the onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase

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ELSEVIER Molecular and Biochemical Parasitology 68 (1994) 103-117

MOLECULAR AND

BIOCHEMICAL PARASITOLOGY

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase

Wallace R. Wilson a, Rocky S. Tuan b, Kenneth J. Shepley b, David O. Freedman a, Bruce M. Greene a,*, Kwablah Awadzi c, Thomas R. Unnasch a,,

a Division of Geographic Medicine, BBRB 206, University of Alabama at Birmingham, Birmingham, AL 35294, USA b . • • Department of Orthopedtc Surgery and Btochemtstry, and Department of Molecular Biology, Thomas Jefferson University, Philadelphia,

PA. 19107, USA c Onchocerciasis Chemotherapy Research Centre, Hohoe Hospital PO Box 144, Hohoe, Ghana

Received 9 June 1994; accepted 29 July 1994

Abstract

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a eDNA encoding the PDI homologue of Onchocerca volvulus is described. This eDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Keywords: Onchocerca; Protein disulphide isomerase; Prolyl-4 hydroxylase; Collagen; Cuticle; Filariasis

Abbreviations: ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; MBP, maltose binding protein; ORF, open reading frame; OvAg: Onchocerca volvulus soluble adult parasite antigen; PCR, polymerase chain reaction; PDI, protein disulfide isomerase

Note: Nucleotide sequence data reported in this paper have been submitted to the Genbank TM data base with the accession number U12440.

* Corresponding author. Tel.: (205) 975-7601; Fax: (205) 933- 5671; E-mail: [email protected].

t Deceased

Elsevier Science B.V. SSD1 0166-6851(94)00161-8

1. Introduct ion

The cuticle and epicuticle form the external surface of parasitic nematodes, thus forming the inter- face between the parasite and its host. The cuticle and epicuticle are therefore important potential tar- gets for chemotherapeutic and immunotherapeutic attack. From studies of a number of parasitic nematodes, as well as of the free-living nematode

104 W.R. Wilson et al. / Molecular and Biochemical Parasitology 68 (1994) 103-117

Caenorhabditis elegans, the cuticle has been found to consist primarily of a network of collagen molecules [1]. The cuticular collagen is highly cross-linked, which is thought to be important in maintaining the structural rigidity of the cuticle [2]. Since it is possible to solubilize much of the cuticular network by treatment with reducing agents [3], much of the cross-linking appears to be mediated through intermolecular disulfide bonds. In addition to the reducible disulfide bonds, the network is stabilized through a number of intermolecular tyrosine-tyrosine bonds, as well as through glutamyl- lysine amide linkages [2].

Synthesis of the cuticle components occurs in the hypodermis, a metabolically active tissue located just to the interior of the cuticle [4]. In the case of the cuticular collagen network, the procollagen molecules are synthesized as 30-kDa monomers [1]. Following synthesis, the monomers are first cross-linked through the non-reducible Tyr-Tyr intermolecular bonds to form multimers containing 2-4 monomers. The multimers are then further cross-linked by the formation of intermolecular disulfide bridges, resulting in the final formation of the cuticular network (for reviews see [1] and [2]) Proper formation of the final disulfide bridges appears to be critical to the proper development of the cuticle, since mutations disrupt- ing this process result in abnormal morphology, such as is found in the roller mutants of C. elegans [5].

The central enzyme involved in the biosynthesis of collagen is prolyl 4-hydroxylase [6]. This protein catalyzes the post-transcriptional oxidation of proline to 4-hydroxyproline in nascent collagen chains. Be- cause of its central role in the biosynthesis of collagen, prolyl 4-hydroxylase has been a subject of intense interest as a potential chemotherapeutic target, since molecules inhibiting this activity might be expected to be relatively specific inhibitors of collagen biosynthesis [7]. Prolyl 4-hydroxylase has been characterized from a variety of vertebrate and inver- tebrate organisms. In all but one case (in Chlamy- domonas reinhardti [8]) the enzyme consists of a heterotetramer containing two ot and two /3 subunits. The initial cloning and DNA sequence analysis of the /3 subunit of prolyl 4-hydroxylase resulted in the surprising discovery that the derived amino acid sequence of the isolated cDNA was identical to the enzyme protein disulfide isomerase [9]. Protein disul-

fide isomerase (PDI) is the enzyme responsible for the formation of correct disulfide bonds in nascent peptide chains. Subsequent to the discovery that the fl subunit of prolyl 4-hydroxylase and PDI were identical, several additional activities were found to be associated with this polypeptide. The multifunctional protein (hereafter referred to for simplicity as PDI) is identical to a human [10] and bovine [11] membrane associated thyroid hormone binding activity. PDI has also been shown to be identical to iodothyronine 5'-monodeiodinase, the activity responsible for formation of triiodothyronine, the active form of thyroid hormone [12], and is identical to glycosylation site binding protein, a component of the oligosaccharyl transferase complex [13]. Finally, PDI is identical to R-cognin, a protein involved in adhesion of retinal cells in vitro, and which also may be involved in neuronal differentiation [14]. PDI is also a major plasma membrane protein of human corneal fibroblasts [15].

As described above, the nematode cuticular network consists of a network of collagen molecules, which is primarily held together by intermolecular disulfide bonds. PDI, with its dual functions as the/3 subunit of prolyl 4-hydroxylase and the activity responsible for the correct formation of disulfide bonds, is likely to play a central role in cuticular biosynthesis in parasitic nematodes. In the experiments described below, the isolation and characterization of a complete cDNA encoding this multifunctional protein from Onchocerca volvulus is described.

2. Materials and methods

2.1. Parasite material and human sera

Adult O. volvulus were collected from infected individuals residing in the villages in the vicinity of Hohoe, Ghana and Asserekro, C6te d'Ivoire. Para- sites were freed from surrounding host tissue by digestion with collagenase, as previously described [16]. Sera from O. volvulus infected individuals were collected from the villages in the vicinity of Hohoe, Ghana and in Bong County, Liberia. Clinical classi- fication of the O. volvulus infected individuals was undertaken as previously described [17]. Informed

W.R. Wilson et al. / Molecular and Biochemical Parasitology 68 (1994) 103-117 105

consent was obtained from all individuals participat- ing in the study.

2.2. Preparation of parasite DNA, RNA and antigen

Parasite DNA was prepared from collagenase freed parasites by proteinase K digestion, followed by phenol extraction, as previously described [18]. Parasite RNA was prepared by guanidium isothio- cyanate extraction, followed by centrifugation over a cesium chloride step gradient, as previously described [19]. Soluble parasite antigen (OvAg) was prepared by homogenization of collagenase freed parasites in phosphate-buffered saline, followed by centrifugation, as previously described [20].

2.3. Isolation of cDNA clones

The initial cDNA encoding the O. volvulus PDI was isolated by screening an adult O. volvulus expression cDNA library with a rabbit antiserum prepared against O. volvulus infective larvae. The preparation of the anti-larval antiserum, preparation of the cDNA library, and procedures for screening the library with the antiserum have been described in previous work by this laboratory [19]. The inserted cDNA present in the original clone was then used as a hybridization probe to identify additional cDNA clones in the library, following published procedures [21].

2.4. Polymerase chain reaction amplification of the 5' end of the mRNA encoding O. volvulus protein disulfide isomerase

A cDNA representing the 5' end of the mRNA encoding the O. volvulus PDI was isolated by polymerase chain reaction (PCR) amplification using two primers derived from the sequence of the original incomplete cDNA clone, and a 5' primer based upon the sequence of the spliced leader present on some O. volvulus mRNAs [22]. Adult O. volvulus total RNA (1 /xg) was used to prepare first strand cDNA by annealing a primer derived from the internal sequence of the original cDNA. The sequence of the primer used to prepare the first strand cDNA was 5' ACCAGCAAGATACATTTCCG 3'. The first strand cDNA then served as a template for PCR, using a nested primer located upstream of the sequence used

to prepare the first strand cDNA, and a primer derived from the O. volvulus spliced leader. The sequence of these primers was 5' CT'FATATI'TG- GTCATATCTTCT 3' and 5' GGGAAGC'ITGG'I~- TAATrACCCAAGTTTGAG 3', respectively. The first strand cDNA was denatured for 5 min at 95°C in a buffer containing 10 mM Tris-HCl, pH 8.3/50 mM KC1/1.5 mM MgCI2/0.01% gelatin/200 /.~M dATP/200 /~M dCTP/200 /xM dGTP/200 ~M dTI'P/0.5 /xM of each primer. Following denatura- tion, 5 units TaqI DNA polymerase (Perkin-Elmer) and i unit of PCR specificity enhancer (Perfect Match, Stratagene, San Diego, CA) were added to the reaction, and the PCR initiated. Cycling parame- ters for the PCR included 30 cycles of 1 min at 94°C, 1 min at 60°C and 2 rain at 72°C, followed by 7 min at 72°C. Following amplification, the PCR product was purified by separation on a 1.5% agarose gel, followed by adsorption to a glass slurry (Bio 101, San Diego, CA). The purified product was then sub-cloned into a T-tailed blunt-end cloning vector ( T / A cloning vector, InVitrogen, San Diego, CA).

2.5. Overexpression and purification of ARAL6 recombinant antigen

The cDNA insert encoded in hRAL6, which encoded the carboxy terminal 18 kDa of the O. volvulus PDI, was sub-cloned into the bacterial expression vector pMALCR1 (New England Biolabs, Beverly, MA). Cultures containing this plasmid were grown to mid-log phase at 31°C, and induced to produce fusion protein by the addition of isopropyl /3-D- thiogalactoside (IPTG) to a final concentration of 1 mM. Cultures were grown in the presence of IPTG for 3 h at 31°C, and the cells harvested by centrifugation for 10 min at 1600 × g. The cells were resus- pended in a buffer containing 20 mM Tris-HC1, pH 7.5/200 mM NaC1/1 mM EDTA/1 mM dithio- threitol/1 mM phenylmethyl sulfonyl fluoride/2 mg m1-1 lysozyme, and incubated on ice for 45 min. The cells were lysed by two cycles of freezing and thawing, followed by sonication for 15 min at an 80% duty cycle with a Branson sonicator equipped with a microtip. Cell debris was removed by centrifugation at 27000 × g for 20 min at 4°C. The supematant was then applied to an column prepared with amylose affinity resin (New England Biolabs). The column was washed with a buffer containing 20

106 W.R. Wilson et al. /Molecular and Biochemical Parasitology 68 (1994) 103-117

mM Tris-HCl, pH 7.5/200 mM NaCI/1 mM EDTA until the absorbance at 280 nm of the eluent was below 0.05. Bound protein was then eluted from the column by application of the wash buffer containing 10 mM maltose. The yield of the purified protein ranged from 10-20 mg 1-1

2.6. Preparation of polyclonal antiserum against the O. volvulus protein disulfide isomerase

A total of 100/xg of purified fusion protein, or E. coli maltose binding protein (prepared from a cell culture containing the vector pMALCR1 alone) was emulsified in 1 ml of RIBI adjuvant (RIBI Im- munoChem Research, Inc., Hamilton, MT), and used to immunize a rabbit following the manufacturer's instructions. The animals were boosted with injec- tions of 100 /zg of antigen at 3 and 6 weeks following the primary immunization.

2. 7. Immunological methods

Western blots were prepared by separation of 20 /xg of OvAg on a 8% SDS-polyacrylamide gel [23], followed by electrophoretic transfer to a PVDF membrane (Immobilon, New England Nuclear). Rab- bit antiserum diluted to a 1:500 dilution in 20 mM Tris-HCl, pH 7.5/250 mM NaCI/0.05% Tween 20 /3% bovine serum albumin (TBST/BSA) was used to develop the blots. Bound antibody was detected by alkaline phosphatase conjugated goat anti- rabbit IgG diluted 1:1000 in TBST/BSA (Jackson Laboratories, Jackson, ME). ELISA assays were performed using serum samples were pre-absorbed with a resin consisting of purified MBP coupled to cyanogen bromide-activated Sepharose CL-6B. The amount of antigen and serum dilution used in the ELISA were optimized based upon results obtained from an initial checkerboard titration using a pool of sera collected from O. volvulus-infected individuals. ELISA assays were performed in parallel with equimolar amounts of fusion protein and MBP (2 pmol/weU). Sera were used at a dilution of 1:200 in TBST/BSA. Bound antibody was detected using goat anti-human IgG conjugated to alkaline phosphatase, diluted 1:10 000 in TBST/BSA. All assays were performed in triplicate. Results are expressed as

the mean of the net values for each serum sample, minus residual reactivity to purified MBP.

2.8. In situ hybridization and immunohistochemistry

Parasite sections were prepared for in situ hybridization and immunolocalization as previously described [24]. Probes for the in situ hybridization included a sub-clone of the ARAL-6 cDNA in pUC13, or pUC13 alone. The probes were labeled with biotin as previously described [24]. Positive hybridization signals were detected by development with strepavidin conjugated alkaline phosphatase.

Immunolocalizations were carded out using antiserum raised against the RAL-6-MBP fusion protein, or pre-immune rabbit serum from the rabbit used to prepare the anti-RAL-6-MBP antiserum. The condi- tions for the immunolocalization experiments were as previously described [24]. Positive immunoreac- tivity was detected by the development of a deep red horseradish peroxidase reaction product. All speci- mens were viewed by Nomarski differential interfer- ence contrast optics, and photographed using Kodak Ektar 100 film.

3. Results

As part of an ongoing study to identify cDNA clones encoding antigens of O. volvulus L3 that are accessible to the host's immune system, a series of expression cDNA libraries prepared from adult O. volvulus mRNA were exhaustively screened with a rabbit antiserum raised against viable O. colvulus L3. It was possible to divide the clones which were recognized by this antiserum into six classes, based upon cross-hybridization of their cDNA inserts. This was consistent with the finding that the rabbit anti-L3 antiserum recognized 6-8 distinct polypeptides prepared by in vitro translation of O. volvulus adult mRNA [19]. A representative of each on the clone classes was selected for DNA sequence analysis. One such clone, designated ARAL-6, contained a partial open reading frame that encoded a polypeptide which was similar to the fl subunit of prolyl 4-hydroxylase and PDI from a variety of organisms (data not shown). This open reading frame was in frame with the open reading frame of the /3-galac-


tosidase gene of the vector, suggesting that it was responsible for the expression of the epitopes that were recognized by the rabbit antiserum.

The cDNA insert of ARAL-6 was isolated and used to probe a genomic Southern blot, to gain preliminary data on the organization of the gene encoding the putative O. volvulus PDI homologue. The cDNA hybridized to a single band in O. volvu-

lus genomic DNA digested with either EcoRI or HindlII (Fig. 1A). No hybridization was detected to human DNA, confirming that the cDNA was derived from parasite m R N A (Fig. 1A, lanes labeled H). Since the ARAL-6 cDNA insert lacked sites for either EcoRI or HindlII, these results suggested that the O. volvulus PDI homologue is encoded by a single-copy gene. This is consistent with what has been found in other organisms, where PDI is also encoded by a single-copy gene [25]. The ARAL-6 cDNA insert was also used to probe a Northern blot prepared with O. volvulus adult stage mRNA. It was

A

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1.0

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0.4

B 1 2 kb

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2.8

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Fig. 1. Genomic Southern blot and Northern blot analysis of the gene encoding the O. volvulus protein disulfide isomerase homologue. (A) A genomic Southern blot was prepared from genomic DNA samples (10 p.g) digested with the restriction enzymes EcoRI (lanes labeled 1) or HindlII (lanes labeled 2), as described in Materials and methods. The blot was probed with the purified cDNA insert present in ARAL-6. Lanes labeled O contained O. volvulus genomic DNA, while those labeled H contained human DNA. (B) A Northern blot was prepared from total O. volvulus adult stage RNA (20 /xg) as described in Materials and methods. The blot was probed with the purified cDNA insert derived from ARAL-6.

kDa A B C

1 0 1 _ 7 1 _

4 3 _

2 9 _

Fig. 2. Western blot analysis of the O. volvulus protein disulfide isomerase homologue. A Western blot consisting of three identical lanes containing 0vAg was prepared as described in Materials and methods. Following transfer, the lanes were separated, and probed with different serum samples. (A) Blot probed with preimmune serum. (B) blot probed with serum raised against purified E. coli MBP prepared from a culture containing the pMALCR1 vector plasmid alone. (C) Blot probed with serum raised against the purified RAL-6-MBP fusion protein.

found that the cDNA hybridized to a single mRNA of approximately 1700 bases (Fig. 1B).

In order to confirm that the long open reading frame encoding the O. volvulus PDI homolog was expressed in vivo, the inserted cDNA present in ARAL-6 was sub-cloned into the bacterial expression vector pMALCR1. Fusion protein produced by in- duction of cell cultures carrying this construct was then purified by amylose affinity chromatography. This resulted in a preparation which was apparently homogeneous, based upon SDS PAGE analysis (data not shown). The purified fusion protein was then used to immunize a rabbit, in order to produce a polyclonal monospecific antiserum. When this antibody was used to probe Western blots prepared with crude adult O. volvulus soluble antigen (OvAg), a single protein with an apparent molecular weight of approximately 56000 Da was detected (Fig. 2C). This is similar in size to PDI found in other organisms, (approximately 57000 Da) [26]. Preimmune

108 WR. Wilson et al. / Molecular and Biochemical Parasitology 68 (1994) 103-1l 7

Fig. 3. In situ hybridization of the O. volvulus protein disulfide isomerase homologue: Tissue for in situ hybridizations was prepared as described in Materials and methods. Sections were probed with either a plasmid sub-clone containing the RAL-6 eDNA, or the plasmid vector alone. (A and B) Cross-section of an adult female hybridized with RAL-6 cDNA, showing the intrauterine morula (m). Hybridization signal was seen in the hypodermis (h) of the adult female, while the uterine wall (u) and intestine (i) were negative. Expression of the PDI mRNA was detected in the early morula (arrow, panel A), and increased in the late morula (arrow, panel B). PDI mRNA was also present in the 'pretzel stage' microfilariae (C), as well as in the more mature stretched stage (panel B, arrowhead). A low level of expression was also seen in mature skin dwelling microfilariae (D). (E) Control section hybridized to labeled vector plasmid DNA. Bar = 25 /zm in all panels.


serum, or an antiserum raised against E. coli maltose binding protein purified from cultures containing the vector alone did not recognize a specific antigen in OvAg (Fig. 2A and B).

In order to gain further insight into the physiolog- ical role of the putative O. volvulus PDI homologue, the site of transcription of the mRNA encoding this protein was examined by in situ hybridization. If a

major function of the Onchocerca volvulus PDI homologue was to participate in cuticle biosynthesis, it could be hypothesized that expression of this protein would be increased in those parasite tissues which were actively involved in cuticle biosynthesis. Consistent with this prediction, high levels of mRNA hybridizing to the ARAL-6 cDNA were found in developing microfilariae (Fig. 3A-C) . mRNA hy-

Fig. 4. Localization of O. volvulus protein disulfide isomerase protein by immunohistochemistry. Parasite sections were prepared as described in Materials and methods. (A) Low magnification cross-section exposed to anti-RAL6-MBP fusion protein antiserum, showing staining of the adult hypodermis (h), uterine microfilariae (mr) and morula (m). (B) Control cross-section exposed to pre-immune antiserum. (C) Higher magnification of the anti-RAL-6-MBP antiserum treated cross-section, showing immunostaining localized to the periphery of the early morula. (D) Higher magnification of a section exposed to anti-RAL-6-MBP fusion protein antiserum, showing intense staining of the periphery of late morula. Bar = 100 /xm in panel A, 50 p,m in panel B, and 25 /,~m in panels C and D.


Fig. 5. Localization of O. volvulus protein disulfide isomerase protein by immunohistochemistry: Sections were prepared as described in Materials and methods. (A) Longitudinal section of an adult female probed with the anti-RAL-6 antiserum, showing staining localized to the periphery of early stage microfilariae. (B) Cross-section containing more developed coiled microfilariae. Intense immunostaining was noted to the periphery of the microfilariae (arrows) The hypodermis of the adult female (h) was also recognized by the antibody. Anti-RAL-6-MBP fusion protein immunostaining continued to be detected, but at a lower level, in mature uterine microfilariae (C), and in mature skin derived microfilariae (D).

W.R. Wilson et al, /Molecular and Biochemical Parasitology 68 (1994) 103-117 111

bridizing to ARAL-6 was detectable in the early morula stage of development (Fig. 3A), and reached maximal levels in late morula (Fig. 3B). mRNA hybridizing to ARAL-6 was also detected in immature uterine microfilariae (Fig. 3C), and in mature skin-dwelling microfilariae (Fig. 3D), although message levels in the mature microfilariae appear to be lower than those in the developing parasites. In the adult female, mRNA hybridizing to ARAL-6 was localized to the hypodermis, the tissue responsible for the synthesis of cuticle components (Fig. 3A). In contrast, neither the cuticle itself, nor the uterine wall or the intestine of the adult female, contained mRNA which hybridized to ARAL-6 (Fig. 3A). No hybridization signal was seen in parasite sections hybridized with labeled vector plasmid lacking the ARAL-6 cDNA (Fig. 3E).

Native Onchocerca volvulus PDI was localized by immunohistochemistry using the polyclonal anti-

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Fig. 6. Analysis of sera from individuals exposed to O. volvulus for the presence of antibodies recognizing parasite protein disulfide isomerase. Sera were assayed for the presence of antibodies recognizing the RAL-6-MBP fusion protein as described in Mate- rials and methods. Values presented are the mean of triplicate determinations for each serum sample, minus reactivity to MBP alone. Patient groups are as follows: Endemic normal; Infected, O. volvulus-infected; Non-endemic control, individuals from North America who have never been exposed to O. volvulus, The dotted line indicates a cutoff value corresponding to the mean plus three standard deviations of the non-endemic control population.

serum raised against purified ARAL-6 fusion protein. The pattern of expression of the protein corre- sponded well with the results obtained from in situ hybridization of the mRNA (Figs. 4 and 5). The most intense immunostaining was localized to late morula (Fig. 4C and D) and immature uterine microfilariae (Fig. 5B and C), although immunostaining was also found in the hypodermis of the adult female (Fig. 4A and Fig. 5A). Within the developing microfilariae, immunostaining was concentrated around the periphery of the developing parasites (Figs. 4D and 5B). As expected, no staining was seen in control sections treated with preimmune serum (Fig. 4B).

The initial isolation of ARAL-6 was accom- plished by immunoscreening an expression cDNA library with an antiserum raised against Onchocerca volvulus L3. It was therefore possible that this protein might be recognized by the immune system of individuals exposed to the parasite. To test this hypothesis, sera collected from"a total of 123 patients residing in two areas hyperendemic for onchocerciasis were tested for the presence of antibodies recognizing the purified ARAL-6 fusion protein by ELISA. None of the serum samples collected from endemic normals (defined as individuals that remained free of O. volvulus infection for a period of at least three years, despite having resided in an area hyperendemic for onchocerciasis) contained antibodies which recognized the ARAL-6 fusion protein. In contrast, approximately 16% of the sera collected from O. volvulus-infected individuals contained antibodies that recognized the fusion protein (Fig. 6).

The cDNA encoded in ARAL-6 did not encode a full length open reading frame. Therefore, a series of experiments were performed to isolate a full length cDNA encoding the putative O. volvulus homologue of PDI. The cDNA present in ARAL-6 was first used as a hybridization probe to identify more complete cDNA clones in the available cDNA libraries. The sequence of a putative full length cDNA was then completed by PCR amplification of the 5' end of the message, as described in Materials and methods. The sequence of the putative full length cDNA sequence is shown in Fig. 7. The putative full length cDNA was 1628 bp in size, consistent with the finding that the original cDNA recognized a single message of approximately 1700 b in a Northern blot prepared

1 1 2 W.R. Wilson et al. / Molecular and Biochemical Parasitology 68 (1994) 103-117

from O. volvulus adult stage RNA (Fig. 1B). The first 22 nucleotides of the cDNA sequence were identical to the spliced leader present on some O. volvulus mRNAs. This was expected, since the

spliced leader primer was used as the 5' primer to isolate a cDNA corresponding to the 5' end of the message. At position 26, an initiating AUG codon began the single long open reading frame (ORF)

ggtttaa~tacccaagtttgagatc 25

ATGTTCAGGTTGGTCGTCGTCCTTAGCCTGTCGCTTCAGTTTGTACTGTACTCCGCGGCTCAAGATGCAAGCATA 100 M F R L V V V L S L S L Q F V L Y 8 A A Q D A S I 25

GAGGAGGATGAC GGGGTC CTTGTCC T CACAAAGAATAATTTCGATGACGC AGTAC, CAGCACATGAGTTTATTC TG 175 E E D D G V L V L T K N N F D D A V A A H E F I L 50

GTTGAATTCTATGCACCGTGGTGTGGACACTGCAAGGCTTTGGCACCTGAATATGCAA~GCAGCC, CACGTGTTG 250 V E F Y A P W C G H C K A L A P E Y A K A A H V L 75

A~GAAGACAGTCCAATTAAAC TTGGGAAATGCGAC GC GAC CGTACATGGTGAGC TTGC TTCA~TACGAG 325 K K E D S P I K L G K C D A T V 14 G E L A S K Y E i00

GTGCGTGGTTACCCGACGCTAAAGTTATTCCGTTCGGGCAAACCACAGGAATATGGTGGTGGACGAGATGCAGCG 400 V R G Y P T L K L F R S G K P Q E Y G G G R D A A 125

TCC ATC GTTGCTTGGC TAAAAAAGAAGACAGGTC CAGCAGC CAAAACTATGC TCTCAGCTGATGATGTAAAGGAT 475 S I V A W L K K K T G P A A K T M L S A D D V K Ik 150

TTTC AGGA~TAATGAAGTGTGCGTAATTGG TTACTTTAAGGATACTGAAAGCGCGGATGC AAAAGTT~ C TG 550 F Q E N N E V C V I G Y F K D T E S A D A K V F L 175

GAAGTAGC TC~ATTCGATGATATACCATTCGGTATTACTACTGAAATTGATGCAGCAAAAC AGC TCGGGC TG 625 E V A G G F D D I P F G I T T E I D A A K Q L G L 200

GA~TGAC GGAATTGTATTGTTGAAGA~TTCGATGAAC-GACGAGCCGAATTTGGTGAGAAGTTAGTAGCTGAT 700 E N D G I V L L K K F D E G R A E F G E K L V A D 225

GCGC TTAGATCTTGGGTTCAGGTCGA~GATTACCAC TTGTGAGTGAA'/"rCAC GCAAGATAC TGCAC CAATTATT 775 A L R S W V Q v E • R L P L V S E F T Q D T A P I I 250

TTTGGTGGTGATATTAAATCACACAACCTC TTATTCATTTCTAAAGAGAGTTCCGAATTCGAGAAAC TAGAGAAA 850 F G G D I K S H N L L F I S K E S S E F E K L E K 275

GAATTC C GAGCAGCTGCCAAGA~TTTAAGGGCAAGGTT~C GTCATCATTGATACGGATGTAGAAGACAAT 925 E F R A A A K K F K G K V F F V I I D T D V E D N 300

0

GC TCG TATC CTGGAATTCTTC GGTC TCAAAAAAGAAGATTTAGCAGCATTGAGACTGATCAGTTTGGAAGAAGAT 1000 A R I L E F F G L K K E D L A A L R L I S L E E D 325

ATGAC C AAATATAAGCC C GATTTTAAAGAAATTATAGC TGAAAATATTGTGCAATTCAC GGAAATGTATC TTGC T 1075 M T K Y K P D F K E I I A E N I V Q F T E M Y L A 350

GGTAAATTAAAACC ACATC TAATGACTCAAGATATTC C CAGTGACTGGGATAAGAATCCTGTGAAGATACTAGTT 1150 G K L K P H L M T (~ D I P S D W D K N P V K I L V 375

GGAAAGAAC TTTGAAGATGTCGCAAAGAATGC TAAGAAGGACGTTC TTG TGTTGTTCTATGC TC C ATGGTGTGGA 1225 G K N F E D V A K N A K K D V L V L F Y A P W C G 400

CATTGC AAAC AG TTGAT GC CAACTTGGGATAAATTGGGTGAAAAATATAAAGAC CATGATAC TATATTGATTGCT 1300 H C K Q L M P T W D K L G E K Y K D H D T I L I A 425

AAAATGGATGCAAC AGCAAATGAAGTGGAAAACGTTAAAGTGC AGTC ATTTC CAACAATCAAGTTTTTCC CTGCC 1375 K M D A T A N E V E N V K V Q S F P T I K F F P A 450

AGTTCTAATAAGGTAATTGATTTTACTGGTGAAAGGACATTAGAAGGGCTGACGAAATTTTTGGAAAGCGGAGGT 1450 S S N K V I D F T G E R T L E G L T K F L E S G G 475

0 AAAGATGG TGC TGG TTT ATC AGACGAAG AAAAAGC AAAAGAAGAAAGAAAAGTAAAAAAAAATTAAaaaaaagga 1525 K D G A G L S D E E K A K E E R K V K K N * 497

agaaaaagaaataaaggaaatacaaaaagaaagaaagaaaaagaaaaccagaaaaccaaaaaaaaaaagaaaaag 1600

aaaaaagt gaaaaaaaaaaaaaaaaaaa 1628

Fig. 7. DNA sequence of the O. volvulus protein disulfide isomerase homologue: The DNA sequence and derived open reading frame for the full length cDNA encoding the O. volvulus PDI homologue was obtained as described in the text. The putative ORF is indicated by capital letters, while putative untranslated regions are indicated by small letters. The sequence encoding the spliced leader is indicated by italics. The putative signal sequence is indicated by bold lettering. Boundaries of the cDNA encoded in ARAL-6 are indicated by diamonds over the nucleic acid sequence.

W.R. Wilson et al. /Molecular and Biochemical Parasitology 68 (1994) 103-117 113

~ l v u l u s human b o v i n e m o u s e

r a t r ~ i t a l f a l f a y e a s ¢

v o l v u l u s huma n

~ v i n e = ~ u s e r a t r a b b i t a l f a l f a y e a s t

v o l v u l u s hmean bovine

mouse

rat

r~it

alfalfa

y e a l t

v o l v u l u a

human bovine

.~uee

rat

r a b b i t a l f a l f a y e a s t

v o l v u l u s

human b o v i n e mouse

rat

rabbit

a l f a l f a y e a s t

v o l v u l u s

h u m a n b o v l n e

~ o u a e

r a t rabbit


v o l v ~ l u s

human b o v i n e m o u s e r a t r a b ~ i ~ a l f a l f a yeast

v o l v u l u s human b o v i n e mouse

ra~

rabbit


I~RLVVVLSL ~ y S A A : L : R A . . C L A VAAL=RAD:- : L : R A . . C L A LTAL~RAG:G

:L,SRA..CLA LAN~tARVG:D ;L :RA : . ~ LA----VT:G

: A . M : A . ~ G : L P S L . : LVPS

:KFSAGA.LS WSSL. : A : S V

P E Y ~ I ( I (E30~PIKI~ KCDATVEGE- : : : : : : : G ~ : : A : G : E : . : & : V : : : - - E : - : : : : : : : G ~ : : A : G : E : . : A : V : : : - - E : - : = : : : R : A K : :A:G:E:.:A :V:::--E:-

: : : : : : : A K : : A : G : E : . : A : V : : : - - E : - : : : : : : : G K : : A : G : D : . : A : V : : : - - E : - : : : E : : : E . : S T H , P : . V : A : V : : ~ - N E : H : : : V : : : E T : V - - . ~ : T : A Q I : C : - - ~ O

ANLK]~TGpAAKTNLF~DDV KDFODKqEVC N : z t = . : : = : :T:LREGAAA ESLV:SS: :A N : = : : . : : : = :S:LSDGAAA EALV:SS: :A N : : : : . : : : : : T : L S D T A A A E S L V . S S : : T

N : : : : . : : : : :T:LSDTAAA E S L V . S S : : T

N = : : : . : : | : :T:LADSARA ESLV:SS: :A E Y : = : Q S : : : S T Z I K s : : z A TA:VGD:K:V

~ F N I : ~ : : VAWADLPAY LA-.:TFVTP

LGL~IV- -LLK~E~RAEFC~LVAD

Y Q : . K : : , : - - : F : : : : : : : ~ : ~ G ~ . T K . Y Q : . K : : . : - - : P : : : : : : : ~ : E G E . T K . Y Q : . X : : . : - - : F : : : : : : : I~ I : :GE.TK. Y Q : . X : : , : - - : F : : : : : : : ~ : I ~ E , T K . Y Q . H . : : . : - - : F : : : : : : : ~ : E G E . T K . GOSSVS:P:V R : T : p : : = L F V.-SKDFHV. :S.YLpSAMD EP.-V~GK. : . I A D - - - : :

FISKESSE~E

:.P:SV:.Y.

:.P:SV:.Y: G::SMFK.-:

:.PISV:.Y. G::SSFK:-:

:.P:SV:.Y. G;:SNFK.-:

: .P.SAA.H. G : : SGFKQ- : : : h r A - r ~ A : SFKT:YHE-V : Y - N . E E : L : EY'KPLFTE- L

S L - - ~ Y K I : ~ F K E I I A

T : - - ~ : . : : = : : : . S E : : T :

T : - - : : . : : : : : : . S D : . T : T : - - : : . : : : :::.SD:.T:

T : * - t : . : : : : : : , S D : . T : T : - - : : . : : : :::.SD:.T:

Z.Q~,G~KF F::N---.EL

Q:S-~:.AFD E~S:KIV.ES

DVLVI~yA~rrmCtqLMFT

N : F : E : : : : : : : : = : : : A I I

N : F : E : : : : : : : : ~ : : : A : I N : F : £ : : : : : : : : = : : : A : I N:F:E::::: : : : : : : : A : I N : F : E : : : : : :::::::A:I

N : : . E : : : : : : : : : : : : A : I : : : : : Y : : : : :::::R:A::

~-A---SIE EDDG~V-LT KNNFDDAVAA HEFILVEFYA P ~ A L A 65

........ 9: :.:H:::-:R :S::A.=.=: :KY.::=::: :::::=:::: 60

- - . : . . . . p . : . : H : t : - : H : G : : : . : . : : : K Y : : : : : : : 62 . - - : . . . . .= : . : N : : : - : K : S : : , . : - : : : K Y . : : : : : : : : : : : : : : : : 62 ....... A.: .:N:::-:K :S::A.: :N~.::::: :::::;: 62

61 .... WC~t%A: :.=N:::-:K SS::A.E.:: :KH.:::::: ;::::;::::

:IF:~S:T. AK.F::T-:D NT::H:T:KK :.::.::::: :::::::K:: 69

F--:QQEA.A p.:SA.:K:ATOS:~.YIQS :.L.:At:F: :::::::MM: 68

---LASKYEV RGYPTLKLFR SG---KPQEY GGGRDAASIV 128

-SD::QQ:G: :::::.:F:: N:DTAS:K:: TA::.:DD:: 126

-SD::QQ:G: :::::.:F:, N:DTAS:K:: TA::.:DD:: 128

oSD::QQ:G: :::::.:F:. N:DTAS:K:: T&::.:DD:: 128

-SD::OQ:G: :::::.:F:. N:DTAS:K:: TA::.:DD:: 128

-SD::QQ:G: :::::.:F:. NG:::::K:: TA::.:DD:: 127

NKD:::EN.: .:F::.:.:: N:GKNI-::: K=P:.:EG:: 136

--D:CMEHN, P:F:S::.:, NSDVNNSI.: E:P:T:EA:: 132

VIGY~TES ADAKVFLEVAGG-FDDIPWG ITTEIDAAKQ 197

:::F:::V:: DS::Q::QA: EAI-:::::: ::SNS:VFSK 195

:::F:::N:: DS::Q::LA: EVI-:::::: ::SNS:VFSK 197

: : : F : : : V : : D S : : Q : : L A : E A I - : : : : : : : :SI;~"VFSK 197

:::F:::AG: DS::Q::LA: EAV-:::::: :SNS:VFSK 197

:::F:::V:: DA::Q::LA: EAT-:::::: .:ASS:VFSR 196

..:V:PKFSG E.YD~:.A.: EXLRS:YD:A H:LNAKHLPK 206

::VQSGKI.A DFNAT:YSM: NKH:N:YD=V SAE~A:DDFK 201

ALRSWVQVER LPLVSEFTQD TAPIIF .... GGDIKSHNLL 261

N:LDF.K~Q ::;:I:::EQ :::K:: .... ::.::T:I:: 259

K:LDP.K~Q ::I:I:::EQ :::K:: .... ::.::T:I:: 261

K:LDF.KMNQ ::::1:::SO :::K:: .... ::.::T:I:: 261

K:LDF.K~Q ::::I:::EQ :::K:: .... ::.::T:I:: 261

KILDF.K~Q ::::I:::EQ :::::: .... ::.::T:I:: 260

: :EKF.EESS T:. :TV:N.. PS~P:VVKF FNSPNAKAM: 275

VF~:.:::A :: .... YFG. IDGSV:AQYV-ESGLPLGY: 262

-Fd~KEFRAA AKKFKGKVFF VII0--TDVE .... DNARIL EFFGL--KKE DLAALR--LI 320

G::SNFKT-: :ES::::,L: .F::--S:HT .... ::Q::: :::::--::: .CP:.:--:: 318

:ES::::.L: .F::--S:HT .... ::Q::: :=:::--::: .CP:.:--:: 320

:EG::::.L: .F::--S:HT .... ::Q::: :::::--::: .CP:.:--:: 320

:EG::::.L: .F::--S:HT . . . . ::Q::: :::::--::: .CP:.:--:: 320

:EG::::.L: .F::--S:HA .... ::Q::: :::::--::: -CP:::--:: 319

=EQY:0~GV- SF,V--G::: SS~3A----F QY:::--::: Q,P ..... :: 330

::--:N,GL.qNF.SIDARKF GRHAGNLN~ :Q:P:FAIH. MTED:.YG:P 328

~IVQFTI~ LAGKLKpHLM TQDIPSDWDK NPVKILVGKN FEDVA~AKK 388

:R:TE:CHRF :E::.::::: S:.RAG:::: .:::.P:::: :::::FDE:: 386

:K:TE:C~RF :E~::::::: S:..:D:::: .:::.::::: ::.::FDE:: 388

:K:TE:CMRF :E~:.::::: S:..:E:::: .:::.:::A: ~:.::FDE:: 388

:K :T : :Ch~F : E : : : : : : : : S : . , : E : : : : , : : : . : : : ; : : : . : : F D E : : 388 :G:TE:CQRF : E : : , : : : : : S : . . : E : : : . . : : : , : : : : : : : . : : F D E : : 387 . . .PTNLKA: K D : : . E : F . K $EP: :ET-NN E : : : . . : : Q T L : : : V F K S G : 397 KA:ESLVKDF :K:DAS:I.K S:.:F~-QD SS:FQ::::: H,,.VNDP:: 396

W D K L G I ~ K D HD-TILIAKM DATANEV--E NVKVQSFPTI KFFPASSNK- 454

::::::T::: :.-N:.:::: :S:::::--: A:::H::::. ::::::AD.T 453

::::::T::: :.-N:.:::: :S:::::--: A:::H::::. ::::::AD.T 455

::::::T::: :.-N:.:::: :S:::::--: A:::H::::. ::::::AD.T 455

::::::T::: :.-N:.:::: :S:::::--: A:::H::::. ::::::AD.T 455

::::::T::, :Q-D:.:::: :S:::::--: A:::H::::, :::::GPG,T 4S4

L:E.AVSFQS DA-D..:::L :::::.,PT. TFD::GY::. Y:RS::GK-- 464

YQE:A.T:AN ATSD.::::L :H:E:.:R-- G:V.EGY::: VLY:GGKKSE 464

VZOt-Lx=~'z'~ BGLTt~I,ESG GK-~?,AGLSD . . . . . . . . . E E K A - - - - K E E R - - - X - V K - X N 4 9 6

: = : Y N : = : : : . = F K : : : : : : = Q * : : : : D - : - - -DDLEDL: : - :EEPDM: : DDDQ:A:IDII L 508 = : : Y N = : = : : . = I ~ K : : : : : : : Q - : : : : D - - - D - D D L E D L : : - : E E P D L : : D D D Q : A : t D I L 5 1 0

:=:YM=:::: .:~:::::: =Q-::::D-: EDL-DLE--- :-:LEPDM:: DDDQ:A:SDI L 509

::=YM:=::: .=FK:::::: :Q~::::D .... NDDL-DL: :-:LEPDM:: DDDQ:A:tD| L 509

=:=YW=: .=FK::::: :Q'::::D-. ---DGLEDL: :-AEEPDL:: DDDQ:A:.IW L 509

.SQYD:G::K =D.IE:.:-- KNK:KT:AA- --HOEVEQPK A-:AOPEA:Q P ...... =DI L 513

S.VYO:S:S= .S:FD:.K~ :HF:VD:KAL YEEAQEKAA: :-:DADAELA DEED-A.~R L 523

B volvulus

human

mouse

rat

- - rabbit

bovine

alfalfa

yeast

Fig. 8. Comparison of the derived amino acid sequence of the O. volvulus protein disulfide isomerase homologue to PDI from other organisms. (A) Sequences were aligned using the CLUSTAL 5 algorithm [42], followed by final adjustment of the alignment by eye. ':', amino acid identity to the O. volvulus sequence,'.', a similar amino acid substitution and '-', gap introduced to maximize the alignment. Groups of amino acids considered similar include: Leucine, isoleucine, and valine; Glutamine and asparagine; Lysine and arginine; Glutamic acid and aspartic acid. The two putative active sites for isomerase activity are indicated by underlining. The canonical ER retention signal is indicated by bold type. The GenBank accession numbers for the sequences shown are: human, P07237: bovine, P05307; Mouse, P09103; rat, P04785; rabbit, P21195; alfalfa, P29828; yeast, P17967. (B) Phenogram of the sequences shown in Panel A. The phylogeny was developed with maximum parsimony methods using the computer program PAUP [43]. The data were exhaustively searched to identify the single most parsimonious tree, with the yeast sequence designated as the outgroup. Gaps in the sequence were not considered to be a new character state. The phenogram was rooted by designating the ingroup sequences as monophyletic. The small numbers over the branches indicate the branch lengths, while the large numbers below the branches indicate the percentage of times a given branch to the fight of the number was supported in 1000 bootstrap replications of the data. The data were also analyzed by the numerical methods included in the PHYLIP program package [44]. Distance matrices were prepared using the algorithms of Kimura [45] and Dayhoff [46]. Phylograms were then derived from the distance data using the algorithm devised by Fitsch and Margoliash [47], and by the Neighbor Joining method [48]. The distance methods all resulted in phylogenies in which the placement of the O. volvulus sequence was identical to that determined by parsimony.


found in the cDNA clone. This long ORF included the ORF present within the ARAL-6 cDNA. Up- stream of the putative start codon, a stop codon was found in frame with the long ORF, suggesting that the ATG present at position 30 represents the bona fide translation start. The open reading frame initiated by this ATG extended for 1491 bp. Following the stop codon, a putative poly(A) addition signal (AATAAA) [27] was found, located approximately 70 bp upstream of the start of a poly(A) tail.

The derived amino acid sequence of the long ORF is shown in Fig. 8A, compared to the derived amino acid sequences of the PDI homologues from a variety of other organisms. The O. volvulus sequence was found to be most similar to human PDI. Overall, the derived amino acid sequence of the O. volvulus PDI homologue was 50% identical to human PDI. Allowing for conservative amino acid substitutions, the sequence was 62% similar to human PDI. The O. volvulus sequence contained 2 regions with the sequence WCGHCK (underlined in Fig. 8A). These sequences have been shown to represent the active sites for protein disulfide isomerase activity in other organisms [28]. The blocks of sequence surrounding the two active sites appear to be the most highly conserved portions of the O. volvulus PDI homologue. A phylogram based upon the sequences shown in Fig. 8A is shown in Fig. 8B. As might be predicted from the evolutionary position of the parasitic nematodes, the O. volvulus PDI homologue appears to be distinct from the vertebrate PDI sequences, but is more closely related to the vertebrate PDIs than it is to the plant or yeast sequences.

The amino terminal domain of PDI generally encodes a signal sequence which is removed from the mature protein [28]. In a similar manner, the first 19 amino acids of the O. volvulus protein have properties consistent with a role as a signal sequence. This sequence is hydrophobic, and terminates with the sequence SAA, which is consistent with the - 3 , - 1 rule for signal peptidase cleavage sites [29]. Assuming that the carboxy terminal A of the sequence SAA represents the carboxy terminal amino acid of the cleaved signal peptide, the mature protein would have a molecular weight of 53 200 Da. This is consistent with the finding that antibodies raised against the purified ARAL-6 fusion protein recognized a single protein with an apparent molecular weight of approximately 56 000 Da in OvAg.

The derived amino acid sequence of the O. volvulus PDI cDNA was also rather divergent at its carboxy terminal end. Most significantly, the O. volvulus sequence lacks the conserved KDEL sequence found in all other known examples of PDI, and which serves as an ER retention signal [30]. Instead, the O. volvulus protein appears to terminate with a rather basic highly charged stretch of amino acid residues (Fig. 8A).

4. Discussion

The results presented above suggest that the PDI homologue of Onchocerca volvulus has many properties similar to those of PDI from other organisms. These include the size of the mature protein, the fact that it appears to be encoded by a single-copy gene, and the presence of two conserved amino acid motifs which in other organisms have been shown to represent the active sites for isomerase activity. Similarly, 0. volvulus PDI homologue begins with a hydrophobic amino acid sequence whose properties are consistent with a signal sequence. This suggests that the O. volvulus PDI is post-translationally processed through the endoplasmic reticulum (ER). Interest- ingly, the O. volvulus PDI homologue does not end with the canonical ER retention signal KDEL or HDEL, which are found in the derived amino acid sequences of PDI from other organisms (Fig. 8A and [30]). Instead, the parasite homologue terminates with a stretch of basic, charged amino acids. This is similar to what has been previously described in O. volvulus calreticulin, a protein that is also normally localized to the ER [31]. Like the O. volvulus PDI homologue, the parasite calreticulin also lacks a KDEL retention signal, terminating with a stretch of highly charged basic amino acids [32]. There are two possible explanations for this finding. The first is that, unlike the situation found in other organisms, the O. volvulus PDI is not necessarily an ER resident protein. Interestingly, in situ hybridization and immunolocalization studies have localized the expression of O. volvulus calreticulin to the periphery of O. volvulus infective larvae, a location which would not be predicted for an ER resident protein (R. Tuan and T.R. Unnasch, unpublished). It is possible that a similar situation exists in the case of O. volvulus PDI, in that the protein is not localized exclusively to


the ER. If this was the case, one would predict that a second, ER resident version of PDI containing the ER retention signal may also be present in O. volvulus. However, since the O. volvulus PDI homologue appears to be encoded by a single-copy gene, alternative versions of the protein must be encoded by a single genomic sequence. Thus, if alternative versions of the protein exist, they would have to be produced by a mechanism such as alternative splic- ing. A second explanation for the lack of the canonical KDEL ER retention signal in the O. volvulus PDI homologue is that O. volvulus uses a unique version of the ER retention signal, where a stretch of basic amino acids substitutes for the more common KDEL retention signal.

PDI is a multifunctional protein which is associated with at least three different functions. At least one of the functions of PDI is essential for viability, since knockout mutations of the PDI gene in yeast result in a lethal phenotype [33]. However, it is possible that most important function of this multifunctional protein might vary from organism to organism, and from cell type to cell type. The evidence presented above is consistent with the hypothesis that one of the major functions of O. volvulus PDI is in cuticle biosynthesis. Maximal expression of the O. volvulus PDI appears to be found in developing uterine microfilaria, and appears to be localized to the periphery of the developing embryos. The late embryos of nematodes are the stage where synthesis of the first cuticle occurs [34], and cuticle synthesis is thought to proceed in the tissues directly underly- ing the cuticle [4]. Furthermore, in adult females, PDI expression is maximal in the hypodermis, the tissue responsible for cuticle biosynthesis in adult parasites. The expression of PDI in the adult hypodermis is less than what is found in the developing microfilariae. This is consistent with the fact that maximal cuticle biosynthesis occurs during periods of active parasite development [35]. Thus, both the time and place of expression of the O. volvulus PDI homologue are consistent with the hypothesis that this protein is involved in cuticle biosynthesis.

PDI acts both as the /3 subunit of prolyl 4-hydroxylase as well as the activity responsible for the correct formation of disulfide bonds. Both of these activities may be important in cuticle biosynthesis. The data presented above cannot distinguish which

of these two roles PDI might play in this process. Localization of the a subunit of prolyl 4-hydroxylase might be useful in determining if the parasite PDI is acting primarily as the /3 subunit of this enzyme.

Antibodies recognizing the ARAL-6 fusion protein were found in approximately 16% of the individuals with patent O. volvulus infection. In contrast, none of the 15 individuals in the endemic control group produced antibodies which recognized the ARAL-6 fusion protein. Thus, there was a marginally significant association between the presence of antibodies to the ARAL-6 fusion protein and the presence of patent O. volvulus infection (p = 0.09, Fisher's Exact Test). It is possible that the lack of antibodies to the O. volvulus PDI homologue in these patients is a specific manifestation of the low titer of parasite specific antibody found in endemic normal individuals [36-38]. Alternatively, the association may be an artifact of the relatively small number of individuals which could be included in the endemic normal group. Such individuals are rare in areas that are hyperendemic for onchocerciasis [38].

Human PDI is one of the major antigens exposed on the surface of cultured retinal epithelial cells [15], and has recently been shown to be an autoantigen in Lewis rats [39]. It is therefore possible that the development of an immune response to O. volvulus PDI might lead to a cross-reacting immune response to the human version of the protein. Evidence has been accumulating suggesting that such a cross reacting immune response may play an important role in the pathogenesis of onchocerciasis [40,41]. Addi- tional studies examining the immune recognition of constructs expressing the entire parasite protein, as well as its human homologue, will be necessary in order to determine if PDI is one of the antigens involved in inducing a potentially damaging cross- reacting immune response in patients with onchocerciasis.

Acknowledgements

We would like to thank Drs. L. To~ and C. Back of the Onchocerciasis Control Programme in West Africa, and Dr. Brika of the Onchocerciasis National


Team of C6te d'Ivoire for providing parasite material used in this study. We would also like to thank Dr. N. Lang-Unnasch and Dr. C. Wilson for critical reading of the manuscript. This research received financial support from the Edna McConneU Clark Foundation (EMCF 02190) and from the National Institutes of Health (Projects AI 29693 and AI 28780).

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the onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase

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