Purification and characterization of cathepsin B‐like cysteine protease from cotyledons of daikon radish, Raphanus sativus

Abstract

Genome analysis has indicated that plants, like animals, possess a variety of protease genes. The Arabidopsis thaliana genome has 680 protease (known and putative peptidase) sequences representing all six catalytic types: aspartic, cysteine, glutamic, metallo, serine and threonine peptidases (MEROPS peptidase database; http://merops.sanger.ac.uk/), reflecting the diverse functions of plant proteases. In A. thaliana, cDNAs encoding 10 members [vignain, brassicain, RD19A, pseudotzain, aleurain Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE-cellulose, hydroxyapatite and organomercurial-Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS ⁄ PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO 2 ). Daikon CBCP is less sensitive (1 ⁄ 7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination.Abbreviations

Insecticide Resistance Mechanisms in the Green Peach Aphid Myzus persicae (Hemiptera: Aphididae) II: Costs and Benefits

“…In aphids, including M. persicae , PIs interfere with aminoacid assimilation, having a negative impact on fitness [74], [80]. In addition, these defense mechanisms have been reported in Brassica plants [74], [81], [82]. Our results show a significant up-regulation of the cathepsin B gene in both S genotypes.…”

Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

“…Structural features may explain this result. As previously shown [23], the occluding loop of cathepsins B from animals is longer to that of plants. Besides, an insertion in the propeptide sequence of cathepsins B from plants is not present in that from animals.…”

Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination

“…Cysteine protease activity that increased during germination could be isolated by cystatin-Sepharose and the enzyme was identified by amino acid sequencing. Using this approach, the expression profiles of active CBCP and RD21 were investigated in cotyledons during germination at the protein level (13,14). The level of CBCP transcript in cotyledons was highest at 2 days after imbibition and decreased gradually during days 3 8, whereas CBCP enzyme activity reached a maximum at 5 days after imbibition and subsequently decreased.…”

“…Major and minor products obtained following digestion with DRCP26 are indicated by thick and thin lines, respectively. As a reference, cleavage products and cleavage sites of CBCP (13) and RD21 (14) are also shown. DRCP26, whereas 27 and 11 kDa proteins were digested with DRCP26.…”

“…In an effort to delineate the activity profiles of papain-like cysteine protease in cotyledons of daikon radish during germination, E-64-sensitive protease activity was isolated from Responsive to dessication-21 (RD21) and CBCP by various chromatographic procedures and changes in enzyme activity of each fraction were analysed (13,14). Cysteine protease activity that increased during germination could be isolated by cystatin-Sepharose and the enzyme was identified by amino acid sequencing.…”