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

Abstract

Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

Fig. 1

Identification of E-64-sensitive Z-Phe-Arg-MCA activity transiently expressed in the early phase of germinating embryo. (A) DEAE-cellulose column chromatography of daikon radish cotyledon extract at 1 and 5 days after imbibition. Extract of cotyledons (100 g) was applied to a DEAE-cellulose column (0.9 × 16 cm) equilibrated with buffer A [20 mM acetate buffer (pH 6.5) containing 1 mM β-mercaptoethanol and 0.5 mM EDTA] and washed with the same buffer. Oligopeptidase B was eluted with buffer A containing 50 mM NaCl (indicated by arrow), and RD21 and CBCP were eluted using a linear gradient of NaCl (0.05–0.5 M) in the same buffer. The elution starting points with buffer A containing 50 mM NaCl and the linear gradient of NaCl are indicated by arrows. (B) Sephacryl S-100 gel filtration of fractions I and II. Fractions I and II separated by DEAE-cellulose chromatography were applied to a Sephacryl S-100 column (2.0 × 100 cm) and eluted with buffer A containing 0.1 M NaCl. The column was calibrated using BSA (68 kDa), ovalbumin (45 kDa), soybean trypsin inhibitor (25 kDa) and cytochrome c (12 kDa).

Fig. 2

Identification of Z-Phe-Arg-MCA cleaving activity in fractions I and II. Fractions I (A) and II (B) obtained from DEAE-cellulose column chromatography were applied to a phenyl-Sepharose column and eluted using a linear gradient of ammonium sulfate (1-0 M) in buffer A as described in ‘Experimental procedures’ section. (C) The fraction with activity (indicated by a horizontal bar) was concentrated and incubated with cystatin-Sepharose. After washing with buffer A containing 0.5 M NaCl and 0.1% Triton X-100, the gel was rinsed with buffer A. Cystatin-Sepharose-bound proteins were eluted by SDS-heat treatment and then analysed by SDS–PAGE as described in ‘Experimental procedures’ section. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue (CBB) R-250. For sequence analysis, cystatin-Sepharose-bound proteins resolved by SDS–PAGE were electroblotted onto a PVDF membrane and stained with Ponceau 3R. The 26 kDa band of fractions I and II was subjected to peptide sequence analysis.

Fig. 3

Change in E-64-sensitive and E-64-insensitive Z-Phe-Arg-MCA cleaving activity in cotyledons during the germination of embryo. (A) Cotyledon (20 g) extracts prepared from germinating embryo at 8, 24, 36, 48, 72 and 144 h after imbibition were fractionated by DEAE-cellulose chromatography as described in ‘Experimental procedures’ section. Enzyme activity of unbound (circles) and bound (squares) fractions was assayed in the absence (opened circles, opened squares) or presence (closed circles, closed squares) of E-64. (B) Sephacryl S-100 gel filtration of Z-Phe-Arg-MCA cleaving activity in the DEAE-cellulose unbound fraction. Cotyledon (20 g) extracts prepared from germinating embryo at 24, 36, 48 and 72 h after imbibition were fractionated by DEAE-cellulose chromatography and the unbound fraction was applied to a Sephacryl S-100 column as described in ‘Experimental procedures’ section. (C) Expression profile of DRCP26 transcript during germination. Total RNA was isolated from daikon cotyledons at 0, 1, 2, 3, 4, 5, 6 and 8 days after imbibition and analysed by RT-PCR using DRCP26-specific primers as described in ‘Experimental procedures’ section. Actin-2 transcript was amplified as an internal control.

Fig. 4

Amino acid sequence alignment of the predicted DRCP26 catalytic domain (DDBJ accession number: AB721307) with sequences of Arabidopsis GCP1 (DDBJ accession number: AY043294) and Brassica COT44 (UniProt ID: P25251). Active sites (Cys, His and Asn) are boxed. Identical amino acid residues among all aligned sequences are indicated in bold type. Possible Cys residues involved in disulfide bridge are marked with closed circles.

Fig. 5

Purification of DRCP26 by Mono-Q column chromatography. DRCP26 was finally purified to homogeneity by Mono-Q column chromatography as described in ‘Experimental procedures’ section. Elution profile of protein (A) and Z-Phe-Arg-MCA cleaving activity (B). The fraction with activity indicated by a horizontal bar in (A) was subjected to SDS–PAGE (12% gel) and stained with CBB R-250 (C).

Fig. 6

Cleavage of glucagon, ACTH (1–24) and somatostatin-28 by DRCP26. Glucagon, ACTH (1–24) and somatostatin-28 were digested with DRCP26 and digests were analysed as described in ‘Experimental procedures’ section. Cleavage products and major cleavage sites are indicated. 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.

Fig. 7

Digestion of storage proteins by DRCP26. (A) Homogenate of daikon radish cotyledon at 14, 40, 52 and 72 h after imbibition was subjected to SDS–PAGE (10% gel) and stained with CBB R-250. The protein band (31 kDa) decreased during germination was indicated by arrow. (B) Digestion of storage proteins by DRCP26. Homogenate of daikon radish cotyledon at 14 h after imbibition was incubated with DRCP26 at 37°C for 24 h in the absence and presence of E-64 as described in ‘Experimental procedures’ section. After centrifugation, the reaction mixture (12 µg protein) was subjected to SDS–PAGE (12% gel) and stained with CBB R-250. Two protein bands (27 and 11 kDa) disappeared by the incubation with DRCP26 were indicated by arrows.