Cloning of a Schistosoma japonicum gene encoding an antigen with homology to calreticulin

MOLECULAR

Molecular and Biochemical Parasitology 71 (1995) 81-87

EHEMICAL PARASITOLOGY

Cloning of a Schistosoma japonicum gene encoding an antigen with homology to calreticulin Maureen C. Huggins *, James Gibbs, N. Anne Moloney Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT3 UK

Received 9 September 1995; accepted 27 January 1995

Abstract A recombinant Agtll clone, IVGM, encoding part of a 55-kDa antigen was isolated from an adult Schistosoma cDNA library. The protein expressed by this clone was recognised strongly by serum from rats that had been vaccinated with irradiated cercariae (VrS) rendering them highly immune to a challenge infection. Antibodies in VrS which were specific for IVGS3 did not recognise adult worm antigens of S. mansoni, suggesting that the recombinant antigen contains species-specific epitopes, although IVGS3 was weakly recognised by rat serum raised against irradiated S. mansoni cercariae, indicating the presence of a related antigen in this species. A further clone, AMl(p), was obtained which, together with IVGS3 encompasses the entire coding region of the gene which has been called Sj55. Sequence analysis revealed similarities with murine calreticulin, a protein resident in the endoplasmic reticulum. As with murine calreticulin, Sj55 was shown to be a calcium-binding protein. Antigens with homologies to calreticulin have also been described in two other helminths, S. mansoni and Onchocerca ~~oluulus.

juponicum

Keywords: Schistosoma japonicum; Sj55; RAL-1; Calreticulin; Recombinant antigen; Parasite

1. Introduction Schistosoma japonicum is the causative agent of schistosomiasis in the Far East. In one approach to developing a recombinant vaccine against this dis-

Abbreviations: CIS, serum from mice with a chronic S. japonicum infection; ER, endoplasmic reticulum; SmVrS, SmVRS

sera from rats and rabbits. resoectivelv. vaccinated with rramma. irradiated S. mansoni cercariae; VrS, VRS, VMS, sera from rats, rabbits and mice, respectively, vaccinated with ultraviolet-irradiated S. japonicum cercariae. Nucleotide sequence data reported in this paper are available in the GenBankTM data base under the accession No. M80.524. * Corresponding author. Tel.: (44-171) 427-2467; Fax: (44-171) 436-5389; e-mail: [email protected] I

ease we have looked for antigens recognised by serum from animals that have been vaccinated with cercariae attenuated by exposure to ultra violet radiation, this renders the animals highly immune to infection with normal cercariae [l]. It is known that antibodies present in these sera are capable of protecting mice against infection [2]. In this paper we describe the cloning and the initial characterisation of a gene isolated from cDNA libraries constructed using mRNA from adults of the Chinese strain of S. japonicum. The recombinant was selected because it was preferentially recognised by irradiated vaccine sera. Its nucleotide and predieted amino-acid sequence has homology with calreticulin, a protein described by others as a resident

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)00038-O

82

M.C. Huggins et al. /Molecular

calcium binding protein of the endoplasmic lum (ER) (reviewed in Ref. 3).

and Biochemical Parasitology 71 (1995) 81-87

reticu-

2.4. Sequence analysis

2. Materials and methods

Sequencing was carried out on single-stranded double-stranded templates using the Sequenase (US Biochemical).

2.1. cDNA library construction

2.5. Calcium-binding assay

cDNA was prepared from adult S. japonicum total RNA using oligo(dT) primers and the Amersham cDNA synthesis system. A hgtll cloning kit (Amersham) was used to construct a library containing 3.5 X lo5 plaque forming units of which 99% were recombinants. The library was screened with serum from highly immune animals and positive clones plaque purified. One such clone, called IVGS3, was found to be part of a gene encoding calreticulin. In order to obtain DNA encoding the 5’ end of the calreticulin gene an oligonucleotide complementary to the coding strand of IVGS3 was used as a primer for the synthesis of cDNA. After ligation with EcoRI adaptors this cDNA was ligated with phosphatased, EcoRI-digested hgtl0 arms (Amersham) and packaged. The AgtlO library consisted of 1.8 X lo4 plaque forming units which were screened with biotinylated IVGM DNA. Approx. 30% of the plaques were positive, the inserts of several of these clones were amplified by PCR and the clone with the largest insert was named AMl(p). 2.2. Antigen preparation Recombinant hgtll phage, plated onto agar, were expressed overnight, the top agar scraped off, boiled for 10 min in SDS gel sample buffer and then while molten loaded onto SDS-polyacrylamide gels. A Agtll lysogen was expressed in liquid culture, pelleted and resuspended in gel sample buffer. 2.3. Sera and Western blotting Irradiated vaccine sera were used at a dilution of 1: 50. All sera used in library screening or probing of Western blots were preabsorbed extensively with a lysed preparation of an expressed hgtll lysogen. Western blots were probed using goat anti rat, rabbit or mouse Ig biotinylated whole antibody followed by streptavidin-biotinylated horseradish peroxidase complex (Amersham).

or kit

The DNA insert from IVGS3 was amplified by PCR and sub cloned into the plasmid vector pTrcHis (Invitrogen). The expressed fusion protein, purified on a nickel chelated sepharose column, was electrophoresed on a polyacrylamide gel and then transferred to nitro-cellulose paper by standard methods. The nitro-cellulose paper was incubated with [45Ca]CaCl 2 (Amersham) and autoradiographed 141. Part of S. japonicum paramyosin expressed in pTrcHis and then purified was used as a negative control.

3. Results and Discussion

Three of the clones isolated from an adult worm cDNA library which were strongly recognised by serum from rats vaccinated with irradiated cercariae (VrS) were found by DNA hybridisation to be related. They had an insert size of approx. 1.3 kb, and one of the clones, IVGS3, was characterised. 3.1. Recognition of IVGS3 by vaccination sera Fig. 1 shows the recognition of IVGS3 by various sera. None of the anti-schistosome sera used recognises an expressed lysogen of Agtll, showing that they are not reacting with the P-galactosidase portion of the fusion protein. Part of S. japonicum myosin expressed in Agtll was included as a positive control as it is recognised by all sera used. It is thought that the antigen expressed by this clone is initially produced as a fusion with P-galactosidase but is rapidly broken down. One filter probed with rabbit serum raised against P-galactosidase confirmed adequate transfer of all three antigens. IVGS3 is recognised strongly by VrS, less well by serum from rabbits vaccinated with irradiated cercariae (VRS) but not at all by serum from mice vaccinated with irradiated cercariae (VMS). It is thought that VrS and VRS recognise different protective antigens

M.C. Huggins et al. /Molecular ABCAEC

66

and Biochemical Parasitology 71 (1995) 81-87

ABCABC

Thus IVGS3 is recognised by a set of vaccine sera which are most effective in passive transfer experiments when given at the time of challenge [l]. Recognition by immune sera is not species specific although antibodies in VrS specific for IVGS3 are. IVGS3 specific antibodies recognise a 55-kDa antigen in S. japonicum adult worms (data not shown) therefore this S. japonicum antigen has been named Sj5.5.

1 AB

2 C

3

ABCAB

4

205-

-

97.466

5

6

3.2. Sequence

analysis

C

KD

116

83

7

Fig. 1. Recognition of IVGS3 by vaccination and chronic infection sera. Western blots of lanes A, hgtll lysogen; B, IVGS3 expressed in hgtll; C, part of S. japonicum myosin expressed in hgtll. Filters were probed with 1, VrS; 2, VRS; 3, VMS; 4, CIS; 5, SmVrS; 6, SmVRS; 7, rabbit anti-P-galactosidase serum. Molecular masses in kDa are shown on the left.

to those recognised by VMS as they are effective against different stages of the parasite [l]. IVGS3 is not recognised by serum from chronically infected mice (US), we have found that resistance to reinfection in chronically infected mice is not immunologically mediated [S]. The effectiveness of vaccination with irradiated cercariae is species restricted [6,7] and therefore the filters were probed with serum from animals vaccinated with attenuated S. mansoni cercariae. Serum from S. mansoni vaccinated rabbits does not recognise IVGS3; however, the antigen is weakly recognised by serum from S. mansoni vaccinated rats (SmVrS). Antibodies in VrS eluted from the IVGS3 fusion protein do not recognise an antigen in extracts of adult S. mansoni worms (data not shown) suggesting that although a cross reacting protein is present in S. mansoni it does not possess epitopes recognised by VrS.

Examination of the nucleotide sequence revealed that IVGS3 starts at a natural EcoRI site and no ATG codon which could code for an initiating methionine was evident. Total adult RNA was Northern blotted and radio labelled IVGS3 DNA hybridised with a transcript of approx. 1.6 kb in length (data not shown). It was therefore deduced that IVGS3 consists of only part of the gene encoding SjSS. A AgtlO cDNA library specific for the 5’ end of the gene encoding Sj55 was constructed and the recombinant with the largest insert (AMi( was sequenced. This clone has an identical 97-bp overlap with IVGS3, and extends 101 bp 5’ of it. Sequence data from both of the recombinants identified a 1188-bp open reading frame preceded by a 5’ untranslated region and including a putative initiating ATG codon. The open reading frame is followed by a stop codon and a 3’ untranslated region of 129 nucleotides, including a putative polyadenylation signal sequence and eight adenines presumed to be the poly(A) tail. A Southern blot of genomic DNA suggested that Sj55 is encoded by a single-copy gene (data not shown) as is the gene encoding the S. mansoni homologue, see below [S]. 3.3. Deduced teins

amino-acid

homologies

to other pro-

A search of the Swissprot data base revealed that the deduced amino-acid sequence of Sj55 is 52% homologous to murine calreticulin, a calcium binding protein found in the ER [9]. Other recombinant parasite proteins have homologies with calreticulin. A recombinant clone, RAL-1, from Onchocerca ~~oluulus, encodes part of a 42-kDa antigen which is 65% homologous to murine calreticulin [lo]. Serum

M.C. Huggins et al./Molecular

84

MLLSVPLLLGLLGLAAAEPAIYFKEQFLDGDAWTNR-WESKHKSD-FGKFVLSSG * * * ** * * * *t ***

Mu

1 MIL-VLICF-LLSYRVSGQKVWFSQTF ** t l ** * ****

Sj55 Sm

KFYGDLEKDKGLQTSQDARFY~S~F-RPFSNKGQTLWL * * ** * ****** * ***** 53

*****

***

Mu

FPSGLDQKDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHL l ** ** ******** * ** t** **** * ********* **

112

LGSDIDPKKFHGESPYKIMFGPDICGMATKKVHVIFNYKOKNHLIKKEIPCKDDLKTHL

Mu

YTLIVRPDNTYEVKIDNSQVESGSLEDD~FLPPKKIKDeL?AAKPEDWDERAKIDDPTD ********* *** ** **** ** ** * ** * ** *** * 171

YTLIVRPDNSYEVLVDNEKVESGLLEEDWNMLAP ***** * * ******* *** * ** ** **

****

MU

SKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGE~PRQIDNPDYKGTW ** ***** l ******* ** * ** *** ***** *** ***** * -...~......__.............-.........................-... X~~~RW~lPKTIRR~~~~~R~RD~E~~EK~PEYKGEWSP~IENPKYKGQW ** l * ****** l ********* ***** *t * ****t***

****

***

IHPEIDNPEYSPDANIYAYDSFAVLGLDLWQVKSGTIFDNFLITNDEAYAE

289

* **** t * * ***** ** **** *** . . . . . . . . .**** . . . . .._..__KPAQIDNPDYKPDPELYIQDDIGYVGFDLWQVDSGSIFDNILITDSPDFAKQEGERLW** ***II* ** ***** ****************t**************

*

VTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKEDDDDRDEDE-DEEDEKE~DEE **** ** ** * * * * 348

---------RKRHDNE--LAEDQSATKS-------DSDKETD~EPTEEDED~P~ l ** * l * ** * * l **

Sm

---------RKRYDAE--VAKEQSSAK---------DKED

MU

ESPGQAKDEL =ZZZZ=Z * *** 388

Sm Fig. 2. Homology

*

*

*

*

**

NPSGD-HDEL = **** **** DPSGD-HDEL ==X.ZZZ

between the predicted amino-acid

sequences of the murine (Mu), S. japonicum (Sj55) and S. mansoni (Sm) calreticulins.

identity with Sj5S are indicated by an asterisk (*).

leader sequences

are shown in bold. The first set of amino-acid

indicated by dots above the amino-acid

Amino

acids are numbered for Sj55 on the left. The putative

repeats in SjS5 is underlined with dots and the second set is

codes. A region in Sj55 with a high PEST score is indicated by a single line above the amino-acid

Putative nuclear targeting signals in murine and S. mansoni calreticulins

underlined twice.

l

******

Mu

Positions of amino-acid

*

******

KPVQIDNPEYKHDPELYVLNDIGWGFDLWQVDSGSIFDNILITDSPDF~EEGE~W-

Sj55

*

DDKKPDDWDEQFIDDPDD

Sm

Sj55

*

KKPDNWDQPKTIPDNDAKKPDDWDDAMDGEWERPQKDNPEYKGE~P~IDNPKYKGEW

Mu Sj55

***

KM~~~.?~.~KK~.~.~.~.~ERY~~~.~~.D ** *******t*** * ****

YTLIVNPNNKYEVLVDNAKVREGSLEDDWDNL

Sm

**

****

Sm

230

*

LGSDIDPKTFHGETPYKIMFGPDICGMATKRIHVIFNYKGQNHLXKKDIPCKDDQKTHL ******** *II** ***t************ l ******* l ***** ******

Sm

Sj55

codes.

***

KSPVDPIEDLGLKTTQDARFYGIARKISETFSNRGKTMVLIKL

Sj55

***

KSPVNPIEDLGLKTTQDARFYGIARKISEPFSNRDKTLVLQFTVKFDKTVTCGGAYIKL **** ************************ II*** ** ************ ********

Sm

Sj55

hydrophobic

*

--PDQKSIDGWIQSTKNGDKQGQFKIEAG ** * ** * *** * *** l *

M-LSILLTL-LLSKYALGHEVWFSETF--PNE-SIENWVQSTYNAEKQGKFKVEAG

Mu Sj55

and Biochemical Parasitology 71 (1995) 81-87

are underlined once. ER retention signal sequences

are

M.C. Huggins et al. /Molecular

and Biochemical Parasitology 71 (1995) 81-87

raised against a 60-65-kDa fraction of S. mansoni proteins recognised a clone which was 52% identical to murine calreticulin [11,12]. The recombinant S. mansoni calreticulin was recognised by 50% of sera tested from patients with Systemic Lupus Erythematosus, as would be consistent with calreticulin being a component of the Ro/SS-A auto-antigens [12,13]. However, we found that neither IVGS3, nor any adult S. japonicum antigen of the correct molecular weight was recognised by sera from patients with Systemic Lupus Erythematosus and Sjogrens syndrome (data not shown) and it has recently been shown that human calreticulin is not a component of Ro/SSA auto antigens as previously thought [14]. Comparison of the deduced amino-acid sequence of the S. mansoni recombinant calreticulin, with Sj55 (Fig. 21 shows that there is approx. 75% identity, Sj55 being more divergent at the N and C termini. Another S. mansoni protein, SmIrVl, which was reported to have similarities with calreticulin [ 151 has less amino-acid identity with our clones than the S. mansoni calreticulin. Murine calreticulin possesses a 5’ hydrophobic leader sequence that directs its transportation across the membrane of the ER into the lumen 191, hydrophobic regions are also found at the N termini of the schistosome proteins. In addition murine calreticulin has a signal peptide &DEL) at the C terminus that causes the mature protein to be retained in the ER [16]. A similar group of amino acids (HDEL) is present in the schistosome proteins which is identical to the retention signal sequence found in resident ER proteins of some other species [17]. Like murine calreticulin Sj55 has two sets of degenerate internal repeats within a proline rich region (between residues 207 and 299) the functional significance of which are not known. Amino acids 372-383 give a high PEST score of 18.68. This algorithm ranks protein sequences based on characteristics that are thought to be indicative of a rapidly degraded protein in eukaryotic cells [18]. As well as being resident in the ER, calreticulin has been found to occur in the nucleus [19] where it is involved in the regulation of gene transcription [20,21]. Nuclear targeting signal sequences are present in S. mansoni and murine calreticulin. Amino acids at the corresponding position in Sj55, APKMIDDPN, do not comply with the consensus se-

85

quence nor is there a nuclear targeting sequence elsewhere in the S. japonicum protein. Furthermore, localisation of Sj55 by immuno-cytochemistry of electron micrographs of adult worms (to be reported elsewhere) has not as yet revealed a nuclear location for this antigen. The predicted molecular mass of IVGS3 is less than that of the observed molecular mass of the native or recombinant protein when run on reducing polyacrylamide gels. This discrepancy is most likely due to the high content of charged residues which can lead to a change in mobility during electrophoresis. 3.4. Calcium binding Calreticulin binds calcium [22] and polyacidic clusters towards the C terminus have been suggested as being the site for low-affinity calcium binding [9]. Sj55 has a high proportion of acidic residues, some of which are clustered, but these are not confined to the C terminus. A Western blot of IVGS3 expressed in the plasmid vector pTrcHis, together with a fragment of S. japonicum paramyosin expressed in the same vector as a negative control, was incubated

KD

A

6

A

6

674330-

2 Fig. 3. Calcium binding to IVGS3. (1) An autoradiograph of a Western blot incubated with 45Ca (2) The same western blot stained with Amido black. A, IVGS3 expressed in pTrcHis; B, a fragment of S. japonicum paramyosin expressed in pTrcHis. Molecular masses in kDa are shown on the left.

86

M.C. Huggins et al. /Molecular

and Biochemical

with 45Ca and binding detected by autoradiography. Calcium binds to IVGS3 but not to recombinant paramyosin (Fig. 3) indicating that calcium was not binding to the bacterial part of the fusion protein, Therefore, either the carboxy region of IVGS3 is able to bind calcium despite its reduced concentration of polyacidic clusters, or alternative calciumbinding sites are present elsewhere in IVGS3. There are high-affinity calcium-binding sites in the proline rich domain of murine calreticulin [23] although the motifs responsible for this are not the classical EF hand type and as yet have not been defined. Calcium-binding studies using different fragments of Sj55 need to be done to define the calcium-binding domains of S. japonicum calreticulin. In this paper we have described the initial characterisation of a recombinant antigen that is strongly recognised by irradiated vaccine sera and which has homologies to calreticulin, an intracellular calciumbinding protein. Further studies will determine where the antigen is located in the parasite and whether it has a role in inducing immunity.

Acknowledgements We are grateful to Dr. Q.D. Bickle for S. mansoni vaccination sera; to Dr. Isenberg, Dept. Rheumatology Research, UCSM, London and Dr. Dillon, Medical Molecular Biology Unit, Dept. Biochemistry, UCL, London for supplying us with auto immune sera; and to David Judge, School of Biological Sciences, University of Cambridge for help with sequence data analysis. This work was supported by grants from the Medical Research Council and the European Community.

References [l] Moloney, N.A., Hinchcliffe, P. and Webbe, G. (1987) Passive transfer of resistance to mice with sera from rabbits, rats or mice vaccinated with ultraviolet-attenuated cercariae of Schistosoma japonicum. Parasitology 94, 497-508. [2] Moloney, N.A. and Webbe, G. (1990) Antibody is responsible for the passive transfer of immunity to mice from rabbits, rats or mice vaccinated with attenuated Schistosoma japonicum cercariae. Parasitology 100, 235-239.

Parasitology

71 (1995) 81-87

[3] Michalek, M. Milner, R.E., Burns, K. and Opas, M. (1992) Calreticulin. Biochem. J. 285, 681-692. [4] 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. 95, 511-519. [5] Moloney, N.A. and Webbe, G. (1984) Factors affecting the acquisition of resistance to Schistosoma japonicum in the mouse. II. Evidence that resistance to reinfection is not mediated by specific effector mechanisms. Parasitology 89, 361-367. [6] Cheever, A.W., Hieny, S., Duvall, R.H. and Sher, A. (1983) Lack of resistance to Schistosoma japonicum in mice immunized with irradiated S. mansoni cercariae. Trans. R. Sot. Trop. Med. Hyg. 77, 812-814. [7] Moloney, N.A., Bickle. Q.D. and Webbe, G. (1985) The induction of specific immunity against Schistosoma japonicum by exposure of mice to ultraviolet attenuated cercariae. Parasitology 90, 3 13-323. [8] Khalife, J., Pierce, R.J., Godin, C. and Capron, A. (1993) Cloning and sequencing of the gene encoding Schistosoma mansoni calreticulin. Mol. Biochem. Parasitol. 62, 313-316. [91 Smith, M.J. and Koch, G.L.E. (1989) Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO J. 8, 35813586. 1101 Unnasch. T.R., Gallin, M.Y., Soboslay, P.T., Erttmann, K.D. and Greene, B.M. (1988) Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca uoluulus infective larvae. J. Clin. Invest. 82, 262-269. 1111 Khalife, J., Grzych, J.M., Pierce, R., Ameisen, J.C., Schacht, A.M., Gras-Masse, H., Tartar, A., Lecocq, J.P. and Capron, A. (1990) Immunological crossreactivity between the human immunodeficiency virus type 1 virion infectivity factor and a 170kD surface antigen of Schistosoma mansoni J. Exp. Med. 172, 1001-1004. [121 Khalife, J., Trottein, F., Schacht, A., Godin, C., Pierce, R.J. and Capron, A. (1993) Cloning of the gene encoding a Schistosoma mansoni antigen homologous to human Ro/SSA autoantigen. Mol. Biochem. Parasitol. 57, 193-202. t131 McCauliffe, D.P., Lux, F.A., Lieu, T-S., Sanz, I., Hanke, J., Newkirk, 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 location of a human Ro/SS-A autoantigen. J. Clin. Invest. 85, 13791391. [141 Lu, .I., Willis, A.C. and Sim, R.B. (1993) A calreticulin-like protein co-purifies with a ‘60kD’ component of Ro/SSA but is not recognised by antibodies in Sjligren’s syndrome sera. Clin. Exp. Immunol. 94, 429-434. ml Hawn, T.R, Timothy, T.D. and Strand, M. (1993) Molecular cloning and expression of SmIrVl, a Schistosoma mansoni antigen with similarity to calnexin, calreticulin and OvRall. J. Biol. Chem. 268, 7692-7698. [16] Munro, S. and Pelham, H.R.B. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899-907. [17] Pelham, H.R.B. (1990) The retention signal for soluble pro-

M.C. Huggins et al. /Molecular

and Biochemical Parasitology

teins of the endoplasmic reticulum. Trends Biochem. Sci. 5, 483-486. [18] Rogers, S. Wells, R. and Rechsteiner, M. (1986) Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 234, 364-368. [19] Opas, M., Dziak, E., Fliegel, L. and Michalak, M. (1991) Regulation of expression and intracellular distribution of calreticulin. a major calcium binding protein of non muscle cells. J. Cell Physiol. 149, 160-171. [20] Burns, K., Duggan, B.. Atkinson, E.A., Famulski, KS., Nemer, M., Bleackley, R.C. and Michalek, M. (1994) Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367, 476-480.

71 (1995) 81-87

87

[21] Dedhar, S., Rennie, P.S., Shago, M., Leung Hagesteijn, C.Y., Yang, H., Filmus, J., Hawley, R.G., Bruchivsky, N., Cheng, H., Matusik, R.J. and Gig&e, V. (1994) Inhibition of nuclear hormone receptor activity by calreticulin. Nature 367, 480-483. [22] Mater, D.R.J. and Koch, G.L.E. (1988) Identification of a set of calcium-binding proteins in the reticuloplasm, the luminal content of the endoplasmic reticulum. J. Cell Sci. 91, 61-70. [23] Baksh, S. and Michalak, M. (1991) Expression of calreticulin in Escherichia coli and identification of its Ca’+ binding domains. J. Biol. Chem. 266, 21458-21465.