Rice Aspartic Proteinase, Oryzasin, Expressed During Seed Ripening and Germination, has a Gene Organization Distinct from Those of Animal and Microbial Aspartic Proteinases

Abstract

The gene organization and nucleotide sequence of an aspartic proteinase (AP) of plant origin were first disclosed by cDNA and genomic DNA cloning of a rice AP (oryzasin). The deduced amino acid sequence of oryzasin 1 was significantly similar to those of other APs (34-85%), with highest similarity (85%) to barley AP (HvAP). Oryzasin 1, as well as HvAP, is distinct from animal and microbial APs in that the plant APs contain a unique 104-amino-acid insertion in the C-terminal region. The oryzasin 1 gene spans approximately 6.6 kbp and is composed of 14 exons and 13 introns. The exon-intron organization of the oryzasin 1 gene is totally different from those of genes for animal and microbial APs such as human cathepsin D, rat renin, bovine chymosin, aspergillopepsin A of Aspergillus awamori, proteinase A of Saccharomyces cerevisiae and rhizopuspepsin of Rhizopus niveus, despite the fact that oryzasin 1 shows overall sequence similarity to these APs. , it is expected that some other proteinases exist which are involved in protein processing and down-regulation at this stage. Meanwhile, information has been provided as to the involvement of aspartic proteinases (APs) in the proteolysis of seed proteins during ripening [6]. We undertook experiments to find such an enzyme in rice seeds. Using ripened seed samples, we found a proteolytic activity that bounds to a pepstatinconjugated affinity column (Asakura, T., Watanabe, H., Abe, K. and Arai, S., unpublished result), although the significance and characteristics of this activity remain to be clarified. KeywordsA great deal of information is available on APs of animal and microbial origins [7, 81. Physiological

Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

“…This hydroxyethylamine-containing inhibitor was found to inhibit recombinant cyprosin, albeit weakly (Table IV) at 140 nM. In contrast, Indinavir (compound 24 in Table IV), Boc -Phe-His-ACHPA-Leu-Phe-NH 2 …”

“…Six cysteine residues are predicted to be present within the plant-specific insert (Fig. 1) of cyprosin, and these are all conserved in the insert sequences of aspartic proteinase precursors from other plants (1)(2)(3)(4). A putative assignment of these into disulfide-bonded pairs (3I-97I, 28I-69I, and 34I-66I) has been proposed (12) on the basis of the swaposin similarity (see Introduction).…”

Suppression of a Phospholipase D Gene,OsPLDβ1, Activates Defense Responses and Increases Disease Resistance in Rice

“…The OsPLDb1-RNAi vector was constructed by generating an inverted hairpin loop into the pZH2Bik binary vector, which was modified to contain the rice ubiquitin promoter (OsmUbiP), the intron of a rice aspartic protease gene (RAP intron; Asakura et al, 1995), and the Nos terminator (T-Nos), in the sequence from pPZP202 (Hajdukiewicz et al, 1994). A 343-bp fragment corresponding to nucleotides 2,323 to 2,665 of OsPLDb1, which does not overlap with the amplified OsPLDb1-specific fragment (1,973-2,314) in the real-time PCR experiments described in Supplemental Table S4, was amplified using the following oligonucleotides: 5#-GAATGCATGAGACGAGTTCGC-3# (forward) and 5#-CCTTTTCTTGTCATGTCCTACT-3# (reverse).…”

The Glycosylation of the Aspartic Proteinases from Barley (Hordeum Vulgare L.) and Cardoon (Cynara Cardunculus L.)

“…In plants, an inserted sequence of approximately 100 amino acids separates the homologous aspartic proteinase sequences into two regions (Runeberg-Roos et al, 1991;Cordeiro et al, 1994;Asakura et al, 1995). Although this plant-specific insert Correspondence to J. Costa, Instituto de Tecnologia Quimica e BioIbgica, Universidade Nova de Lisboa, Apart.…”