Molecular cloning of a cystatin from parasitic intestinal nematode, Nippostrongylus brasiliensis Teruki Dainichi, Yoichi Maekawa, Kazunari Ishii, and Kunisuke Himeno Department of Parasitology and Immunology, The University of Tokushima School of Medicine, Tokushima, Japan Abstract: A novel member of the cystatin family, nippocystatin (NbCys), was identified from excretory-secretory (ES)-products of a nematode Nippostrongylus brasiliensis, and the cDNA was cloned and sequenced. The mRNA of NbCys was confirmed to be expressed in both larvae and adults of the parasite. NbCys was translated as a proform with a single domain for secretion and was detected as a 14-kDa mature form in ES-products of the adult worm. Recombinant protein of NbCys profoundly inhibited the activity of cysteine proteases such as cathepsin L and B, but not that of cathepsin D, an aspartic protease. Furthermore, the ES-products had also been confirmed to inhibit cysteine proteases. Taken together, NbCys may play a role in evasion of N. brasiliensis from host defense systems, since cysteine proteases are known to participate in immune systems of infected hosts. J. Med. Invest. 48:81-87, 2001 Keywords:Cystatin, Nippostrongylus brasiliensis, cloning INTRODUCTION Natural cysteine protease inhibitors are part of the cystatin superfamily, which is subdivided into families 1, 2 and 3, or the stefin family, cystatin family and kininogen family, respectively (1). There is high sequence homology among all of these families, particularly with respect to a highly conserved, proposed reactive site consisting of 5 amino acids, Gln-Val-Val-Ala-Gly (QVVAG) (2). Members of family 2, the cystatin family, are secretion-type proteins that have a single domain with a molecular weight of 13-15-kDa. In nematodes, members of this family have been identified from Caenorhabditis elegans (3) and some species of filaria (4-7). Nippostrongylus brasiliensis, from the order Rhabditida, generally produce acute infections in rodents, and have life cycles similar to trichostrongyle parasites that infect the small intestines of humans and livestock (8). Although N. brasiliensis is supposed to express cystatin, there is no report of cystatin derived from this nematode. We here describe a newly defined, 14 -kDa nippocystatin (NbCys) belonging to the cystatin family 2 and derived from N. brasiliensis. This was a secretion-type cystatin, and its recombinant protein selectively inhibited cysteine proteases. Cysteine protease is indispensable for antigen-specific immunity because of its function in antigen processing and presentation (9,10). Therefore, this parasite may require this protease inhibitor to evade the host defense system via modulation of the host immune responses. MATERIALS AND METHODS 1. Parasites The strain of Nippostrongylus brasiliensis was provided by Dr. M. Yamada, Kyoto Prefectural University of Medicine, Kyoto, Japan (11). Parasites were maintained by serial passage in SD rats. Excretory-secretory (ES)-products of adult N. brasiliensis were collected by the protocol described below. Adult worms of N. brasiliensis, collected from the small intestine of rats that had been infected 7 d previously with 4,000 infective-stage larvae per animal, were sterilized by repeated washing with PBS containing penicillin and streptomycin. Worms were cultured with PBS at37°C for 6 hr. Culture supernatant was collected and concentrated using Centricon Plus 20 PL-10 centrifuge (Millipore, Bedford, MA). 2. cDNA cloning of NbCys Total RNA was isolated from adult worms of N. brasiliensis, prepared by the method above, using Trizol reagent (Life Technologies, Rockville, MD). A fragment of NbCys cDNA was obtained by RT-PCR. The total RNA was reverse-transcribed using hexanucleotide random primers with Superscript II reverse transcriptase (Life Technologies). Then the cDNA was amplified with Taq DNA polymerase (Takara Shuzo, Shiga, Japan). The thermocycle conditions were 35 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 30 sec. The sense and antisense primer sequences were 5'-TCATCTCAAGTTGTCGCTGGT-3' and 5'-AAATTTTCCCATGGTTTCTCCCA-3', the designs of which were based on conserved sequences among previously defined cystatin from other nematodes, Onchocerca volvulus (4), Brugia malayi (7), Acanthocheilonema viteae (6) and Caenorhabditis elegans (3). Amplified DNAs were resolved by 2-3% agarose gel electrophoresis and stained with ethidium bromide. The DNAs were extracted using Qiaex II gel extraction kit (Qiagen, Hilden, Germany) and then subcloned into pGEM-T easy vector (Promega, Madison, WI). Plasmids were extracted using a Qiaprep spin miniprep kit (Qiagen). The DNA sequences were defined using ABI PRISM BigDye terminator cycle sequencing ready reaction kit (Perkin Elmer, Norwalk, CT) and ABI PRISM 377 DNA sequencer (Perkin Elmer). On the basis of the nucleotide sequence of this cDNA fragment, specific primers were then synthesized for 3'- and 5'-RACE. For 3'-RACE, total RNA from adult N. brasiliensis was reverse-transcribed using Not I primer-adapter with dT-polymer (Life Technologies). The adapter-added cDNA was amplified with Taq DNA polymerase. The thermocycle conditions were 35 cycles of 94°C for 1 min, 64°C for 1 min and 72°C for 1 min. The sense primer sequence was 5'-GCAAGCGAACTTACGGCGACGA-3', and the antisense primer was the adapter primer. For 5'- RACE, total RNA from adult N. brasiliensis was reverse-transcribed using the gene specific primer, 5'-TGGCAGTTCGTCGCCGTAAGTTCG-3'. The dC-polymer was added to the resultant RT-product with terminal deoxynucleotidyltransferase (TdT) and was amplified with Taq DNA polymerase. The thermocycle conditions were 30 cycles of 94°C for 1 min, 54°C for 1 min and 72°C for 1 min. The sense primer was the adapter primer with dG-polymer (Life Technologies) and the antisense primer was 5'-TCGCCGTAAGTTCGCTTGCAG-3'. The DNA sequences of the RACE-PCR products were defined as written above. 3. Evaluation of cystatin mRNA expression Total RNA from adult worm and from third stage larvae were extracted with Trizol reagent and were reverse-transcribed using hexanucleotide random primers with Superscript II. Then the cDNAs were amplified with Taq DNA polymerase. The thermocycle conditions were 35 cycles of 94°C for 1 min, 54°C for 1 min and 72°C for 1 min. The sense and antisense primer sequences were 5'-ATGCCATCTGCGTTCGTTCTG-3' and 5'-TTAAACCTGCTCTCCTGGAGC-3'. The amplified DNAs were resolved by 2% agarose gel electrophoresis, stained with ethidium bromide and visualized with UV light. 4. Expression of rNbCys The NbCys cDNA was subcloned into the expression plasmid vector pET and expressed in E. coli strain BL21 as a protein fused to a leader sequence of influenza virus HA epitope and six histidines. rNbCys was purified from E. coli lysate by affinity chromatography using TALON resin (Clontech Laboratories Inc., Palo Alto, CA). The purity of rNbCys was determined by SDS-PAGE and staining with Coomassie brilliant blue (CBB). Purified rNbCys was washed and dialyzed with PBS. 5. Measurement of protease activities and their inhibition The activity of cysteine proteases was measured as follows. Protease was incubated with or without NbCys in 0.1 M sodium acetate buffer [pH 5.5] containing 1 mM EDTA, 8 mM cysteine and 20 μM Z-Phe-Arg-MCA (Peptide Institute, Osaka, Japan) as substrate at 37°C for 15 min. The reaction was stopped by addition of 100 mM monochloroacetate [pH4.3]. The amount of product was monitored fluorometrically with excitation at 370 nm and emission at 460 nm in a fluorescence spectrometer (Hitachi, Ibaraki, Japan). Activity of cathepsin D was measured in a Folin-Lowry reaction, as reported previously (12). RESULTS The cystatins identified from parasitic nematodes are all from species of the superfamily Filarioidea, namely filaria. Although cystatin has also been identified in C. elegans by analysis of genomic sequence, this nematode is free living. All of the cysteine protease inhibitors from nematodes reported to date belong to the cystatin family. To obtain a homologue of cystatin from N. brasiliensis, we amplified cDNA by PCR using primers of a consensus sequence found among nematode cystatins. A PCR product of 174 bp was amplified as expected. On the basis of the sequence of this cloned cDNA fragment, the full length of the cDNA was cloned by using 3'-RACE and 5'-RACE (Fig. 1). The length of the open reading frame was 435 bp and the protein consisted of 144 amino acids. An amino acid alignment of the protein with cystatins from other nematodes using the BLASTP program with the sequence databases at NCBI revealed the following similarities:two cystatins from C. elegans (72% and 63%), onchocystatin from Onchocerca volvulus (55%), Acanthocheilonema viteae cystatin (33%), and Brugia malayi cystatin (30%). The amino acid sequence of NbCys showed a lesser similarity to rat cystatin C (27%). This cloned protein had a stretch of 5 amino acids, QVVAG, which is highly conserved in the cystatin superfamily, and had a hydrophobic signal peptide like other members of cystatin family 2 (Fig. 2). We concluded that this newly defined molecule belongs to family 2, and named this molecule nippocystatin (NbCys). We evaluated whether NbCys mRNA is expressed in infective third stage larvae as well as in adult worms by RT-PCR. The open reading frame of NbCys cDNA was amplified as a 435 bp band from total cDNA of both larvae and adult worm (Fig. 3). These findings indicate that NbCys was expressed at the mRNA level at both developmental stages. Since the cDNA of NbCys encoded a hydrophobic signal peptide with 22 amino acids, NbCys was expected to be secreted as a 14-kDa mature form without a signal sequence. To confirm that NbCys is expressed as a mature form, we analyzed ES-products by immunoblotting with anti-recombinant NbCys (rNbCys) antiserum (Fig. 4). We detected NbCys as a 14-kDa band in ES-products, suggesting that NbCys is actually expressed as a protein of mature form. To investigate the function of NbCys as a cystatin, we used rNbCys to examine the ability to inhibit several cathepsins (Fig. 5). rNbCys strongly inhibited the enzymatic activity of two cysteine proteases, cathepsin L and cathepsin B. Cathepsin L was inhibited at a lower concentration of rNbCys than cathepsin B. On the other hand, aspartic protease cathepsin D was not inhibited by rNbCys even at extremely high concentrations. These findings indicate that NbCys has the ability to inhibit cysteine proteases, like other members of the cystatin family. To evaluate whether ES-products from N. brasiliensis actually have a component to inhibit cysteine proteases, we examined the ability of ES-products to inhibit cathepsin L as an index. When cathepsin L was incubated with ES-products, cathepsin L activity was inhibited in a dose-dependent manner (Fig. 6). This inhibitory activity in ES-products was not due to the large amount of protein, since an equivalent amount of BSA did not suppress the cathepsin L activity. NbCys was expressed in ES-products as a mature form (Fig. 4). These findings strongly suggest that the cysteine protease inhibitory component in ES-products is NbCys. DISCUSSION We newly cloned a cystatin from ES-products of intestinal nematode, N. brasiliensis. It consisted of 144 amino acids with a MW of 14-kDa and was named NbCys. NbCys was expressed in both third stage larvae and adult worm. Further, we synthesized its recombinant protein and confirmed that this protein exclusively inhibited cysteine proteases selectively. Immunoblot analysis showed that ES-products from N. brasiliensis contained NbCys. Furthermore, ES-products were confirmed to inhibit cysteine protease activity in vitro. These findings suggest that NbCys is secreted as a mature form and acts in the host. Onchocystatin from the filarial parasite, Onchocerca volvulus, is the first cystatin to be identified in nematode (4). Onchocystatin belongs to cystatin family 2, having signal peptide and being expressed in third stage larvae and adults, suggesting that this protein is a secretion-type and has an extrasomatic function. Analysis of the genomic sequence in the free living nematode, C. elegans, indicated that this nematode has a cystatin encoded by two genes (3). The amino acid sequence of NbCys showed greater similarity to the sequence of two cystatins from C. elegans than to that of onchocystatin. These similarities do not contradict taxonomical findings, since both N. brasiliensis and C. elegans belong to the order Rhabditida, while O. volvulus belongs to the order Spiruria. Recently there have been several reports that cystatin modulates immune responses. For example, chicken cystatin up-regulates NO-release from IFN-γ-activated mouse peritoneal macrophages via cytokine synthesis (14, 15). Cystatins from parasites have also been reported to show immunomodulatory effects. Recombinant cystatin from Acanthocheilonema viteae down-regulates T-lymphocyte proliferation and enhances IL-10 production in vitro (6). Furthermore, Garraud et al. reported that recombinant cystatin of Onchocerca volvulus induces either polyclonal or antigen-specific IgE and IgG4 antibodies in vitro in an IL-4-and/or IL-13-dependent manner (5). Thus, it is quite conceivable that NbCys also has immunomodulatory functions. We previously reported that treatment of mice with CA074, a synthetic and specific inhibitor for cathepsin B, changes the immune response from a Th2- to Th1-type in BALB/c mice infected with Leishmania major (16) or immunized with ovalbumin (17). That is, treatment with CA074 suppresses IL-4-production and augments IFN-γ-production. In general, host defense against helminth infection is mediated by Th2-type responses with IgE production, eosinophilia and mastocytosis (8). That NbCys has a similar effect as CA074 is quite reasonable since N. brasiliensis can defeat the host immunity by impairing the Th2-type responses. ES-products of N. brasiliensis are well known as antigens that evoke an immune response in hosts infected with N. brasiliensis (11). Some functional proteins that induce the production of specific antibodies in infected rats have been isolated from ES-products of N. brasiliensis (18, 19). At present, however, although these products have been regarded as immunodominant antigens, their immunomodulatory effects have not been elucidated (18, 19). Our results suggest that NbCys is a novel candidate for the immunomodulatory effector secreted by N. brasiliensis. ACKNOWLEDGEMENTS This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science, Sport and Culture (10044297, 10470067, 10770140, 11147219, 11153219, 11877046 and 12217101) and Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists. REFERENCES 1. Turk V, Bode W : The cystatins. protein inhibitors of cysteine proteinases. FEBS Lett 285:213-219, 1991 2. Abrahamson M, Ritonja A, Brown MA, Grubb A, Machleidt W, Barrett AJ : Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J Biol Chem 262:9688-9694, 1987 3. Wilson R, Ainscough R, Anderson K:2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature 368 : 32-38, 1994 4. Lustigman S, Brotman B, Huima T, Prince AM, McKerrow JH : Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus. J Biol Chem267:17339-17346, 1992 5. Garraud O, Nkenfou C, Bradley JE, Perler FB, Nutman TB : Identification of recombinant filarial proteins capable of inducing polyclonal and antigen-specific IgE and IgG4 antibodies. J Immunol 155:1316-1325, 1995 6. Hartmann S, Kyewski B, Sonnenburg B, Lucius R : A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production. Eur J Immunol 27:2253-2260, 1997 7. Gregory WF, Blaxter ML, Maizels RM : Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi. Mol Biochem Parasitol 87:85-95, 1997 8. Finkelman FD, Shea-Donohue T, Goldhill J, Sullivan CA, Morris SC, Madden KB, Gause WC, Urban JF Jr : Cytokine regulation of host defense against parasitic gastrointestinal nematodes:lessons from studies with rodent models. Annu Rev Immunol 15:505-533, 1997 9. Watts C : Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Immunol 15:821-850, 1997 10. Nakagawa TY, Rudensky AY : The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol Rev 172 :121-129, 1999 11. Yamada M, Nakazawa M, Matsumoto Y, Arizono N : IgE antibody production in rats against multiple components of excretory-secretory products of the nematode Nippostrongylus brasiliensis. Immunology 72:104-108, 1991 12. Turk V, Lah T, Kregar I. Cathepsin D, Cathepsin E : In : Bergmeyer HU eds. Methods of enzymatic analysis, 3rd edn. Verlag Chemie GmbH, Weinhaim, 1984, vol. 5, pp. 211-222 13. Nielsen H, Engelbrecht J, Brunak S, von Heijne G : Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1-6, 1997 14. Verdot L, Lalmanach G, Vercruysse V, Hartmann S, Lucius R, Hoebeke J, Gauthier F, Vray B : Cystatins up-regulate nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages. J Biol Chem 271:28077-28081, 1996 15. Verdot L, Lalmanach G, Vercruysse V, Hoebeke J, Gauthier F, Vray B : Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis. Eur J Biochem 266:1111-1117, 1999 16. Maekawa Y, Himeno K, Ishikawa H, Hisaeda H, Sakai T, Dainichi T, Asao T, Good RA, Katunuma N:Switch of CD4+ T cell differentiation from Th2 to Th1 by treatment with cathepsin B inhibitor in experimental leishmaniasis. J Immunol 161:2120-2127, 1998 17. Zhang T, Maekawa Y, Hanba J, Dainichi T, Nashed BF, Hisaeda H, Sakai T, Asao T, Himeno K, Good RA, Katunuma N : Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain in ovalbumin-immunized mice. Immunology 100:13-20, 2000 18. Kamata I, Yamada M, Uchikawa R, Matsuda S, Arizono N : Cysteine protease of the nematode Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1 antibody response in rats. Clin Exp Immunol 102:71-77, 1995 19. Nakazawa M, Yamada M, Uchikawa R, Arizono N : Immunocytochemical localization of secretory acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis. Cell Tissue Res 280 :59-64, 1995 Received for publication November 30, 2000;accepted January 12, 2001. Address correspondence and reprint requests to Kunisuke Himeno, Department of Parasitology and Immunology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7114.