PROTEINASE AND PROTEIN PROTEINASE INHIBITORS FROM SWEET POTATO
We have studied the physiological roles of trypsin inhibitors (TIs), the storage proteins of sweet potato, since 1980. In recent years, we focused on the anti-insect properties of TIs. Large scales of farm experiments for trypsin inhibitors in Chiayi were done, including effect of sweet potato leaves against insect pests, the anti-insect properties of transgenic plants of cauliflower, etc. We found that TIs has the potential as anti-insect agent.
ON-GOING PROJECTS
1. Expression and function of a cysteine proteinase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. (D. J. Huang and Y. H. Lin)
2. Expression and localization of protein disulfide isomerase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots (D. J. Huang and Y. H. Lin).
3. Sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots mucilage with antioxidant activities in vitro (D. J. Huang and Y. H. Lin)
4. Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leave. (H. J. Chen and Y. H. Lin)
Expression and function of a cysteine proteinase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots.
Cysteine proteinase (CPR) cDNA clone (SPCPRPP) of sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots was isolated by differential display. The open reading frame in this cDNA encodes a pre-pro-protein of 371 amino acids with conserved catalytic amino acids of papain. Examination of the expression patterns in sweet potato by northern blot analyses revealed that the transcripts of SPCPRPP were specifically induced in the storage roots. Recombinant SPCPRPP protein overproduced in E. coli (M15) was purified by Ni2+-chelated affinity chromatography. Active recombinant SPCPRPP protein was able to digest the 22 kDa sweet potato trypsin inhibitor (TI) protein when the latter was reduced by DTT (dithiothreitol) or NTS (NADPH/thioredoxin system). A smaller peptide (14 kDa) was obtained as a digestion product. We showed that polyclonal antibodies of TI and sporamin could 52 53 Annual Report 2005 cross-react with the other antigen hence suggested that TI and sporamin were identical in sweet potato. These results suggest that CPR is responsible for initiation of degradation and remobilization of stored 22 kDa TI during sprouting of SP storage roots after the reduction of 22 kDa TI by the NTS.
Expression and localization of protein disulfide isomerase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots.
A protein disulfide isomerase (PDI) coding sequence was cloned by differential display from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. The cDNA is 1869 bp in length and encodes for a protein of 503 amino acids with molecular weight of 56 kDa. Primary structure analysis of the deduced protein revealed two thioredoxin-like active sites (CGHC), an endoplasmic reticulum-retention signal at its Cterminus (KDEL), which are also found in other PDIs in plants and animals. One amidation site (PGKK) was found in the sequence. Although there is only about 65% identity between the sweet potato enzyme and other plant PDIs, the active site regions are almost identical. The corresponding mRNA level was found highest in the mature flower; followed by storage roots, sprouts and full expanded green leaves; while lowest in sprouted roots and vein. A recombinant protein expressed from the sweet potato storage root cDNA clone (SPPDI1) effectively catalyzed by glutathioneinsulin transhydrogenation. In western blot analysis, SPPDI1 level was the highest in the storage roots, followed by sprouts, full expanded green leaves and vein and no signal at all in sprouted roots. Immunohistological analyses of storage root for PDI show that the protein is localized in epidermal cell, xylem vessels, nucleus and starch granule of cells between xylem vessels and pith. In the sweet potato vein, PDI is localized in epidermal cell, laticifer and vascular cambium. These results suggested that the protein coded for by the sweet potato gene is a novel member of the PDI family in plants. SPPDI1 gene of sweet potato storage roots display differential gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.
Sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots mucilage with antioxidant activities in vitro.
Sweet potato storage root mucilage was extracted and purified by SDS and heating treatments. Total antioxidant activity, DPPH (1,1-diphenyl-2-picrylhydrazyl) staining, reducing power method, metal ion-dependent hydroxyl radicals, FTC (ferric thiocyanate) method, and protection of calf thymus DNA against hydroxyl radical-induced damage were studied. The crude and purified mucilage protein with a concentration of 0.1 mg/mL exhibited highest inhibition activity (half-inhibition concentration, IC50, was 0.08 mg/mL and IC50 > 0.1 mg/mL) in total antioxidant activity test. In the DPPH staining, the crude and purified mucilage appeared as white spots when it was diluted to 50 µg and 100 µg. Like total antioxidant activity, the reducing power, metal iondependent hydroxyl radicals, FTC activity and protection against hydroxyl radical-induced calf thymus DNA damage were found with the mucilage. It was suggested that the mucilage might contribute its antioxidant activities against both hydroxyl and peroxyl radicals.
Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leave.
Granulins are a family of evolutionarily ancient proteins that are involved in regulating cell growth and division in animals. In this report a full-length cDNA, SPCP3, was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP3 contains 1389 nucleotides (462 amino acids) in its open reading frame, and exhibits high amino acid sequence homologies (ca. 64% to 73.6%) with several plant granulincontaining cysteine proteases, including potato, tomato, soybean, kidney bean, pea, maize, rice, cabbage, and Arabidopsis. Gene structural analysis shows that SPCP3 encodes a putative precursor protein. Via cleavage of the N-terminal propeptide, it generates a protein with 324 amino acids (from the 139th to the 462nd amino acid residues), which contains two main domains: the conserved catalytic domain with the putative catalytic residues (the 163rd Cys, 299th His and 319th Asn) and the C-terminal granulin domain (from the 375th to the 462nd amino acid residues). Semi-quantitative RT-PCR and protein gel blot hybridization showed that SPCP3 gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was almost undetectable in mature green leaves, veins, and roots. Phylogenic analysis showed that SPCP3 displayed close association with a group of plant granulin-containing cysteine proteases which have been implied to be involved in programmed cell death. In conclusion, sweet potato SPCP3 is a functional, senescence-associated gene. Its mRNA and protein levels were significantly enhanced in natural and induced senescing leaves. The physiological role and/or function of SPCP3 associated with programmed cell death during leaf senescence were also discussed.
RECENT PUBLICATIONS
(A) Refereed Papers
1. Hou WC, Wang YT, Lin* YH, Shiao LJ, Chen TE, Wang CW and Dai* W. 2000. A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan No.5) seedling. Journal of Expermental Botany 51: 713-719. (SCI) NSC-88-2311-B001-043
2. Hou, W.C., H.J. Chen, and Y.H. Lin*. 2000. Dioscorins from Dioscorea species all exhibit both?carbonic anhydrase and trypsin inhibitor activities. Bot. Bull. Acad. Sin. 41: 191-196. (SCI)
3. Chen, H.J., W.C. Hou, W.N. Jane, andY.H. Lin*. 2000. Isolation and characterization of an isocitrate lyase gene from senescent leaves of sweet potato (Ipomoea batatas cv. Tainong 57) J. Plant Physiology 157: 669-676. (SCI)
4. Hou, W.C., H.J. Chen, and Y.H. Lin*. 2000. Dioscorins from Dioscorea species all exhibit both? carbonic anhydrase and trypsin inhibitor activities. Bot. Bull. Acad. Sin. 41: 191-196. (SCI)
5. Chen, H.J., W.C. Hou, and Y.H. Lin*. 2001. Ca2+-dependent and Ca2+-independent excretion modes of salicylic acid in tabacco cell suspension culture. Journal of Experimental Botany 52: 1219-1226. (SCI: Impact factor: factor=3.180; 5/135=11% in Plant Science)
6. Hou, W.C., Y.C. Chen, H.J. Chen, Y.H. Lin*, L.L. Yang, and M.H. Lee. 2001. Antioxidant activities of trypsin inhibitor, a 33 kDa root storage protein of sweet potato Ipomoea batatas (L.) Lam cv. Tainong 57. J. Agri. Food Chem. 49(6): 2978-2981. (SCI: Impact factor: 2.101; 4/92=4.35% in Food Science & Technology)
7. Hou, W.C., H.J. Chen, Y.H. Lin*, Y.C. Chen, L.L. Yang, and M. H. Lee. 2001. Activity staining of isocitrate lyase after electrophoresis on either native or sodium dodecyl sulfate polyacrylamide gels. Electrophoresis 22: 2653-2655. (SCI: Impact factor: 4.325; 2/68=2.9% in Analytical Chemistry)
8. Hou, W.C., M.H. Lee, H.J. Chen, W.L. Liang, C.H. Han, Y.W. Liu, and Y.H. Lin*. 2001. Antioxidant activities of dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber. J. Agri. Food Chem. 49: 4956-4960. (SCI: Impact factor: 2.101; 4.35% in Food Science & Technology)
9. Hou, W.C., D.J. Huang, and Y.H. Lin*. 2002. An aspartic type protease degrades trypsin inhibitors, the major storage proteins of sweet potato Ipomoea batatas (L.) Lam cv. Tainong 57. Bot. Bull. Acad. Sin. 43: 271-276. (SCI: Impact factor: 0.506; 73% in Plant Science)
10. Hou, W.C. and Y.H. Lin*. 2002. Sweet potato (Ipomoea batatas (L.) Lam) trypsin inhibitors, the major storage proteins, inhibit one endogenous serine protease activity. Plant Science 163: 733-739.? (SCI: Impact factor: 1.652; 25% in Plant Science)
11. Hsu, F.L., Y.H. Lin*, M.H. Lee, C.L. Lin, and W.C. Hou. 2002. Both dioscorin, the tuber storage protein of yam (Dioscorea alata cv. Tainong No. 1), and its peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activities. J. Agri. Food Chem. 50(21): 6109-6113. (SCI: Impact factor: 2.101; 4.35% in Food Science & Technology)
12. Hou, W.C., Y.L. Lu, S.Y. Liu and Y.H. Lin*. 2003. Activities of superoxide dismutase and glutathione peroxidase in leaves of different cultivars of Liriope spicate L. on 10% SDS-PAGE gels. Bot. Bull. Acad. Sin. 44 (1): 37-41. (SCI: Impact factor: 0.506; 73% in Plant Science)
13. Hou, W.C., H.J. Chen and Y.H. Lin*. 2003. Antioxidant peptides with angiotensin concerting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. J. Agri. Food Chem.51 (6): 1706-1709. (SCI: Impact factor: 2.101; 4.35% in Food Science & Technology)
14. Chen, H.J., W.C. Hou, C.Y. Yang, D.J. Huang, J.S. Liu and Y.H. Lin*. 2003. Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas (L.)Lam.) leaves. J. Plant Physiol. 160 (5) 547-555. (SCI: Impact factor: 1.149; 31% in Plant Science)
15. Hou, W.C., M.H. Lee, F.L. Hsu and Y.H. Lin*. 2003. Inhibitory activities of semicarbazide-sensitive amine oxidase and angiotensin converting enzyme of pectin hydroxamic acid. J. Agri. Food Chem. 51: 6362-6366. (SCI: Impact factor: 2.101; 4.35% in Food Science & Technology)
16. Huang D.J., H.J. Chen, W.C. Hou, T.E. Chen and Y.H. Lin*. 2004. In vitro reduction of trypsin inhibitor by purified NADPH/thioredoxin system from sprouts of sweet potato (Ipomoea batatas (L) Lam.) storage roots. Plant Science 166/2: 435-441. (SCI: Impact factor: 1.652; 25% in Plant Science)
17. Huang D.J., H.J. Chen, W.C. Hou and Y.H. Lin*. 2004. Isolation and characterization of thioredoxin h cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. Plant Science 166/2: 515-523. (SCI: Impact factor: 1.652; 25% in Plant Science)
18. Chen, H.J., W.C. Hou, J.S. Liu, C.Y. Yang, D.J. Huang, and Y.H. Lin*. 2004. Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.)Lam.) senescent leaves. Journal of Expermental Botany 55 (#398): 825-835. (SCI: Impact factor: 3.180; 15/135=11% in Plant Science)
19. Chen T.E., D.J. Huang, and Y.H. Lin*. 2004. Isolation and characterization of a serine protease from the storage roots of sweet potato (Ipomoea batatas [L.] Lam) Plant Science 166: 1019-1026. (SCI: Impact factor: 1.652; 25% in Plant Science)
20. Huang D.J., C.D. Lin, H.J. Chen, and Y.H. Lin*. 2004. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') constituents. Bot. Bull. Acad. Sin. 45: 179-186 (SCI: Impact factor: 0.506; 73% in Plant Science)
21. Hou W.C., W.C. Wu, C.Y. Yang, H.J. Chen, S.Y. Liu, and Y.H. Lin*. 2004. Antioxidant activities of methanolic and hot-water extracts from leaves of three cultivars of Mai-Men-Dong (Liriope spicata L.) Bot. Bull. Acad. Sin. 45: 285-290 (SCI: Impact factor: 0.506; 73% in Plant Science)
22. Huang D.J., H.J. Chen, W.C Hou, C.D. Lin, and Y.H. Lin*. 2004. Active Recombinant Thioredoxin h Protein with Antioxidant Activities from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. J. Agri. Food Chem. 52: 4720-4724 (SCI: Impact factor: 2.101; 4.35% in Food Science & Technology)
23. Hou, W.C., H.J. Chen, C.H. Han, C.Y. Lin, and Y.H. Lin*. 2004. Glutathione peroxidase-like activity of 33 kDa trypsin inhibitor from roots of sweet potato (Ipomoea batatas (L.) Lam 'Tainong 57'). Plant Science 166: 1541-1546. (SCI: Impact factor: 1.652; 25% in Plant Science)
24. Huang D.J., H.J. Chen, C.D. Lin, and Y.H. Lin*. 2005. Antioxidant and antiproliferative activities of water spinach (Ipomoea aquatica Forsk) constituents. Bot. Bull. Acad. Sin. 46: 285-290 (SCI: Impact factor: 0.506; 73% in Plant Science)
25. Hou, W.C., C.H. Han, H.J. Chen, C.L. Wen, and Y.H. Lin*. 2005. Storage proteins of two cultivars of sweet potato (Ipomoea batatas (L.) Lam) and their protease hydrolysates exhibited antioxidant activity in vitro. Plant Science 168: 449-456. (SCI: Impact factor: 1.652; 25% in Plant Science)
26. Huang D.J., H.J. Chen, W.C Hou, C.D. Lin, and Y.H. Lin*. 2005. Expression and function of a cysteine proteinase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57' storage roots. Plant Science. (SCI: Impact factor: 1.652; 25% in Plant Science)
Huang D.J., H.J. Chen, W.C. Hou, T.E. Chen and Lin, Y.H.*. 2004. In vitro reduction of trypsin inhibitor by purified NADPH/thioredoxin system from sprouts of sweet potato (Ipomoea batatas (L) Lam.) storage roots. Plant Science 166/2: 435-441.
Huang D.J., H.J. Chen, W.C. Hou and Lin, Y.H.*. 2004. Isolation and characterization of thioredoxin h cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. Plant Science 166/2: 515-523.
Chen, H.J., W.C. Hou, J.S. Liu, C.Y. Yang, D.J. Huang, and Lin, Y.H.*. 2004. Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam.) senescent leaves. Journal of Expermental Botany 55 (#398): 825-835.
Chen T.E., D.J. Huang, and Lin, Y.H.*. 2004. Isolation and characterization of a serine protease from the storage roots of sweet potato (Ipomoea batatas [L.] Lam) Plant Science 166: 1019-1026.
Huang D.J., C.D. Lin, H.J. Chen, and Lin, Y.H.*. 2004. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') constituents. Bot. Bull. Acad. Sin. 45: 179-186.
Huang D.J., H.J. Chen, W.C Hou, C.D. Lin, and Lin, Y.H.*. 2004. Active Recombinant Thioredoxin h Protein with Antioxidant Activities from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. J. Agri. Food Chem. 52: 4720-4724
Hou, W.C., H.J. Chen, C.H. Han, C.Y. Lin, and Lin, Y.H.*. 2004. Glutathione peroxidase-like activity of 33 kDa trypsin inhibitor from roots of sweet potato (Ipomoea batatas (L.) Lam 'Tainong 57'). Plant Science 166: 1541-1546.
Huang D.J., H.J. Chen, C.D. Lin, and Lin, Y.H.*. 2004. Antioxidant and antiproliferative activities of water spinach (Ipomoea aquatica Forsk) constituents. Bot. Bull. Acad. Sin. 46: 285-290
Hou, W.C., W.C. Wu, C.Y. Yang, H.J. Chen, S.Y. Liu, and Lin, Y.H.*. 2004. Antioxidant activities of methanolic and hot-water extracts from leaves of three cultivars of Mai-Men-Dong (Liriope spicata L.). Bot. Bull. Acad. Sin. 46: 285-290
Hou, W.C., C.H. Han, H.J. Chen, C.L. Wen, and Lin, Y.H.*. 2005. Storage proteins of two cultivars of sweet potato (Ipomoea batatas (L.) Lam) and their protease hydrolysates exhibited antioxidant activity in vitro. Plant Science 168: 449-456.
Huang D.J., H.J. Chen, W.C. Hou, T.E. Chen, W.Y. Hsu, and Lin, Y.H.*. 2005. Expression and function of a cysteine proteinase cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots. Plant Science 169: 423-431.
Huang D.J., H.J. Chen, and Lin, Y.H.*. 2005. Isolation and expression of protein disulfide isomerase cDNA from sweet potato (Ipomoea batatas) storage roots. Plant Science 169: 776-784.
Huang D.J., H.J. Chen, W.C. Hou, C.D. Lin, W.Y. Hsu, and Lin, Y.H.*. 2005. Sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage root mucilage with antioxidant activities in vitro. Food Chemistry (in press).
Chen, H.J., D.J. Huang, W.C. Hou, J.S. Liu, and Lin, Y.H.*. 2005. Molecular cloning and characterization of a granulin-containing cysteine protease SPCP3 from sweet potato (Ipomoea batatas) senescent leaves. Journal of Plant Physiology (in press)
