishmaniu sources. One to five pg of recombinant calreticulin proteins were separated in a 10% SDS-PAGE gel, transferred to nitrocellulose membranes and analyzed by Western blot [29] using calreticulin antibody ( l/l000 dilution). The proteins were visualized by the ECL light reaction system (Amersham, Inc.). The binding assay for 4s Ca + + was performed according to Maruyama et al. [30]. Briefly, proteins on the filters were incubated with Ipci/ml 45Ca in buffer C (60 mM KCl, 5 mM MgCl,, 10 mM imidazole-HCl, pH 7.0) for 10 min. The filters were washed with distilled water. dried and exposed to x-ray film. 2.6. RNA electrophoretic
mobility
shift assay
57
brane in nondenaturing running buffer (25 mM Tris and 200 mM glycine). The membrane was stained with 0.1% Ponceau S and photographed. The membrane was then washed 3 times with TBS-T (20 mM Tris-HCl pH 7.4, 100 mM NaCI, 1 mM EDTA, 0.1% Tween 20) and incubated in 2 ml TBS-T with 1~10~ cpm in vivo labeled total L. donovani RNA at room temperature for 30 min. The membrane was washed 4 times with TBST-T and visualized by autoradiography. Labeled RNA was prepared from 3x10’ promastigotes of cultured L. donovani, first incubated for 1 h in phosphate free RPM1 1640, followed by addition of 2 mCi of “‘P-orthophosphate and incubated for 3 h [32]. RNA was extracted with RNA STAT-60 according to the manufacturer’s protocol (TELTEST ‘B’. Inc.).
3. Results 3.1. Isolation and expression of the Leishmania calreticulin gene and comparison to human calreticulin.
(REMSA)
RNA-protein interactions by REMSA were performed as described before [20]. Radiolabeled [(a-“P) CTP] RV 3’ ( + ) SL RNA was transcribed from a T7 promoter as described [20]. 2.7. In vitro autokinase phosphatase
activity
and alkaline
treatment
In vitro autokinase activity was measured as described earlier using Y-~‘P ATP [19]. The labeled proteins were separated in 10% SDS-PAGE and visualized by autoradiography. The alkaline phosphatase (AP) treatment was done according to Nakhasi, et al. [22]. After the treatment, proteins were used directly in REMSA [22]. 2.8. RNA binding assay for endogenous Leishmania
RNA
Purified recombinant proteins were separated on non-denaturing, discontinuous polyacrylamide gel [31] and transferred to nitrocellulose mem-
We used a PCR based strategy to isolate a calreticulin homologue of the human gene. A 4.5-kb fragment from the Leishmunia cosmid library was cloned and nucleotide sequence of - 1.4 kb was determined. The nucleotide sequence of the 1.4-kb DNA fragment has been deposited in the GenBank with an accession number U49191. The first AUG codon was assigned on the basis of a largest open reading frame (ORF) originating from such an AUG and on the size of the protein coded in an in vitro translation system by this ORF that had a calculated molecular weight similar to known calreticulins. The deduced amino acid sequence of the ORF revealed a putative signal sequence of 18 hydrophobic amino acids at the N-terminus followed by 379 amino acids (Fig. 1). The ORF terminates with KEDL, a putative ER retention signal similar to the KDEL found in human calreticulin [9]. Two putative glycosylation sites were identified at residues 126 and 154 in the mature L. d. cal protein (Fig. 1). In addition, two putative protein kinase C phosphorylation sites at Leishmania
58
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
residues 55-51, 74-76; and two casein kinase sites at residues 170-174, 304-308 were identified (Fig. 1). A putative nuclear localization site at residues 179-186 w’as also identified in the mature L. d. cal protein (Fig. 1). A comparison of the deduced amino acid sequence of L. d. cal with human calreticulin showed an overall homology of 42% (Fig. 1). Amino acid sequences comparison between the three putative domains revealed a 52% homology in the P-domain, 47% in the N-terminal domain, and 29% in the C-terminal domain. Even though there is a limited amino acid sequence homology between the human and Leishmania calreticulins, it was of interest to test structural similarities between the two calreticulins. The ORF of L. d. cal was expressed in E. coli and western blot analysis of the recombinant protein was performed using polyclonal antibodies against human calreticulin (Fig. 2). The L. d. cal showed two Coomassie stained bands (Fig. 2, lane 2) and both forms reacted with the human antibody (Fig. 2, lane 4). Recombinant human calreticulin expressed as a maltose binding fusion protein (H. Cal-MBP) was used as a positive
+
H.cal-Ab
+
kDa
12
34
Fig. 2. SDS-PAGE and western blot analysis of recombinant human and recombinant Leishmania calreticulin. H. Cal.MBP, human calreticulin expressed as fusion protein with maltose binding protein. L. d. Cal.. Leishmania donouani calreticulin expressed as histidine tagged protein. Lanes 1 and 2 represent Coomassie stain of the two proteins on SDS-PAGE. Lanes 3 and 4 represent immuno-reactive bands of the two proteins using polyclonal antibody against human calreticulin.
L. don. RNA Promastigote Amastigote
-1.9 kb-
Fig. 3. Northern blot analysis of total RNA from L. donovani pro- and in vitro grown ‘amastigotes’. Twenty fig of total RNA from amastigotes, lane 1, and from promastigote. lane 2, were separated on 15% agarose gel containing 6% formaldehyde. The approximate size of the L. d. cal mRNA is indicated by an arrow.
control for human calreticulin antibody (Fig. 2, lanes 1 and 3). The specificity of human calreticulin antibody was demonstrated by its inability to react with other proteins such as maltose binding protein, MBP (data not shown). To demonstrate that calreticulin gene was in fact expressed in the parasite, Northern blot analysis of the total RNA isolated from both L. donovani pro- and in vitro grown ‘amastigotes’ was performed and probed with cloned L. d. cal DNA (Fig. 3). Calreticulin mRNA was present in both Leishmania forms (Fig. 3, lanes l-2). Further, the size of the L. d. cal mRNA ( - 1.9 kb) was similar to that reported in other organisms [7-121 and corresponds to the size predicted from the Leishmania genomic clone. 3.2. Leishmaniu calreticulin is a glycoprotein. To test whether putative glycosylation sites in the mature L. d. cal have functional significance, we translated L. d. cal RNA in a rabbit reticulocyte lysate in the presence or absence of canine pancreatic microsomes. L. d. cal RNA translated in rabbit reticulocyte lysates lacking microsomes synthesized two polypeptides of - 60 and 63 kDa
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
59
Microsomal Mem.: k;;
97 -
431
2
3
4
5
6
7
8
9
10
Fig. 4. Glycosylation of in vitro synthesized L. d. cal. SDS-PAGE analysis of proteins synthesized from transcripts of plasmid PTA L.Cal. which contains the entire L.Cal ORF, and were incubated in rabbit reticulocyte lysates alone (lanes l-3, 7-8) or in the presence of canine pancreatic microsomes (lanes 4-6, 9-10). Translation products were treated with either NP40 buffer and thermolysin protease, Prt-ase + NP40 (lanes 3 and 6) or thermolysin protease (Prt-ase) alone (lanes 2 and 5). Translation products were treated with either N-glycanase F, Buffer + N-gly-ase (lanes 8 and IO) or without, Buffer (lanes 7 and 9). The location of molecular weight standards (kDa) are shown at left.
(Fig. 4, lane 1). In contrast, L. d. cal RNA translated in the presence of microsomal membranes yielded two proteins of - 70 and 73 kDa (Fig. 4, lane 4). To establish that the L. d. cal signal sequence led to the import of the L. d. cal protein into the lumen of the microsomes, the translation products were treated with the protease thermolysin. Protease treatment of L. d. cal translated in the absence of microsomes with or without detergent (NP40) led to the complete digestion of the translation product (Fig. 4, lanes 2-3). Following translation of L. d. cal in the presence of microsomes, the microsomes were pelleted by centrifugation and then subjected to various treatments. In the absence of NP40, the microsomes protected the majority of the 70-73 kDa L. d. cal translation product from being digested by the protease (Fig. 4, lane 5). In contrast, addition of NP40 to the latter resulted in the degradation of the - 70-73 kDa proteins to an - 60 kDa species (Fig. 4, lane 6). These results indicate that the L. d. cal contains
a functional signal sequence for import into microsomes. Presumably glycosylation of the L. d. cal protein within such microsomes imparts resistance to its complete digestion with thermolysin. To determine whether the addition of carbohydrates was responsible for higher molecular weight of L. d. cal, we treated L. d. cal translation products with N-glycanase in the presence or absence of microsomes. Treatment of L. d. cal translated in the absence of microsomes with the N-glycanase had no effect on its mobility (Fig. 4, compare lane 8 with lane 7). Incubation of L. d. cal translated in the presence of microsomes with N-glycanase led to a reduction in the size of the labeled proteins from - 70-73 kDa to - 60-63 kDa (Fig. 4, lane 10); the later molecular size being identical to calreticulin observed in translation reactions without microsomes (Fig. 4, lane 1). Thus. confirming that translation of L. d. cal in the presence of microsomes resulted in the glycosylation of the protein.
60
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
3.3. L. Cal is a Ca + + binding protein.
3.4. L. Cal is an autokinase
To determine whether L. d. cal binds Ca+ +, SDS-PAGE gel analysis of a fusion protein consisting of human calreticulin and MBP (H. CalMBP), the Leishmania his-tag calreticulin (L. d. cal-6His) and a his- tag DHFR (DHFR-6His) protein was performed. Proteins were transferred to a nitrocellulose membrane and incubated with 45Ca. Both H. Cal-MBP (Fig. 5, lane 1) and L. d. cal (Fig. 5, lane 2) bound labeled calcium. It was previously shown that MBP alone does not react with 45Ca [19]. No reactivity was observed with the control DHFR-6His protein (Fig. 5, lane 3). On the contrary, it appeared to block background binding of Cat + to membrane.
Recombinant DHFR, L. d. cal, Bovine serum albumin and MBP-human Cal proteins were analyzed on a SDS-PAGE and stained with coomassie blue (Fig. 6, Lanes l-4). Upon incubation with 32Py-ATP in a kinase reaction buffer, only L. d. cal (Fig. 6, lane 6) and H. Cal-MBP (Fig. 6, lane 8) were labeled. Some cleavage of the H. Cal-MBP fusion protein occurs during isolation and the slight radioactivity associated with a lower molecular weight band in Fig. 6, lane 8, corresponds to free calreticulin resolved under these conditions. No significant radioactivity was associated with the DHFR-6His protein (Fig. 6, lane 5). These results demonstrate that L. d. cal, like H. Cal-MBP, can be phosphorylated in vitro in the absence of cellular extract. 3.5. L. d. cal is an RNA binding protein.
45Ca overlay: Mr kDa
97 68 4330 18 -
Fig. 5. Autoradiogram of 45Ca labeled proteins after SDSPAGE and transfer to nitrocellulose membrane. Lane 1, human calreticulin expressed as a fusion protein with maltose binding protein (H. Cal.-MBP); lane 2, Leishmania calreticulin expressed as a His-tag protein (L. d. cal-6His); lane 3, DHFR6His protein. Protein molecular weight markers are shown.
Since the only known RNA ligand for human calreticulin is RV 3’ ( + ) SL RNA [21-231, we first tested the RNA binding activity of the L. d. cal protein with the RV 3’ ( + ) SL RNA in EMSA in the presence or absence of alkaline phosphatase (AP) treatment (Fig. 7A). L. d. cal in the absence of AP treatment bound RV RNA (Fig. 7A, lane 2) and this interaction was abrogated by pretreatment of the protein with AP (Fig. 7A, lane 3). The interaction of L. d. cal with RV RNA was determined to be specific since non-specific RNAs such as poly I-C and poly AGC did not compete with binding to RV RNA (data not shown). These results showed that the recombinant L. d. cal had the capacity to bind RV RNA and that a phosphorylated form of L. d. cal is necessary for such an interaction. In order to determine whether L. d. cal binds to an endogenous leishmanial RNA, we metabolically labeled RNA in cultured Leishmania donouani promastigotes with 32P-orthophosphoric acid. The labeled RNA was tested for binding to calreticulins immobilized on a nitrocellulose membrane. Recombinant L. d. cal, drosophila/L. Cal (D/L. Cal), and MBP-human calreticulin (H. CalMBP) were resolved in non-denaturing polyacrylamide gels and transferred to nitrocellulose
M. Joshi et al. / Molecular and Biochemical Parasito1og.v 81 (1996) 53-64
61
Coomassie Stain: 32P r_ATP: Mr kDa
20097-
+
H.caCMBP
*
L.d.cal-GHis
66-
43-
30-
16-
1
2
3
4
5
6
7
6
Fig. 6. Autokinase activity of recombinant Leishmania calreticulin. DHFR-6His (lanes 1 and 5), L. d. cal-6His (lanes 2 and 6), BSA (lanes 3 and 7), H. cal-MBP (lanes 4 and 8). Proteins were either separated by SDS-PAGE and stained with Coomassie blue, lanes l-4, or incubated with y-‘*ATP alone in a kinase reaction prior to electrophoresis and the dried gel autoradiographed, lanes 5-8. Arrows denote the phosphorylated proteins. Protein molecular weight markers are shown.
membranes. Under these nondenaturing conditions, the recombinant proteins which migrate as a single band in denaturing SDS-PAGE (data not shown), are resolved as multiple bands as seen by Ponceau-S staining of these membranes (Fig. 7B). Labeled promastigote RNA bound only to the slowest migrating form of both L. d. cal and D./L. cal (Fig. 7C, lanes 1 and 3) whereas significantly less binding was observed with H. CalMBP (Fig. 7C, lane 4). Interestingly, pretreatment of L. d. cal with AP resulted in its enhanced binding to Leishmania RNA (Fig. 7C, lane 2). On the contrary, phosphatase treatment of H. CalMBP did not enhance binding to Leishmania RNA (data not shown). Further, other recombinant proteins such as MBP or glutathione-s-transferase (GST) proteins did not bind labeled L. d.
RNA in this assay (data not shown). These results demonstrate that L. d. cal: (1) binds to its cognate RNA in Leishmania, and (2) that phosphorylated isoforms of leishmania calreticulin are not needed for RNA binding activity.
4. Discussion Previously, we showed that both human and simian calreticulin are RNA binding proteins and that they bind the RV 3’ (SL) RNA which is necessary for RV replication [19-231. The RV RNA binding activity of human calreticulin resides in the N-terminal domain [20]. The human calreticulin is devoid of the conserved motifs present in most RNA binding proteins [27], how-
M. Joshi et al. 1 Molecular and Biochemical Parasitology 81 (1996) 53-64
62
Protein:
-
L.d.cal
Phosphatase:
-
-
+
A
Phosphatase:
f-
-
4-
-
Complex
-
Fig. 7. (A) REMSA of L. d. cal with a RV 3’ (+ ) SL RNA probe. Lane 1, probe alone. Lane 2 with L. d. Cal protein in the absence of alkaline phosphatase (AP) treatment and lane 3 with L. d. cal protein in the presence of AP. RNA-protein complex is indicated by an arrow. Free probe is also indicated. (B) Ponceau S. staining of the indicated proteins. Roman numerals I-V indicate the position of multiple forms of calreticulin. Lane 1, L. d. cal, without AP treatment; lane 2, the same protein pretreated with AP; lane 3, chimeric drosophila/leishmania calreticulin (D./L. cal) without AP treatment; lane 4, fusion protein of human calreticulin and maltose binding protein (H. Cal-MBP) without AP treatment. (C) The same filter shown in panel B incubated with 1~10~cpm of in vivo “P labeled total Leishmania RNA. The roman numeral-labeled arrows indicate the position of stained bands shown in (B).
ever, its RV RNA binding activity is dependent on the phosphorylation of the protein [19,20]. Thus, from these studies it is evident that RNA binding activity of the phosphorylated form of calreticulin [ 19,201 is an important function of this multifaceted protein for RV replication. Similarities in amino acid sequences among calreticulins from various species has been implicated in the conservation of specific functions [14]. The presence of RNA binding activity of calreticulin may suggest that it has an important role in cellular metabolism. It was of interest, therefore, to ascertain whether this function is conserved in calreticulins from evolutionarily divergent organisms. We choose to study this function in calreticulin from the Leishmania parasite, a unicellular organism, distantly related to multicellular organisms. Since RV RNA is the only RNA ligand known to bind to human calreticulin, we first tested the RNA binding activity of L. d. cal with RV RNA. It was clear from several in vitro experiments that recombinant L. d. cal binding to RV RNA was specific and that such binding was also phosphorylation-dependent, similar to human calreticulin. Further, there is an RNA binding activity in the Leishmania cell extracts that can interact with RV RNA in vitro (data not shown). Even though L. d. cal binds to RV RNA, it is certainly not its physiological ligand. Therefore, it was important to demonstrate whether an endogenous RNA of Leishmania could interact with its own calreticulin. In the present study, we have demonstrated that Leishmania promastigote RNA can be bound by the recombinant L. d. cal and the binding to endogenous RNA does not need phosphorylated isoforms of calreticulin as was observed for its binding with RV RNA [19]. On the contrary, the MBP-human calreticulin fusion protein binds significantly less to Leishmania RNA. Recently, we (Nakhasi, unpublished data) and Cheng et al. [33] have shown similar phosphorylation independent binding of human calreticulin to cellular RNA (hYRNA, a small cytoplasmic RNA of higher eukaryotes). At present, the Leishmaniu RNA ligands for L. d. cal are not known and are the subject of future studies. Thus, it is clear that L. d. cal exhibits dual RNA binding activity depending upon the source of RNA ligand.
M. Joshi et al. I Molecular and Biochemical Parasitology 81 (1996) 53-64
Little is known about the RNA binding proteins or their ligands in Leishmania or other relevant trypanosomatids. The study of RNA protein interaction is of enormous importance in the trypanosomatids because these parasites employ unique biochemical mechanisms such as trans-splicing of leader RNA and RNA editing in gene expression [34,35]. These, in addition to the routine biochemical processes such as translation, RNA processing, transport of RNA from different organelles and storage of RNAs in RNP-complexes to protect from degradation imply a complex regulation of RNA metabolism by cellular proteins [34,35]. Recently, a cellular protein from Leishmania donovani promastigotes was shown to interact with a non-physiological ligand such as antisense RNA of the 5’ untranslated region of /I-tubulin [36]. However, this protein was also shown to interact with several species of small rRNAs and weakly with tRNA [36]. In neither case was the identification of the protein and the mechanism of interaction evident [36]. Besides having conserved the RNA binding activity, L. d. cal has also retained Ca+ + binding and in vitro phosphorylation functions. This is evident by the significant sequence homology in the P-domain and confirmed by in vitro Ca’ + binding. In addition, we demonstrated. in vitro, that L. d. cal unlike human [9], rabbit [8] or chicken liver [37] calreticulins, can be glycosylated. Recently, however, calreticulin isolated from bovine brain [38] and CHO cell [39] has been shown to be glycosylated. In CHO cells, glycosylation of calreticulin occurs in response to acute heat stress and may play role in the cellular thermotolerance [39]. At present, however, it remains to be seen whether L. d. cal is glycosylated in vivo or undergoes change in glycosylation during temperature shift from promastigote to amastigote form [24]. Further, whether such a change in glycosylation plays a role in parasite differentiation similar to the change in thermotolerance in CHO cells [39] remains to be established. In conclusion, the current study demonstrates that the RNA binding activity of calreticulin, besides other functions such as Ca+ + binding and ability to phosphorylate in vitro. has re-
63
mained evolutionarily conserved from this unicellular protozoan to humans. Acknowledgements
We thank Drs. Tosato and Kaplan for critical reading of the manuscript. We also thank Dr. Rokeach for providing anti-human calreticulin antibody and Dr. Buddy Ullman for the Leishmania DNA cosmid library. We also thank S. Baksh for providing protocol for the Ca+ + binding assay. We would like to thank Ms. Smriti (Sumi) Nakhasi for her excellent editorial assistance. References [I] Michalak, M.. Mimer, R.E., Burns, K. and Opas. M. (1992) Calreticulin. Biochem. J. 285, 681-692. [2] Burns, K. and Michalak, M. (1993) interaction of calreticulin with proteins of the endoplasmic and sarcoplasmic reticulum membranes. FEBS lett. 318, 18 I- 185. [3] Opas, M., Dzaik, E., Fliegel, L. and Michalak, M. (1991) Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells. J. Ceil. Physiol. 149, 160&171. [4] Nakamura. M.. Moriya. M., Baba, T., Michikawa, Y.. Yamanobe, T., Arai, K., Okinaga, S. and Kobayashi, T. (I 993) An endoplasmic reticulum protein, calreticulin is transported into the acrosome of rat sperm. Exp. Cell Res. 205, 101~110. [5] Corad, M.E., Umbreit, J.N., Moore, E.G. and Harper, K.P. (1991) Molbiferrin, a homologue of ROSS-A autoantigen and calreticulin. Blood 78. 89a. [6] Burns, K., Duggan, B., Atkinson, E.A., Famulski. K.S., Nemer, M., Bleackely, R.C. and Michalak, M. (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367. 4677480. [7] Sueyoshi, T.. McMullen, B.A., Marnell, L.L., Clos. T.W.D. and Kisiel, W. (1991) A new procedure for separation of protein Z. prothrombin fragment 1.2 and calreticulin from human plasma. Throm. Res. 63. 569575. [8] Fliegel, L.. Burns, K., McLennan, D.H., Reithmeier. R.A.F. and Michalak, M. (1989) Molecular cloning of the high affinity calcium-binding protein(calreticulin) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 264. 21522-21528. [9] McCauliffe, D.P., Lux, F.A., Lieu. T.S.. Sanz. 1.. Hanke. J.. Newkrik, M.M.. Bachinski, L.L., Itoh, Y., Siciliano, M.J., Reichlin, M., Sontheimer, R.D. and Capra, J.D. ( 1990) Molecular cloning, expression, and chromosome 19 localization of a human ROSS-A autoantigen. J. Clin. Invest. 85. 137991391.
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