- Email: [email protected]
Preliminary characterisation of an Onchocerca volvulus aspartic protease
Internat~onalJournalfor 0 1997 Austrahan
Pergamon PII:
RESEARCH
Smety
Parasirolog.v,Vol. for Parasitology.
SOO20-7519(97)00069-6
2l,No.9.p~.
1087-1090, 1997 by Elsetier Scmce Ltd Printed in Great Bntam 00?~7519/97 $17.00+0.00
Published
NOTE
Preliminary
Characterisation of an Onchocerca voZvzdu~ Aspartic Protease ABBAS
JOLODAR
and DAVID
J. MILLER*
Department of Biochemistry and Molecular Biology, James Cook University of North Queensland, Townsville, Queensland 4811, Australia (Received 6 January 1997: accepted 14 May 1997)
Abstract-Jolodar A. SC Miller D. J. 1997. Preliminary characterisation of an Onchocerca uoluulus aspartic protease. International Journal for Parasitology 27: 1087-1090. PCR using 1 primer specific for aspartic proteases and 1 primer annealing to the vector allowed amplification of a 377 bp fragment encoding part of an aspartic protease from an Onchocerca uoluulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone. In common with 2 other nematode aspartic proteases, the 0. uoluulus predicted protein has several of the general characteristics of this class of proteins, but lacks specific determinants of lysosomal localisation. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words: Onchocerca volvulus; aspartic protease; nematodes; lysosomal localisation.
Human onchocerciasis is caused by Onchocercavolvulus, a nematode that infects an estimated 18 million people, mainly in Africa and Latin America. Microfilariae, a larval stage of 0. uoluulus,are responsible for the clinical manifestations of the disease, which range from transient fever or mild itching to extensive oedema of the limbs and blindness. Although direct mortality due to onchocerciasis is low, the disease has a devastating socio-economic impact in endemic areas, as it can incapacitate large sections of the community. The migration of microfilariae through tissues is presumably facilitated by the secretion of proteases. Recent evidence suggests that parasite proteases normally involved in pathological responses may provide important targets for prophylaxis and immunisation (McKerrow et al., 1993). For these reasons we are attempting to characterise the protease complement of 0. volvulus. Aspartic proteases may be of particular significance, as an inhibitor of this class of proteins is
*To whom correspondence should be addressed. Tel: 61-77814473; Fax: 61-77-251394; E-mail: david.miller@jcu. edu.au. GenBank accession number: U81605.
an immunodominant 0. volvulus antigen and is secreted by adult female worms (Lucius et al., 1988). Aspartic proteases are a group of homologous enzymes that includes pepsin, chymosin, renin and cathepsins D and E. With the exception of renin these enzymes have acid pH optima, and they are all thought to have a common mechanism involving 2 aspartic acid residues. Aspartic proteases have common structural features, and are thought to have diverged from a common ancestral sequence. The open reading frame of a typical aspartic protease is composed of a signal peptide of around 14 amino acid residues, a pro-enzyme region of approximately 35 residues, and the mature protein sequence of approximately 330 residues. The pro-enzyme is a zymogen, the N-terminal pro-part apparently being essential for correct folding of the enzyme in viuo (Koelsch et al., 1994). Here we describe the characterisation of a cDNA clone encoding the major part of an aspartic protease from 0. volz~ulus. PCR with the aspartic protease-specific primer (Becker eb al., 1995) in combination with the igtll forward sequencing primer using the Donelson (Donelson et al., 1988) cDNA library (3 ~1; approximately 3 x 106pfu) as template resulted in the ampli-
1087
1088
A. Jolodar and D. J. Miller
fication of a unique 377 bp fragment. The conditions used for PCR were as follows: (10 pmol of each primer; 1.5 U Taq polymerase; 50 ~1 reaction) 5 min denaturation at 94’C, followed by 35 cycles of 94°C (40 s), 59°C (60 s) and 72°C (90 s). Nucleotide sequencing of the 377 bp fragment confirmed that it encoded an aspartic protease-like protein. In order to identify cDNA clones encoding larger fragments of the 0. volvulus aspartic protease, this PCR product was used to probe the cDNA library. Screening of 100000 plaques resulted in the identification of 2 strongly hybridising clones. The insert in 1 of the clones was found to be identical with, but smaller than, the PCR product used to screen the library. However, the insert in the second lambda clone (OVAl) was of approximately 3kb. Nucleotide sequencing indicated that the insert in OVA1 was a concatamer of heterologous cDNAs joined at EcoRI sites; a segment encoding the 0. volvulus aspartic protease is flanked by sequences corresponding to 0. volvulus ribosomal DNA and a fragment with no significant matches in the databases. We have encountered this phenomenon on other occasions when screening the Donelson libraries, suggesting that during the construction of these libraries, the methylation of cDNAs prior to digestion with EcoRI was incomplete. That part of OVA1 encoding the aspartic protease we refer to below as OV7A. Comparison of the predicted amino acid sequence encoded by OV7A with a wide range of aspartic proteases (see below) indicates that the mature 0. volvulus protein is likely to begin at amino acid residue No. 87, i.e. the protein is likely to be synthesised as a precursor, with an 86 amino acids residue N-terminal extension preceding the mature protein. Because Nterminal extensions show conservation of general features rather than at the primary structure level, the 2 parts of the predicted protein sequence were analysed independently (Fig. 1). Comparison of the mature protein sequence (i.e. residues 877304) with the databases indicated that the 0. volvulus protein was most like a putative C. elegans aspartic protease (an ORF on chromosome III; Wilson et al., 1994) and the aspartic protease of AK)>-
lostoma caninum. The OV7A sequence encodes approximately two-thirds of the 0. volvulus aspartic protease, assuming that it is of similar size to the C. elegans and A. caninum enzymes. The points in these latter sequences at which the mature proteins begin was estimated by comparison with mammalian cathepsins; when the presumed precursor regions were excluded from analyses, the degree of identity between the 0. volvulus and C. elegans proteins was approximately 72%, and between the former and the A. caninum sequence was 68%. Interestingly, these nematode proteins are significantly more like higher plant aspartic proteases (e.g., 5.5% identity between the 0. z~olvu1us protein and cynarase from Cynara cardunculus) than they are like typical animal members of this protein family, such as the cathepsins. Of the cathepsins, the 0. ~~olvulus protein was most like chicken cathepsin D (50% identity; see Fig. l), which is an atypical vertebrate cathepsin D because it lacks the b-hairpin. The sequence around the active site (residues 118125 in the 0. volvulus sequence) is highly conserved in all aspartic proteases. Two pairs of cysteine residues which form disulphide bridges in vertebrate cathepsins D, and which are highly conserved in all cathepsin D homologues, are present in the Onchocerca sequence (residues 13 1 and 138. and 296 and 300). A third pair of cysteine residues are conserved between the nematode proteins (position Nos 178 and 183 in the 0. volvulus sequence). The size of the N-terminal extension on the 0. oolvulus protein is atypical of aspartic proteases in general. However, this seems to be a common characteristic of nematode aspartic proteases; that on the C. elegans homologue is 80AA residues, and the Ancylostoma caninum extension is likely to be of a similar size (NB. the available sequence is incomplete in the latter case). As in the precursors of other aspartic proteases, the N-terminal region of the 0. volvulus protein contains an extended (25 amino acid residue) hydrophobic region likely to function as a leader sequence. Several factors suggest that the 0. volvulus protease is not targeted to lysosomes. Proteins are marked for
Fig. 1. Alignment of the protein sequence encoded by 0. tv~luulus OV7A with homologous aspartic proteases. The OV7A protein was aligned with the following sequences (Genbank accession Nos in parentheses): an ORF on C. eleganschromosome III (250755). the A. caninum aspartic protease (U34888), the A. aegypti lysosomal aspartic protease precursor (403168) and the chicken cathepsin D precursor (405744). Note that the A. caninum sequence is incomplete, lacking the N-terminus. The position of an N-glycosylation site present in all lysosomal aspartic proteases, and playing a central role in import into lysosomes, is indicated. The region indicated by the bar is that encoded by the PCR product described in the text. Numbering refers to the 0. ~~olvulus sequence. Shading indicates identity (black) or conservative substitutions (grey) with respect to the 0. volvulus sequence; dashes indicate gaps introduced to optimise the alignment. The alignment was generated using ClustalW 1.6 in conjunction with the BOXSHADE server (http://ulrec3.unil.ch/software/BOX-form.html) running Boxshade 3.21.
O.VOlVUlUS c.ekga!ls A.caninum Axgypti G.gallus
O.VOlVUlUS cetegans Aaninum
C.elegans A.caninum A.eqypti G.gallllS
Primer b
0.v0lvulusMTKTFIT2 I(NR
extensions
Mature proteins
N-terminal
2-O
I
N-glycosylation (calhepsins D)
4.0
FCR
I
product
80
LL ya&#sa__ -_ __ -L.rlC~orADLaI----;------
FE
_- --
-
1090
A. Jolodar and D. J. Miller
export to lysosomes by phosphorylation of high mannose residues (to mannose 6-phosphate) on N-linked oligosaccharides. In human cathepsin D, the residue which is glycosylated in Asn70 in the mature protein sequence (which corresponds to position No. 155 in the 0. z~olvulus sequence; see Fig. l), and an N-glycosylation site at this position is well conserved in all known lysosomal aspartic proteases, including that of Aedes aegypti (Cho & Riakhel, 1992). This site is not present in the 0. volvulus protein, nor is it present in other nematode and plant homologues. Two regions are required for the phosphorylation of Asn70 in human cathepsin D, a lysine residue at a position corresponding to His277 in the 0. volzulus sequence, and amino acid residues 265-292 (Baranski et al., 1991). Again, the lysine residue is highly conserved in lysosomal aspartic proteases, but is not present in the 0. volvulus protein. The complete 0. volvulus sequence is likely to provide more information on the probable location and role of the aspartic protease.
Acknowledgements-The authors thank Dr P. Brindley and Dr S. Harrop for generously providing the aspartic proteasespecific PCR primer and for access to unpublished information We also thank Dr C. Behm for commenting on the manuscript.
REFERENCES Baranski T. J., Koelsch G., Hartsuck J. A. & Kornfeld S. 1991. Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting. Journal of Biological
Chemistry
266: 23365-23372.
Becker M. M., Harrop S. A., Dalton J. P., Kalinna B. H., McManus D. P. & Brindley P. J. 1995. Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation. Journal of Biological Chemistry 270: 244962450 1. Cho W. L. & Riakhel A. S. 1992. Cloning of cDNA for mosquito lysosomal aspartic protease. Journal of Biological
Chemistry
267: 21823-21829.
Donelson J. E., Duke B. 0. L., Moser D., Zeng W., Erondu N. E., Lucius R., Renz A.. Karam M. & Flares G. Z. 1988. Construction of Onchocerca ~oluulus cDNA libraries and partial characterization of the cDNA for a major antigen. Molecular
and Biochemical
Parasitology
31: 241-250.
Koelsch G.. Mares M., Metcalf P. & Fusek M. 1994. Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Letrers 343: 610. Lucius R., Erondu N., Kern A. & Donelson J. 1988. Molecular cloning of an immunodominant antigen of Onchocerca
voluulus.
Journal
of Experimental
Medicine
168:
1199-1204. McKerrow J. H., Sun E., Rosenthal P. J. & Bouvier J. 1993. The proteases and pathogenicity of parasitic protozoa. Annual Review of Microbiology 47: 821-853. Wilson R., Ainscough R., Anderson K. and 50 other authors 1994. 2.2Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nafure 368: 32-38.
Pergamon PII:
RESEARCH
Smety
Parasirolog.v,Vol. for Parasitology.
SOO20-7519(97)00069-6
2l,No.9.p~.
1087-1090, 1997 by Elsetier Scmce Ltd Printed in Great Bntam 00?~7519/97 $17.00+0.00
Published
NOTE
Preliminary
Characterisation of an Onchocerca voZvzdu~ Aspartic Protease ABBAS
JOLODAR
and DAVID
J. MILLER*
Department of Biochemistry and Molecular Biology, James Cook University of North Queensland, Townsville, Queensland 4811, Australia (Received 6 January 1997: accepted 14 May 1997)
Abstract-Jolodar A. SC Miller D. J. 1997. Preliminary characterisation of an Onchocerca uoluulus aspartic protease. International Journal for Parasitology 27: 1087-1090. PCR using 1 primer specific for aspartic proteases and 1 primer annealing to the vector allowed amplification of a 377 bp fragment encoding part of an aspartic protease from an Onchocerca uoluulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone. In common with 2 other nematode aspartic proteases, the 0. uoluulus predicted protein has several of the general characteristics of this class of proteins, but lacks specific determinants of lysosomal localisation. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words: Onchocerca volvulus; aspartic protease; nematodes; lysosomal localisation.
Human onchocerciasis is caused by Onchocercavolvulus, a nematode that infects an estimated 18 million people, mainly in Africa and Latin America. Microfilariae, a larval stage of 0. uoluulus,are responsible for the clinical manifestations of the disease, which range from transient fever or mild itching to extensive oedema of the limbs and blindness. Although direct mortality due to onchocerciasis is low, the disease has a devastating socio-economic impact in endemic areas, as it can incapacitate large sections of the community. The migration of microfilariae through tissues is presumably facilitated by the secretion of proteases. Recent evidence suggests that parasite proteases normally involved in pathological responses may provide important targets for prophylaxis and immunisation (McKerrow et al., 1993). For these reasons we are attempting to characterise the protease complement of 0. volvulus. Aspartic proteases may be of particular significance, as an inhibitor of this class of proteins is
*To whom correspondence should be addressed. Tel: 61-77814473; Fax: 61-77-251394; E-mail: david.miller@jcu. edu.au. GenBank accession number: U81605.
an immunodominant 0. volvulus antigen and is secreted by adult female worms (Lucius et al., 1988). Aspartic proteases are a group of homologous enzymes that includes pepsin, chymosin, renin and cathepsins D and E. With the exception of renin these enzymes have acid pH optima, and they are all thought to have a common mechanism involving 2 aspartic acid residues. Aspartic proteases have common structural features, and are thought to have diverged from a common ancestral sequence. The open reading frame of a typical aspartic protease is composed of a signal peptide of around 14 amino acid residues, a pro-enzyme region of approximately 35 residues, and the mature protein sequence of approximately 330 residues. The pro-enzyme is a zymogen, the N-terminal pro-part apparently being essential for correct folding of the enzyme in viuo (Koelsch et al., 1994). Here we describe the characterisation of a cDNA clone encoding the major part of an aspartic protease from 0. volz~ulus. PCR with the aspartic protease-specific primer (Becker eb al., 1995) in combination with the igtll forward sequencing primer using the Donelson (Donelson et al., 1988) cDNA library (3 ~1; approximately 3 x 106pfu) as template resulted in the ampli-
1087
1088
A. Jolodar and D. J. Miller
fication of a unique 377 bp fragment. The conditions used for PCR were as follows: (10 pmol of each primer; 1.5 U Taq polymerase; 50 ~1 reaction) 5 min denaturation at 94’C, followed by 35 cycles of 94°C (40 s), 59°C (60 s) and 72°C (90 s). Nucleotide sequencing of the 377 bp fragment confirmed that it encoded an aspartic protease-like protein. In order to identify cDNA clones encoding larger fragments of the 0. volvulus aspartic protease, this PCR product was used to probe the cDNA library. Screening of 100000 plaques resulted in the identification of 2 strongly hybridising clones. The insert in 1 of the clones was found to be identical with, but smaller than, the PCR product used to screen the library. However, the insert in the second lambda clone (OVAl) was of approximately 3kb. Nucleotide sequencing indicated that the insert in OVA1 was a concatamer of heterologous cDNAs joined at EcoRI sites; a segment encoding the 0. volvulus aspartic protease is flanked by sequences corresponding to 0. volvulus ribosomal DNA and a fragment with no significant matches in the databases. We have encountered this phenomenon on other occasions when screening the Donelson libraries, suggesting that during the construction of these libraries, the methylation of cDNAs prior to digestion with EcoRI was incomplete. That part of OVA1 encoding the aspartic protease we refer to below as OV7A. Comparison of the predicted amino acid sequence encoded by OV7A with a wide range of aspartic proteases (see below) indicates that the mature 0. volvulus protein is likely to begin at amino acid residue No. 87, i.e. the protein is likely to be synthesised as a precursor, with an 86 amino acids residue N-terminal extension preceding the mature protein. Because Nterminal extensions show conservation of general features rather than at the primary structure level, the 2 parts of the predicted protein sequence were analysed independently (Fig. 1). Comparison of the mature protein sequence (i.e. residues 877304) with the databases indicated that the 0. volvulus protein was most like a putative C. elegans aspartic protease (an ORF on chromosome III; Wilson et al., 1994) and the aspartic protease of AK)>-
lostoma caninum. The OV7A sequence encodes approximately two-thirds of the 0. volvulus aspartic protease, assuming that it is of similar size to the C. elegans and A. caninum enzymes. The points in these latter sequences at which the mature proteins begin was estimated by comparison with mammalian cathepsins; when the presumed precursor regions were excluded from analyses, the degree of identity between the 0. volvulus and C. elegans proteins was approximately 72%, and between the former and the A. caninum sequence was 68%. Interestingly, these nematode proteins are significantly more like higher plant aspartic proteases (e.g., 5.5% identity between the 0. z~olvu1us protein and cynarase from Cynara cardunculus) than they are like typical animal members of this protein family, such as the cathepsins. Of the cathepsins, the 0. ~~olvulus protein was most like chicken cathepsin D (50% identity; see Fig. l), which is an atypical vertebrate cathepsin D because it lacks the b-hairpin. The sequence around the active site (residues 118125 in the 0. volvulus sequence) is highly conserved in all aspartic proteases. Two pairs of cysteine residues which form disulphide bridges in vertebrate cathepsins D, and which are highly conserved in all cathepsin D homologues, are present in the Onchocerca sequence (residues 13 1 and 138. and 296 and 300). A third pair of cysteine residues are conserved between the nematode proteins (position Nos 178 and 183 in the 0. volvulus sequence). The size of the N-terminal extension on the 0. oolvulus protein is atypical of aspartic proteases in general. However, this seems to be a common characteristic of nematode aspartic proteases; that on the C. elegans homologue is 80AA residues, and the Ancylostoma caninum extension is likely to be of a similar size (NB. the available sequence is incomplete in the latter case). As in the precursors of other aspartic proteases, the N-terminal region of the 0. volvulus protein contains an extended (25 amino acid residue) hydrophobic region likely to function as a leader sequence. Several factors suggest that the 0. volvulus protease is not targeted to lysosomes. Proteins are marked for
Fig. 1. Alignment of the protein sequence encoded by 0. tv~luulus OV7A with homologous aspartic proteases. The OV7A protein was aligned with the following sequences (Genbank accession Nos in parentheses): an ORF on C. eleganschromosome III (250755). the A. caninum aspartic protease (U34888), the A. aegypti lysosomal aspartic protease precursor (403168) and the chicken cathepsin D precursor (405744). Note that the A. caninum sequence is incomplete, lacking the N-terminus. The position of an N-glycosylation site present in all lysosomal aspartic proteases, and playing a central role in import into lysosomes, is indicated. The region indicated by the bar is that encoded by the PCR product described in the text. Numbering refers to the 0. ~~olvulus sequence. Shading indicates identity (black) or conservative substitutions (grey) with respect to the 0. volvulus sequence; dashes indicate gaps introduced to optimise the alignment. The alignment was generated using ClustalW 1.6 in conjunction with the BOXSHADE server (http://ulrec3.unil.ch/software/BOX-form.html) running Boxshade 3.21.
O.VOlVUlUS c.ekga!ls A.caninum Axgypti G.gallus
O.VOlVUlUS cetegans Aaninum
C.elegans A.caninum A.eqypti G.gallllS
Primer b
0.v0lvulusMTKTFIT2 I(NR
extensions
Mature proteins
N-terminal
2-O
I
N-glycosylation (calhepsins D)
4.0
FCR
I
product
80
LL ya&#sa__ -_ __ -L.rlC~orADLaI----;------
FE
_- --
-
1090
A. Jolodar and D. J. Miller
export to lysosomes by phosphorylation of high mannose residues (to mannose 6-phosphate) on N-linked oligosaccharides. In human cathepsin D, the residue which is glycosylated in Asn70 in the mature protein sequence (which corresponds to position No. 155 in the 0. z~olvulus sequence; see Fig. l), and an N-glycosylation site at this position is well conserved in all known lysosomal aspartic proteases, including that of Aedes aegypti (Cho & Riakhel, 1992). This site is not present in the 0. volvulus protein, nor is it present in other nematode and plant homologues. Two regions are required for the phosphorylation of Asn70 in human cathepsin D, a lysine residue at a position corresponding to His277 in the 0. volzulus sequence, and amino acid residues 265-292 (Baranski et al., 1991). Again, the lysine residue is highly conserved in lysosomal aspartic proteases, but is not present in the 0. volvulus protein. The complete 0. volvulus sequence is likely to provide more information on the probable location and role of the aspartic protease.
Acknowledgements-The authors thank Dr P. Brindley and Dr S. Harrop for generously providing the aspartic proteasespecific PCR primer and for access to unpublished information We also thank Dr C. Behm for commenting on the manuscript.
REFERENCES Baranski T. J., Koelsch G., Hartsuck J. A. & Kornfeld S. 1991. Mapping and molecular modeling of a recognition domain for lysosomal enzyme targeting. Journal of Biological
Chemistry
266: 23365-23372.
Becker M. M., Harrop S. A., Dalton J. P., Kalinna B. H., McManus D. P. & Brindley P. J. 1995. Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation. Journal of Biological Chemistry 270: 244962450 1. Cho W. L. & Riakhel A. S. 1992. Cloning of cDNA for mosquito lysosomal aspartic protease. Journal of Biological
Chemistry
267: 21823-21829.
Donelson J. E., Duke B. 0. L., Moser D., Zeng W., Erondu N. E., Lucius R., Renz A.. Karam M. & Flares G. Z. 1988. Construction of Onchocerca ~oluulus cDNA libraries and partial characterization of the cDNA for a major antigen. Molecular
and Biochemical
Parasitology
31: 241-250.
Koelsch G.. Mares M., Metcalf P. & Fusek M. 1994. Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Letrers 343: 610. Lucius R., Erondu N., Kern A. & Donelson J. 1988. Molecular cloning of an immunodominant antigen of Onchocerca
voluulus.
Journal
of Experimental
Medicine
168:
1199-1204. McKerrow J. H., Sun E., Rosenthal P. J. & Bouvier J. 1993. The proteases and pathogenicity of parasitic protozoa. Annual Review of Microbiology 47: 821-853. Wilson R., Ainscough R., Anderson K. and 50 other authors 1994. 2.2Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nafure 368: 32-38.