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Soybean subtilisin-like protease involved in initiating storage protein degradation

Published online by Cambridge University Press:  22 February 2007

Xiaowen Liu ,
Zheng Zhang ,
Neel Barnaby ,
Karl A. Wilsona  and
Anna Tan-Wilsona
Xiaowen Liu
Affiliation:
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA
Zheng Zhang
Affiliation:
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA
Neel Barnaby
Affiliation:
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA
Karl A. Wilsona
Affiliation:
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA
Anna Tan-Wilsona*
Affiliation:
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY, 13902-6000, USA
*
*Correspondence Fax: 607-777-6521 Email: annatan@binghamton.edu
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Abstract

Abstract A partial cDNA fragment (1.1kb) of protease C1, an enzyme that initiates the proteolytic degradation of the β-conglycinin storage proteins in the soybean (Glycine max [L.] Merrill. cv. Amsoy 71) through limited action at Glurich regions, has been cloned by reverse transcription-polymerase chain reaction (RT-PCR). The sequence was extended toward the 5' end by another 314 bases. The nucleotide sequence shows that protease C1 belongs to the subtilisin family of serine proteases. The sequence encompasses the critical Asp, His and Ser residues of the catalytic triad, as well as the Asn at the binding site. Northern analysis shows the presence of a 2.5kb mRNA not only in seedling cotyledons, but also in developing seeds. The developing seeds, and even dry seeds, exhibit phenylmethylsulfonyl fluoride (PMSF) and EDTA-sensitive protease activity that only cleaves the α- and α'-, but not the β-subunits, of soybean β-conglycinin. The discrete proteolytic intermediates produced are of the same sizes as those produced by pure enzyme, as are the final 50 kDa and 48 kDa products. The activity is also sensitive to inhibition by synthetic poly-L-Glu, all characteristics of purified protease C1. These data suggest that protease C1, or an enzyme very similar to it, is synthesized in a form that is active in vitro. Because seeds do accumulate β-conglycinin and because there is very little evidence of the proteolytic products of protease C1 action in extracts of dry seeds, one can surmise that the protease C1 is not particularly active in vivo during seed maturation. GenBank lists the sequence of the 1.1kb fragment with accession number AAD02075.

Keywords

protease C1subtilisin-like proteaseGlycine maxseed developmentseedling cotyledonssoybeanstorage protein mobilization

Information

Type
Research Article
Information
Seed Science Research , Volume 11 , Issue 1 , March 2001 , pp. 55 - 68
Copyright
Copyright © Cambridge University Press 2001

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References

Akhtaruzzaman, M., Kimura, Y. and Takagi, S. (1993) Purification and characterization of a CM-glycinin digesting protease from soybean seeds. Bioscience, Biotechnology and Biochemistry 57, 1119–1124.CrossRefGoogle ScholarPubMed
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.CrossRefGoogle ScholarPubMed
Batchelor, A.K., Boutilier, K., Miller, S.S., Labbé, H., Bowman, L., Hu, M., Johnson, D.A., Gijzen, M. and Miki, B.L.A. (2000) The seed-coat specific expression of a subtilisin gene, SCS1, from soybean. Planta 211, 484–492.CrossRefGoogle ScholarPubMed
Becker, C., Senyuk, V.I., Shutov, A.D., Nong, V.H., Fischer, J., Horstmann, C. and Muntz, K. (1997) Proteinase A, a storage globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization. European Journal of Biochemistry 248, 304–312.CrossRefGoogle Scholar
Besse, I., Wong, J.H., Kobrehel, K. and Buchanan, B.B. (1996) Thiocalsin: a thioredoxin-linked substrate-specific protease dependent on calcium. Proceedings of the National Academy of Sciences, USA 93, 3169–3175.CrossRefGoogle ScholarPubMed
Bloom, M.V., Freyer, G.A. and Micklos, D.A. (1996) Laboratory DNA science: An introduction to recombinant DNA techniques and methods of genome analysis. Menlo Park, CA, Benjamin Cummings Publishing Co.Google Scholar
Bogacheva, A.M. (1999) Plant subtilisins. Biochemistry (Moscow) 64, 287–293.Google ScholarPubMed
Bond, H.M. and Bowles, D.J. (1983) Characterization of soybean endopeptidase activity using exogenous and endogenous substrates. Plant Physiology 72, 345–350.CrossRefGoogle ScholarPubMed
Bottari, A., Capocchi, A., Galleschi, L., Jopova, A. and Saviozzi, F. (1996) Asparaginyl endopeptidase during maturation and germination of durum wheat. Physiologia Plantarum 97, 475–480.CrossRefGoogle Scholar
Carter, P. and Wells, J.A. (1990) Functional interaction among catalytic residues in subtilisin BPN. Proteins 7, 335–342.CrossRefGoogle ScholarPubMed
Chavan, J.K. and Hejgaard, J. (1981) Detection and partial characterization of subtilisin inhibitors in legume seeds by isoelectric focusing. Journal of the Science of Food and Agriculture 32, 857–862.CrossRefGoogle ScholarPubMed
Cronn, R.C., Small, R.L. and Wendel, J.F. (1999) Duplicated genes evolve independently after polyploid formation in cotton. Proceedings of the National Academy of Sciences, USA 96, 14406–14411.CrossRefGoogle ScholarPubMed
Debarros, E.G. and Larkins, B.A. (1994) Cloning of a cDNA encoding a putative cysteine protease from germinating maize seeds. Plant Science 99, 189–197.CrossRefGoogle Scholar
Derbyshire, E., Wright, D.J. and Boulter, D. (1976) Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 15, 3–24.CrossRefGoogle Scholar
DeVries, F., Hoge, H. and Bisseling, T. (1988) Isolation of total and polysomal RNA from plant tissues. pp. 1–13in Gelvin, S.B.Schilperoort, R.A.Verma, D.P.S. (Eds) Plant molecular biology manual, Section B6. Dordrecht, Kluwer Academic.Google Scholar
Grannell, A., Cercos, M. and Carbonell, J. (1998) Plant cysteine proteinases in germination and senescence. pp. 578–583in Barrett, A.J.Rawlings, N.D.Woessner, J.F. (Eds) Handbook of proteolytic enzymes. San Diego, CA, Academic Press.Google Scholar
Jorda, L., Coego, A., Conejero, V. and Vera, P. (1999) Genomic cluster containing four differentially regulated subtilisin-like processing protease genes is in tomato plants. Journal of Biological Chemistry 274, 2360–2365.CrossRefGoogle Scholar
Kapur, R., Tan-Wilson, A.L. and Wilson, K.A. (1989) Isolation and partial characterization of a subtilisin inhibitor from the mung bean Vigna radiata. Plant Physiology 91, 106–112.CrossRefGoogle ScholarPubMed
Kermode, A. (1994) Regulation of plant gene expression at the posttranslational level: applications to genetic engineering. pp. 317–390in Basra, A.S. (Ed.) Mechanisms of plant growth and improved productivity. New York, Marcel Dekker.Google Scholar
Kobayashi, T., Kobayashi, E., Sato, S., Hotta, Y., Miyajima, N., Tanaka, A. and Tabata, S. (1994) Characterization of cDNAs induced in meiotic prophase in lily microsporocytes. DNA Research 1, 15–26.CrossRefGoogle ScholarPubMed
Kubota, Y., Shoji, S., Yamanaka, T. and Yamato, M. (1976) Carboxypeptidases from germinating soybeans. I. Purification and properties of two carboxypeptidases. Yakugaku Zasshi 96, 639–647.CrossRefGoogle ScholarPubMed
Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle ScholarPubMed
Lee, K.M., Liu, Z.W., Tan-Wilson, A. and Wilson, K. (1997) Transcript levels for a mung bean cysteine protease during early seedling growth. Seed Science Research 7, 359–372.CrossRefGoogle Scholar
Maruyama, N., Katsube, T., Wada, Y., Oh, M.H., de la Rosa, A.P.B., Okuda, E., Nakagawa, S. and Utsumi, S. (1998) The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features. European Journal of Biochemistry 258, 854–862.CrossRefGoogle ScholarPubMed
McIlvaine, T.C. (1921) A buffer solution for colorimetric comparison. Journal of Biological Chemistry 49, 183–186.CrossRefGoogle Scholar
Meichtry, J., Amrhein, N. and Schaller, A. (1999) Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.). Plant Molecular Biology 39, 749–760.CrossRefGoogle ScholarPubMed
Mikkonen, A. (1986) Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris. Physiologia Plantarum 68, 282–286.CrossRefGoogle Scholar
MIPS (Munich Information Center for Protein Sequences) Arabidopsis thaliana database [accessed 4 October 1999] http://websvr.mips.biochem.mpg.de/proj/thal/Google Scholar
Morita, S., Fukase, M., Hoshino, K., Fukuda, Y., Yamaguchi, M. and Morita, Y. (1996) Partial purification and characterization of a novel soybean protease which is inhibited by Kunitz and Bowman-Birk trypsin inhibitors. Journal of Biochemistry 119, 711–718.CrossRefGoogle ScholarPubMed
Muntz, K. (1996) Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. Journal of Experimental Botany 47, 605–622.CrossRefGoogle Scholar
Nielsen, N.C., Dickinson, C.D., Cho, T-J., Thanh, V.H., Scallon, B.J., Fischer, R.L., Sims, T.L., Drews, G.N. and Goldberg, R.B. (1989) Characterization of the glycinin gene family in soybean. Plant Cell 1, 313–328.Google ScholarPubMed
Nong, V.H., Becker, C. and Muntz, K. (1995) cDNA cloning for a putative cysteine proteinase from developing seeds of soybean. Biochimica et Biophysica Acta 1261, 435–438.CrossRefGoogle ScholarPubMed
Okamoto, T. and Minamikawa, T. (1998) A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: characterization, regulation of gene expression, and posttranslational processing. Journal of Plant Physiology 152, 675–682.CrossRefGoogle Scholar
Okamoto, T., Yuki, A., Mitsuhashi, N. and Minamikawa, T. (1999) Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP). European Journal of Biochemistry 264, 223–232.CrossRefGoogle ScholarPubMed
Qi, X.Q., Wilson, K.A. and Tan-Wilson, A. (1992) Characterization of the major protease involved in the soybean β-conglycinin storage protein mobilization. Plant Physiology 99, 725–733.CrossRefGoogle ScholarPubMed
Qi, X.Q., Chen, R.Y., Wilson, K.A. and Tan-Wilson, A.L. (1994) Characterization of a soybean β-conglycinin-degrading protease cleavage site. Plant Physiology 104, 127–133.CrossRefGoogle ScholarPubMed
Ribeiro, A., Akkermans, A.D.L., van Kammen, A., Bisseling, T. and Pawlowski, K. (1995) A nodule-specific gene encoding a subtilisin-like protease is expressed in early stages of actinorhizal nodule development. Plant Cell 7, 785–794.Google ScholarPubMed
Rogers, J.C. (1998) Compartmentation of plant cell proteins in separate lytic and protein storage vacuoles. Journal of Plant Physiology 152, 653–658.CrossRefGoogle Scholar
Sabala, I., Franzen, H. and von Arnold, S. (1997) A spruce gene, af70, constitutively expressed in somatic embryos and induced by ABA and low temperature in seedlings. Physiologia Plantarum 99, 316–322.CrossRefGoogle Scholar
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual (2nd edition). Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.Google Scholar
Sebastiani, F.L., Farrell, L.B., Schuler, M.A. and Beachy, R.N. (1990) Complete sequence of a cDNA of α subunit of soybean β-conglycinin. Plant Molecular Biology 15, 197–201.CrossRefGoogle ScholarPubMed
Senyuk, V., Rotari, V., Becker, C., Zakharov, A., Horstmann, C., Muntz, K. and Vaintraub, I. (1998) Does an asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings? European Journal of Biochemistry 258, 546–558.CrossRefGoogle ScholarPubMed
Seo, S-B., Tan-Wilson, A. and Wilson, K.A. (2001) Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean β-conglycinin seed proteins. Biochimica et Biophysica Acta 1545, 192–206.CrossRefGoogle ScholarPubMed
Shirzadegan, M., Christie, P. and Seeman, J.R. (1991) An efficient method for isolation of RNA from tissue cultured plant cells. Nucleic Acids Research 19, 6055.CrossRefGoogle ScholarPubMed
Strausberg, S.L., Alexander, P.A., Gallagher, D.T., Gilliland, G.L., Barrett, B.L. and Bryan, P.N. (1995) Directed evolution of a subtilisin with calcium-independent stability. Biotechnology 13, 669–673.Google ScholarPubMed
Swanson, S.J., Bethke, P.C. and Jones, R.L. (1998) Barley aleurone cells contain two types of vacuoles. Characterization of lytic organelles by use of fluorescent probes. Plant Cell 10, 685–698.CrossRefGoogle ScholarPubMed
Tan-Wilson, A.L., Liu, X.W., Chen, R.Y., Qi, X.Q. and Wilson, K.A. (1996) An acidic amino acid-specific protease from germinating soybeans. Phytochemistry 42, 313–319.CrossRefGoogle ScholarPubMed
Thanh, V.H. and Shibasaki, K. (1978) Major proteins of soybean seeds. Subunit structure of β-conglycinin. Journal of Agricultural and Food Chemistry 26, 692–698.CrossRefGoogle Scholar
TIGR Soybean Gene Index [accessed 11 July 2000] http://www.tigr.orgGoogle Scholar
Tornero, P., Conejero, V. and Vera, P. (1996) Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: Similiarity of functional domains to subtilisin-like endoproteases. Proceedings of the National Academy of Sciences, USA 93, 6332–6337.CrossRefGoogle Scholar
Tornero, P., Conejero, V. and Vera, P. (1997) Identification of a new pathogen-induced member of the subtilisin-like processing protease family from plants. Journal of Biological Chemistry 272, 14412–14419.CrossRefGoogle ScholarPubMed
Walbot, V., Clutter, M. and Sussex, I.M. (1972) Reproductive development and embryogeny in Phaseolus. Phytomorphology 22, 59–68.Google Scholar
Wilson, K.A. and Tan-Wilson, A.L. (1987) Characterization of the proteinase that initiates the degradation of the trypsin inhibitor in germinating mung beans (Vigna radiata). Plant Physiology 84, 93–98.CrossRefGoogle ScholarPubMed
Wilson, K.A., Rightmire, B.R., Chen, J.C. and Tan-Wilson, A.L. (1986) Differential proteolysis of glycinin and β-conglycinin polypeptides during soybean germination and seedling growth. Plant Physiology 82, 71–76.CrossRefGoogle ScholarPubMed
Yamagata, H., Ueno, S. and Iwasaki, T. (1989) Isolation and characterization of a possible native cucumisin from developing melon fruits and its limited autolysis to cucumisin. Agricultural and Biological Chemistry 53, 1009–1117.Google Scholar
Yamagata, H., Masuzawa, T., Nagaoka, Y., Ohnishi, T. and Iwasaki, T. (1994) Cucumisin, a serine protease from melon fruits, shares structural homology with subtilisin and is generated from a large precursor. Journal of Biological Chemistry 269, 32725–32731.CrossRefGoogle ScholarPubMed
Yamaoka, Y., Ohba, Y., Takeuchi, M. and Morohashi, Y. (1993) Isolation and properties of a carboxypeptidase from cotyledons of germinated mung bean seeds. Plant Science 95, 1–7.CrossRefGoogle Scholar