153 NOTES & TIPS 10. Besant, P. G., Byrne, L., Thomas, G., and Attwood, P. V. (1998) Anal. Biochem. 258, 372–375. 11. Besant, P. G., and Attwood, P. V. (1998) Anal. Biochem. 265, 187–190. 12. Almaula, N., Lu, Q., Delgado, J., Belkin, S., and Inouye, M. (1995) J. Bacteriol. 177, 2524 –2529. 13. MacDonald, N. J., De la Rosa, A., Benedict, M. A., Freije, J. M., Krutsch, H., and Skeeg, P. S. (1993) J. Biol. Chem. 268, 25780 – 25789. 14. Bominaar, A. A., Tepper, A. D., and Veron, M. (1994) FEBS Lett. 353, 5– 8. An In-Gel Assay of a Recombinant Western Corn Rootworm (Diabrotica virgifera virgifera) Cysteine Proteinase Expressed in Yeast Hisashi Koiwa, 1 Matilde Paino D’Urzo, Keyan Zhu-Salzman,* Jose Ignacio Ibeas, Richard E. Shade,* Larry L. Murdock,* Ray A. Bressan, and Paul M. Hasegawa FIG. 4. (A) Autoradiogram of alkaline-hydrolyzed, neutralized samples of GST-SLN1 (lane 1) and NDPK (lane 2) resolved by RPTLC. (B) Ninhydrin-stained image of the RP-TLC plate (A) showing 3-phosphohistidine (lane 1) and histidine (lane 2) standards, GSTSLN1 (lane 3), and NDPK (lane 4) alkaline hydrolysates. The 3-phosphohistidine standard (B, lane 1) comigrates with the radioactive spots of the GST-SLN1 and NDPK alkaline hydrolysates (A, lanes 1 and 2). on histidine. Through this simple, single-step separation technique, SLN1, NDPK, and other potential histidine phosphoproteins can be qualitatively analyzed for phosphohistidine content. REFERENCES 1. Falke, J. J., Bass, R. B., Butler, S. L., Chervitz, S. A., and Danielson, M. A. (1997) Annu. Rev. Cell Dev. Biol. 13, 457– 512. 2. Loomis, W. F., Shaulsky, G., and Wang, N. (1997) J. Cell. Sci. 110, 1141–1145. 3. Ohmori, H., Kuba, M., and Kumon, A. (1993) J. Biol. Chem. 268, 7625–7627. 4. Urushidani, T., and Nagao, T. (1997) Biochim. Biophys. Acta 1356, 71– 83. 5. Steiner, A. W., Helander, E. R., Fujitaki, J. M., Smith, L. S., and Smith, R. A. (1980) J. Chromatogr. 202, 263–269. 6. Noiman, S., and Shaul, Y. (1995) FEBS Lett. 364, 63– 66. 7. Wagner, P. D., and Vu, N.-P. (1995) J. Biol. Chem. 270, 21758 – 21764. 8. Posas, F., Wurgler-Murphy, S. M., Maeda, T., Witten, E. A., Thai, T. C., and Siato, H. (1996) Cell. 86, 865– 875. 9. Wei, Y.-F., and Matthews, H. R. (1991) Methods Enzymol. 200, 388 – 414. Center for Plant Environmental Stress Physiology and *Department of Entomology, Purdue University, West Lafayette, Indiana 47907-1165 Received December 2, 1999 Cysteine proteinases and their inhibitors (cystatins) function, in conjunction, to facilitate numerous biological processes including protein turnover, pathogenesis, and disease mitigation (1, 2). Cysteine proteinases constitute the major digestive proteolytic activity of many phytophagous coleopteran and hemipteran insect pests, including western corn rootworm (WCR) 2 (3, 4). Induction, by insect attack, of cysteine proteinase inhibitors (phytocystatins) that attenuate the activity of these digestive enzymes is presumed to be a significant host plant resistance mechanism (5, 6). Insect countermeasures to this plant defensive strategy include utilization of diverse proteinases for digestion and de novo production of inhibitor-insensitive proteinase(s) (7, 8). We established previously that WCR digestive proteolytic activity is comprised primarily of multiple isoforms of cathepsin L-like cysteine proteinases of the papain superfamily (9). Further dissection of the WCR proteolytic enzyme complement requires sys1 To whom correspondence should be addressed at Center for Plant Environmental Stress Physiology, 1165 Horticulture Building, Purdue University, West Lafayette, IN 47907-1165. Fax: (765) 494-0391. E-mail: koiwa@hort.purdue.edu. 2 Abbreviations used: WCR, western corn rootworm; scN, soyacystatin N; Cbz-F-R-Mec, N-Cbz-Phe-Arg 7-amido-4-methylcoumarin. Analytical Biochemistry 282, 153–155 (2000) doi:10.1006/abio.2000.4591 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 154 NOTES & TIPS tematic biochemical evaluation of structure/function and inhibition kinetics of individual proteinases by specific phytocystatins. Papain family proteinases can be expressed as recombinant proteins but these typically must be purified to near homogeneity and then activated by removal of an autoinhibitory domain (10, 11). Such elaborate methodology is suitable for detailed characterization of a single enzyme but is a major constraint to analysis of protein complements like those of the WCR digestive proteolytic system, which is comprised of at least five different cathepsin L-like proteinases (4, 9). Presumably, each proteinase is inhibited differentially by unique defensive phyto-inhibitors (12, 13). Herein, we report a highly sensitive assay system for evaluation of phytocystatin inhibition of recombinant WCR cysteine proteinases expressed in yeast cells. A WCR cysteine proteinase, DvCAL2, was expressed in Pichia pastoris, and secreted into gelled medium. Cysteine proteinase activity was visualized after hydrolysis of a substrate that produced a fluorescent product, indicating that the proprotein is autoactivated presumably by deletion of the inhibitory domain. DvCAL2 activity was inhibited by inclusion, in the medium, of soyacystatin N (scN), a cysteine proteinase inhibitor from soybean (14). Materials and Methods WCR proteinase expression vector. A cDNA encoding DvCAL2 proenzyme was amplified by PCR using forward P1 (aactcgagaaaagagaggctatttcctttgttgatttggt) and reverse P2 (aaagcggccgcttaaactaaaggataactggcttgagttgcaataccgcaggcattgttcctgtt) primers and a cDNA clone encoding DvCAL2 isolated from a WCR cDNA library (H. Koiwa et al., unpublished) as template. P2 includes a point mutation that eliminates a putative glycosylation site in the mature enzyme. After the fidelity of the nucleotide sequence was confirmed, the XhoI-NotI fragment was cloned into pPIC9 to obtain pCAL2. The pCAL2 was linearized by digestion with NcoI and transformed into P. pastoris GS115 by electroporation. Pichia cells expressing human cathepsin L (phCHL) and Saccharomyces cerevisiae BJ3501␣ cells expressing rat cathepsin B (pCBR22) were provided by Dr. J. S. Mort (11, 15). Recombinant cysteine proteinase assay. Transformed Pichia cells were streaked onto YPD medium for isolation of a single colony that was subsequently streaked onto plates for analysis of proteinase activity. The basal medium consisted of 0.2X YNB, 1 g/L ammonium sulfate, 20 g/ml uracil, 25 mM potassium phosphate (pH 5.0), and 0.3% phytagel (Sigma). The carbon source was either 0.5% glucose or methanol, and 10 M Cbz-F-R-Mec was included as a proteinase substrate (16). Two hundred microliters of methanol FIG. 1. DvCAL2 expression cassette for Pichia pastoris. A cDNA encoding DvCAL2 proenzyme was inserted into the XhoI and NotI sites of pPIC9. Transcription of DvCAL2 was regulated by the promoter and polyadenylation signal of alcohol oxidase (AOX), and the translated product was targeted for secretion by a signal sequence of ␣-factor (␣F) from Saccharomyces cerevisiae fused to the N-terminus. was added daily to the lids of the plates containing the alcohol to compensate for evaporation. After incubation for 2 days at 28°C, proteinase substrate hydrolysis was detected under long-wavelength UV illumination. The scN-sensitive amidolytic activity was analyzed by including soyacystatin in the medium at a concentration of 30 g/ml. Results and Discussion Recombinant cysteine proteinase activity was measured using a fluorescent substrate-based assay that was developed by modifying conventional YNB medium. Bacteriological grade agar was replaced with phytagel (Sigma) and the concentration of YNB constituents was reduced to attenuate background fluorescence and enhance medium clarity. An acidic medium pH (5.0) promoted processing of the proteinase proregion. A reduction in potassium phosphate level lowered the solidification temperature of the medium to 42– 45°C, so that recombinant scN could be added to and distributed evenly in liquidified medium without heat denaturation. The DvCAL2 proenzyme was expressed in P. pastoris cells as a fusion protein with yeast ␣-factor (Fig. 1). Although the recombinant proteinase was not detectable in a stained SDS-PAGE gel (not shown), a distinct fluorescent zone (halo) was evident around cells expressing DvCAL2, or human cathepsin L or B growing on plates impregnated with Cbz-F-R-Mec (Fig. 2). The fluorescent halo is indicative of Cbz-F-R-Mec hydrolysis that releases fluorescent methylcoumarin. Proteinase expression from Pichia cells was methanol dependent and the activity was inhibited by scN, implicating DvCAL2 as a cysteine proteinase of the papain family (1, 17). Differential inhibition of DvCAL2 and human cathepsin L hydrolytic activities by scN may be indicative of variation in recombinant protein expression or relative effectiveness of the phytocystatin as an inhibitor of each specific proteinase. S. cerevisiae cells produced cathepsin B on medium containing glucose, consistent with its expression being driven by the ␣-factor promoter (15). These results indicate that the Pichia NOTES & TIPS 155 and their inhibitors that compose the host plant-pest interaction. Acknowledgments. The authors thank Northup King for WCR cDNA clones, and Prof. J. S. Mort for the yeast clones expressing cathepsin L and cathepsin B. This work was supported, in part, by grants from the Indiana Soybean Board 98-210 and USDA/NRICGP 98-35503-6384. This is journal Article No. 16253 of Purdue University Agricultural Experimental Station. REFERENCES FIG. 2. DvCAL2 encodes a scN-sensitive cysteine proteinase. Single colonies of Pichia pastoris or Saccharomyces cerevisiae cells were streaked onto phytagel test media. Test media were prepared with methanol or glucose to examine specific induction of proteinase activity, and 0 or 30 g/ml scN protein to assay for specific proteinase inhibition. Images were obtained under UV illumination after 2 days of incubation. The fluorescent halo is indicative of amidolytic activity secreted from the cells detected by hydrolysis of Cbz-F-R-Mec. Strains of P. pastoris cells are 1 and 3, GS115 (pPIC9, control); 2, GS115 (pCAL2, WCR DvCAL2); and 4, GS115 (phCHL, human cathepsin CatL). Strains of S. cerevisiae are 5, BJ3501␣ (control); and 6, BJ3501␣ (pCBr22, human cathepsin CatB). cells can express a functional WCR cysteine proteinase that is secreted to the medium where its in-gel activity can be detected. Substantial research and genomics information implicate papain family proteinases and their inhibitors as having substantial involvement in biological processes of diverse organisms, including host-pest/pathogen interactions (18 –20). Typically, invading organisms express families of proteinase genes that encode isoforms with differential sensitivities to host defensive inhibitors. Consequently, defining the function of each proteinase, and identifying the most effective inhibitor of a specific proteinase, is not possible based only on sequence informatics or gene expression data. The proteinase assay system reported here is a simple but reliable adjunct to traditional biochemical characterization techniques that require purification of native enzymes. 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