Molecular cloning and characterization of a cDNA encoding asparaginyl endopeptidase from sweet potato (Ipomoea batatas (L.) Lam) senescent leaves

Abstract

Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxyl side of asparagine residues, and is possibly involved in the post-translational processing of proproteins. In this report one full-length cDNA, SPAE, was isolated from senescent leaves of sweet potato (Ipomoea batatas (L.) Lam). SPAE contained 1479 nucleotides (492 amino acids) in the open reading frame, and exhibited high amino acid sequence homologies (c. 61-68%) with asparaginyl endopeptidases of Vicia sativa, Phaseolus vulgaris, Canavalia ensiformis, and Vigna mungo. SPAE probably encoded a putative precursor protein. Via cleavage of the N- and C-termini, it produced a mature protein containing 325 amino acids (from the 51st to the 375th amino acid residues), the conserved catalytic residues (the 173rd His and 215th Cys amino acid residues), and the putative N-glycosylation site (the 332nd Asn amino acid residue). Semi-quantitative RT-PCR and western blot hybridization showed that SPAE gene expression was enhanced significantly in natural senescent leaves and in dark- and ethephon-induced senescent leaves, but was much less in mature green leaves, stems, and roots. Phylogenic analysis showed that SPAE displayed close association with vacuolar processing enzymes (legumains/asparaginyl endopeptidases), which function via cleavage for proprotein maturation in the protein bodies during seed maturation and germination. In conclusion, sweet potato SPAE is probably a functional, senescence-associated gene and its mRNA and protein levels were significantly enhanced in natural and induced senescent leaves. The possible role and function of SPAE associated with bulk protein degradation and mobilization during leaf senescence were also discussed.

Expression of Sweet Potato Senescence-Associated Cysteine Proteases Affect Seed and Silique Development and Stress Tolerance in Transgenic Arabidopsis

“…Asparaginyl endopeptidase is an atypical cysteine endopeptidase with a reported insensitivity to the inhibitor L-3-carboxy-2,3-trans-epoxypropionyl-leucylamino(4-guanidino)butane (E-64) (Okamoto & Minamikawa, 1999). A cysteine protease activity band with a molecular mass near 36 kDa similar to the protein gel blot results was also detected and exhibited insensitivity to E-64 inhibitor (Chen et al, 2004). These data provide indirect evidence to support the existence of asparaginyl endopeptidase in senescent leaves.…”

“…The conserved catalytic residues (His and Cys) and central -strands that supported the catalytic residues of human and mouse legumains (Chen et al, 1998) were also found in SPAE, plant legumain/asparaginyl endopeptidase, vacuolar processing enzymes, and the other cysteine proteases (Chen et al, 2004).…”

“…SPAE had been cloned from senescent leaves with PCR-selective subtractive hybridization and exhibited high amino acid sequence homologies to seed vacuolar legumains/asparaginyl endopeptidases of kidney bean (Phaseolus vulgaris), spring vetch (Vicia sativa) and jack bean (Canavalia ensiformis) (Chen et al, 2004). The conserved catalytic residues (His and Cys) and central -strands that supported the catalytic residues of human and mouse legumains (Chen et al, 1998) were also found in SPAE, plant legumain/asparaginyl endopeptidase, vacuolar processing enzymes, and the other cysteine proteases (Chen et al, 2004).…”

“…The deduced molecular mass of mature SPAE protein was, thus, likely between 33 and 36 kDa that detected by protein gel blot with polyclonal antibody against putative SPAE protein (Chen et al, 2004). Asparaginyl endopeptidase is an atypical cysteine endopeptidase with a reported insensitivity to the inhibitor L-3-carboxy-2,3-trans-epoxypropionyl-leucylamino(4-guanidino)butane (E-64) (Okamoto & Minamikawa, 1999).…”

“…Several full-length cDNAs encoding putative isocitrate lyase, papain-like cysteine proteases and asparaginyl endopeptidase, have been cloned from senescent leaves (Chen et al, 2000(Chen et al, , 2004(Chen et al, , 2006(Chen et al, , 2008(Chen et al, , 2009(Chen et al, , 2010b, which likely play roles in association with lipid degradation and gluconeogenesis, and protein degradation and re-mobilization. These data support the occurrence of macromolecule and organelle degradation into small molecules for recycling and re-mobilization during sweet potato leaf senescence.…”