Eur. J. Biochem. 228, 456-462 (1995) 0 FEBS 1995

Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds Claudia BECKER', Andrei D. SHUTOV3, Van Hai NONG'.', Vitalyi I. SENYUK;, Rudolf JUNG4, Christian HORSTMANN', Jiirgen FISCHER', Niels C. NIELSEN4 and Klaus MUNTZ'

' Institut fur Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany Institute of Biotechnology, National Center for Natural Science and Technology, Hanoi, Vietnam Laboratory of Protein Chemistry, Moldavian State University, Kishinev, Moldova, CIS, Czech Independant States Department of Agronomy, Purdue University, West Lafayette, USA

(Received 28 November 1994) - EJB 94 1825/2

Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapaptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyle- don extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development.

cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precur- sor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn- specific enzyme from the developing castor bean endosperm which is thought to catalyze the post-transla- tional processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for protein- ase B.

Keywords. Seed development and germination ; asparagine-specific endopeptidase ; cDNA ; Kcia sativa L.

Asn-specific endopeptidases are involved in proteolytic pro- cesses during different stages of plant development. During seed development, they are thought to catalyze final steps in the pro- teolytic processing of several seed storage proteins. In the case of 11s globulins, which are major storage proteins of dicotyle- donous seeds [I] and oat [2], pro-globulin trimers are finally processed by a single proteolytic cleavage at the C-side of an exposed Asn residue located at the C-terminal end of each sub- unit a-chain [3, 41. This maturation cleavage is a prerequisite for the final assembly of 11s globulins into hexamers and for the deposition of storage globulins in protein bodies of develop- ing storage tissue cells [5]. Asn-specific endopeptidases have been isolated from mature castor beans [6, 71, developing [8] and mature soybeans [9, 101 and mature jack beans [Il l . The purified enzymes from soybean display strong differences in their molecular masses and primary structures which suggest that there are a number of Asn-specific proteinases in the devel-

Correspondence to C. Becker, Institut fur Pflanzengenetik und Kul- turpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany

Fax: +49 39482 5 366. Abbreviation. E64, trans-epoxysuccinyl-~-leucylamido(4-guanidino)-

butane. Note. The novel nucleotide sequence data published here have been

submitted to the EMBL Data Library and are available under accession number 234899.

oping seed. Only three corresponding cDNA sequences from de- veloping castor beans [12], developing soybeans [lo] and devel- oping jack beans [13] have been so far cloned. As their analysis revealed, they all encode the same type of enzyme without any sequence similarity to the commonly known cysteine protein- ases.

During seed germination, exhaustive proteolysis of the stor- age globulins occurs to supply the growing seedling with re- duced nitrogen necessary for growth and development [14]. It has been demonstrated that major enzymes involved in storage protein degradation belong to the class of cysteine proteinases [15, 161. In germinating vetch (Vicia sativa L.) seeds, the occur- rence of four different cysteine proteinases has been reported. Proteinase A [17], CPRI and CPR2 [I81 are papain-like cysteine proteinases, whereas proteinase B was suggested to be a cysteine proteinase with some atypical characters [16]. Purified protein- ase B has been shown to cleave peptide bonds at the C-side of Asn residues in both the chains A and B of insulin [19]. The occurrence of Asn-specific endopeptidases have also been re- ported from germinating Vigna aconitifolia [20] and Phaseolus vulgaris [21] seeds. Their function during germination is still unknown. However, in vitro experiments suggest that proteinase B does not act on 11s proteins of ungerminated seeds but hydro- lyzes the proteins after an initial modification by a papain-like

Becker et al. (Eur: J. Biochem. 228) 457

cysteine proteinase or another proteolytic enzyme ([I61 ; Becker et al., unpublished results). Due to this initial modification Asn residues should become accessible to the action of proteinase B. Until now, no data concerning the primary structure of Asn- specific proteinases from germinating seeds have been obtained. However, the reported processing enzymes and proteinase B ex- hibit similar molecular masses, pH optima and cleavage speci- ficity. Therefore, we suppose, that they may represent members of one subclass of cysteine proteinases encoded by a gene fam- ily. The expression of their genes should be highly regulated since different forms of these cysteine proteinases act on storage globulins at different stages of plant development, i.e. embryo- genesis and germination.

In this publication, proteinase B is characterized in more de- tail and the primary structure of the enzyme is presented. The cDNA-derived polypeptide sequence as well as cleavage analy- ses with octapeptides and pro-legumin as substrates revealed that the developmentally regulated proteinase B from germinating vetch seeds belongs to a novel group of Asn-specific cysteine endopeptidases closely related to similar enzymes recently re- ported from developing dicotyledonous seeds.

MATERIALS AND METHODS

Chemicals. Columns and media for chromatography were obtained from Pharmacia. [a-3ZP]dCTP (6000 CUmmol) was purchased from Amersham International. Oligonucleotides were synthesized on an A394 DNA/RNA synthesizer (Applied Bio- systems). Synthesis of octapeptides was camed out using an ABI model 430A peptide synthesizer. PCR was performed on a thermal cycler (Grant) using a GenAmp kit (Perkin-Elmer Cetus). All other chemicals were obtained commercially and were analytical grade.

Plant material. Vetch seeds [K sativa L., cv. cosentini (Guss) Arcang] were germinated in the dark at 25°C. After 8 days, cotyledons were harvested for purification of proteinase B. For cDNA cloning, Northern-blot analysis and immunoblotting samples were taken at different times of germination. To obtain developing seeds, plants were grown in the greenhouse under conditions of 18 h light at 20°C. Harvested tissues were frozen in liquid nitrogen and stored at -70°C until use.

Protein purification. All steps were performed at 4°C. Cot- yledons were homogenized in 7 ml cold H,0/1 g cotyledon fresh matter containing 2 mM dithiothreitol. Insoluble parts were re- moved by repeated centrifugation (20 min, 13000Xg). The su- pernatant was subjected to isoelectric precipitation at pH 4.0 by adding 0.05 M HCl. After removal of the precipitated proteins, the supernatant was loaded onto a carboxymethyl-Sepharose CL-6B column (1.6 cmX40 cm) equilibrated with 0.01 M so- dium acetate, pH 4.0. During subsequent washing of the column with 0.01 M sodium acetate, pH 4.9 (flow rate 164 mVh), the pH increased and proteinase B was eluted in the range pH 4.4- 4.7. Active fractions of carboxymethyl-Sepharose chromatogra- phy were pooled, adjusted to pH 6.5 and applied (flow rate 30 ml/h) to a DEAE-Sepharose C1-6B column (0.9 cmX3 cm), equilibrated with 37 mM sodium phosphate, pH 6.5, containing 0.04 M NaC1, 1 mM EDTA and 12 mM 2-mercaptoethanol. A linear gradient of 0.04-0.4 M NaCl in 37 mM sodium phos- phate, pH 6.5 (flow rate 13.2 mVh) was used for elution.

Enzyme assay. Proteinase activity was assayed using casein as a substrate and 2,4,6-trinitrobenzenesulphonic acid for quanti- fication of amino nitrogen release [19]. The reaction was per- formed at pH 5.6 and at 30°C. One unit was defined as the amount of enzyme needed to liberate 1 pmol amino groups/min. To investigate the effects of various inhibitors on proteinase B,

aliquots of the purified enzyme without addition of 2-mercapto- ethanol or dithiothreitol were incubated with an appropriate in- hibitor for 30 min at 30°C.

SDWpolyacrylamide gel electrophoresis. Analytical gel electrophoresis was performed according to Laemmli [22] in 12.5 % (masshol.) polyacrylamide gels. Proteins were visualized after Coomassie blue staining.

To identify protease bands, gel slabs containing 0.05% (masdvol.) gelatin were used. After electrophoresis, the gel was washed with water twice for 5 min to remove the Laemmli buffer from the surface and was incubated in the assay buffer (McIlvaine, pH 5.6 with the addition of 180 mM NaCl and 2 mM dithiothreitol) overnight at 30°C. After incubation, the proteins were fixed with 10% trichloroacetic acid, stained with Coomassie blue for 2 hours and afterwards destained to visualize active bands.

In vitro cleavage assays. The peptides ETRNGVEE, ETRDGVEE, ETRQGVEE, ETRNIGEE and ETRNEVEE were used to test the cleavage specificity of the proteinase B prepara- tion according to Scott et al. [8]. Each digestion reaction was carried out for 1.5 h at 30°C in 0.01 M citrate/phosphate, pH 5.0, that contained 2 mM dithiothreitol, one of the substrates at 0.1 mg/ml and 1 p1 of the purified proteinase B. TLC analysis of the digestion products was performed on cellulose plates (Al- drich) developed in 60% (by vol.) 2-propanol and visualized by spraying with 0.5 % ninhydrin in acetone.

Pro-legumin B4 subunits were synthesized in Escherichia coli. After solubilization of the inclusion bodies and subsequent dialysis, a part of the pro-legumin B4 subunits assembled into trimers [23]. 1 pl substrate (10 g/l) was incubated with 1 pl pro- teinase B for 3 h at 30°C. The products were separated by SDS/ PAGE.

Production of antibodies and immunoblot analysis. Ap- proximately 1 mg purified proteinase B was subjected to SDS/ PAGE. The two closely migrating proteinase B bands were cut from the gel and eluted using an electro eluter (Bio-Rad). The denatured protein was used to raise antibodies in a rabbit.

Samples for western-blot analyses were obtained by homog- enizing 500 mg cotyledons in 3 ml 37 mM sodium phosphate, pH 6.5. The extracts were centrifuged twice for 15 min at 13000 rpm, 4"C, and the supernatants were used for SDS/ PAGE. The proteins were transferred electrophoretically to a ni- trocellulose membrane (Serva). Blots were blocked overnight in 0.1 M Tris/HCI, 0.15 M sodium chloride, pH 8.0, containing 0.5 % blocking reagent (Boehringer) and 0.05 % Tween 20. The specific antibody raised against proteinase B was diluted 1 : 8000, the anti-IgG, rabbit alkaline-phosphatase conjugate (Serva) was used at a dilution of 1:6000. Staining of the blots was performed with nitro blue tetrazolium chloride/5-bromo-4- chloro-3-indolylphosphate according to the manufacturers proto- col (Promega).

Peptide sequencing. N-terminal Edman degradation of the blotted proteinase B was performed with a Beckman sequencer, LF3400, using a special procedure for sequencing samples on poly(viny1idene difluoride)-type membranes (Beckman proto-

Isolation of total RNA. 300 mg cotyledons were homoge- nized with liquid nitrogen, then in three volumes extraction buffer (100mM Tris/HCl, pH9.5, 100mM NaCI, 2% SDS). The homogenate was extracted twice with phenolkhloroform (1:l) and the RNA from the aqueous supernatant was precipi- tated with one volume 4 M LiCI. After reprecipitation, the puri- fied RNA was dissolved in Et,PyrCO,-treated water giving a final concentration of 1 pg/pl.

cDNA cloning procedure. Poly(A)-rich RNA was purified according to standard protocols [24] and cDNA synthesis was

cols).

458 Becker et al. ( E m J. Biochern. 228)

Table 1. Purification of vetch proteinase B. Proteinase B was from 7 g dry cotyledons. The protein concentrations were measured by the method of Bradford [27] using a Bio-Rad protein assay kit and bovine serum albumin as a standard. n.d., not determined.

Volume Protein Activity Specific Yield Purifi- activity cation

ml mg units unitslmg % -fold - - Crude extract 61.5 178.0 n.d. n.d.

Carboxymethyl-Sepharose 24.9 0.379 1.83 4.83 89.3 86.4 DEAE-Sepharose 15.3 0.261 2.36 9.04 115 162

Isoelectric precipitation 81.4 36.7 2.05 0.0559 ‘I 00 1

performed using a First Strand Synthesis kit (Stratagene). A de- generate oligonucleotide primer [GA(TC)GA(TC)TT(TC)- GA(AG)GGIACIA(CG)ITGGGC] was synthesized on the basis of the N-terminal amino acid sequence (DDFEGTRWA) of pro- teinase B and used in combination with oligo(dT) to amplify partial proteinase B cDNA. The PCR fragments (1.65 kb) were directly cloned into the pCRII vector (Invitrogen). Two clones were sequenced on both strands by an ALF automated DNA sequencer (Pharmacia) and a sequence-specific oligonucleotide primer (TTAAACAATAGTTGTATGAATAGCATATCC) from the region just before the poly(A) site was designed in the anti- sense orientation to obtain full-length cDNA. A second cDNA library was synthesized by means of a 5’RACE System (Gibco BRL) and dC tailed. Using an oligo(dG) primer in combination with the proteinase-B-specific antisense primer, 1.8-kb DNA fragments were amplified and cloned. Five full-length cDNA clones of proteinase B were sequenced on both strands. Analysis of the sequence data was performed by means of PC/GENE soft- ware.

Northern-blot analysis. Total RNA was prepared from cot- yledons of appropriate germination or developmental stages as described above. Samples of 5 pg total RNA were denatured with glyoxal [24], electrophoresed in a 1.2% agarose gel and transferred to a nitrocellulose filter (MSI). Hybridization was carried out overnight at 65°C in 10 ml solution containing 6X NaCVCit (NaCVCit is 0.15 M NaC1, 15 mM sodium citrate, pH7.0), 0.5 % SDS, 5 XDenhardt’s solution, 0.5 % blocking rea- gent (Boehringer Mannheim) and a randomly primed and 32P- labeled (Megaprime DNA labelling systems, Amersham) pro- teinase B cDNA fragment as probe. The membranes were washed twice with 2XNaCl/Cit, 0.1 % SDS at 65°C for 1 h, fol- lowed by several washings with OSXNaCl/Cit, 0.1 % SDS under the same conditions. Membranes were exposed to X-ray film at -70°C for 1-3 days. Northern-blot analysis of the different tissues was performed as described above except that 10 pg total RNAs were used for electrophoresis and the hybridization tem- perature was 55°C.

Southern-blot analysis. Genomic DNA was isolated from roots and shoots of young seedlings by the method of Miller et al. [25] adapted for plant material. After homogenization of 300 mg plant material in liquid nitrogen, 3 ml lysis buffer (TNE = 10 mM Tris/HCl, pH 8.0, 0.1 M NaCl, 10 mM EDTA), 800 p1 10% SDS and 20 pl proteinase K (25 mg/ml) were added. The sample was incubated at 56°C for 2 h. Proteins and some of the carbohydrates were precipitated with 1 ml saturated NaCl and removed by centrifugation (10 min, 13000Xg). The geno- mic DNA was precipitated with ethanol from the supernatant. Purified DNA was treated with RNase in order to remove con- taminating RNA. Samples of 10 pg genomic DNA were digested with appropriate restriction enzymes and the products were sepa- rated by electrophoresis on a 0.8% agarose gel, transferred to a nylon membrane (Nytran-N, Schleicher & Schuell) and hybrid-

ized as described for Northern-blot analysis. Autoradiography was performed at -70°C for 7 days with an intensifying screen.

RESULTS

Purification and characterization of proteinase B. Proteinase B was purified to homogeneity by only three purification steps (Table 1). Since no specific method to measure only proteinase B activity was available, no corresponding value for crude ex- tracts is given. Isoelectric precipitation at pH 4.0 removed 80% of other proteins including other proteinases. The supernatant of isoelectric precipitation was directly loaded onto carboxymethyl Sepharose CL-6B. Proteinase B was eluted as a hell-defined peak in the pH range 4.4-4.7. The pooled peak fractions con- tained three dominant proteins with molecular masses 39, 37 and 28 kDa as observed in SDSPAGE (not shown). Anion-exchange chromatography was used as the final purification step. An ali- quot of the pooled fractions from the DEAE-Sepharose CL-6B chromatography was subjected to SDSPAGE. Two closely mi- grating proteins with molecular masses of 39 kDa and 37 kDa were detected, which we named proteinases B1 and B2 (Fig. 1A). We failed to separate these two proteins chromato- graphically. During DEAE-Sepharose chromatography, an acti- vation of proteinase B took place which was reproducibly ob- tained. The specific activity of the final preparation against ca- sein was determined to be 9 U/mg protein.

The proteolytic activity of proteinase B bands became visible after SDSPAGE on gel slabs containing gelatin with subsequent incubation at appropriate conditions (Fig. 1 B). Dependent on the presence or the absence of 2-mercaptoethanol during SDS treat- ment of the samples, either proteinase E l or proteinase B2 only displayed activity. This phenomen was not further studied. How- ever, the presence of 2-mercaptoethanol changed the conforma- tion of proteinases B1 and B2, which resulted in lower mobili- ties of both bands in SDS/polyacrylamide gels and it seems to be critical for activity of proteinase B2. Proteinase B1 was only active if 2-mercaptoethanol was omitted.

The total enzyme preparation is activated by dithiothreitol and 2-mercaptoethanol and strongly inhibited by N-ethylmalei- mide and iodoacetate. At the inhibitor concentrations of 0.1 mM, residual activities of proteinases B were 4% and 18%, respec- tively. No inhibition took place by pepstatin A (0.005 mM), EDTA (1 mM) and phenylmethylsulfonyl fluoride (1 mM). These results indicate that proteinase B belongs to the class of cysteine proteinases. However, trans-epoxysuccinyl-L-leuc ylam- ido(4-guanidino)butane (E64) only weakly acts on this protein- ase. Even at a concentration of 0.1 mM, 91% of proteinase B activity was retained. A similar weak sensitivity to E64 was also reported for asparaginyl endopeptidase from mature jack bean [26], indicating that both enzymes are, at least, atypical cysteine proteinases or even belong to another protease group.

A

Becker et al. ( E m J. Biochern. 228)

B

459

1 2 M 3 4 1 2 M 3 4

Fig.1. SDSPAGE of purified proteinase B. (A) Without gelatin and (B) with gelatin in the slabs. SDS treatment of the protein samples was carried out either at 80°C or at room temperature. Lane 1, denatured sample + 2-mercaptoethanol; lane 2, native sample +2-mercaptoethanol; lane 3, native sample - 2-mercaptoethanol; lane 4, native sample +2-mercaptoethanol; M, molecular-mass marker. Unheated proteinase B preparation tends to undergo autolysis during SDS treatment.

1 2 3 4 5 6 7 8

Fig. 2. Digestion of the octapeptides ETRNGVEE, ETRDGVEE, ETRQGVEE, ETRNIVEE and ETRNEVEE by protease B. The

2 3 4 5 6 m 8 h 2 4 5 6 7 8 1 0 1 2 1 5

cotyledon size days after imbibition (DAI)

spots were identified by comparing their mobilities and staining charac- teristics to corresponding chemically synthesized peptides. Digestion of ETRNIGEE (lane I), ETRNGVEE (lane3), ETRQGVEE (lane 3, ETR- NEVEE (lane 7) and ETRDGVEE (lane 8). Undigested substrates ETRNGVEE (2), ETRQGVEE (4) and ETRNEVEE (6).

Fig. 3. Immunoblot analyses of proteinase B during development and germination of vetch seeds. Cotyledons of different developmental stages were homogenized in equal amounts of 37 mM sodium phosphate, pH 6.5. Numbers indicated the sizes (in mm) of the cotyledons during seed development and the different germination days, respectively. m = mature seeds.

Analysis of cleavage specificity of proteinase B. The octapep- tide ETRNGVEE contains four amino acids on either side of the Asn-Gly peptide bond which represents the cleavage site in G2 glycinin. In ETRDGVEE and ETRQGVEE, the Asn residue was replaced by Asp and Gln, respectively. Fig. 2 shows the diges- tion products separated by TLC. The peptide ETRNGVEE was cleaved most efficiently. The tetrapeptides ETRN and GVEE appeared as final reaction products. Although with lower ef i - ciency, the proteinase B preparation also cleaved the peptide ETRDGVEE at the C-terminal side of Asp and the peptides ETRNIGEE and ETRNEVEE. No cleavage was found when Asn was replaced by Gln.

In preliminary experiments, we investigated the action of proteinase B on pro-legumin B4 formed after expression of the corresponding cDNA in E. coli. The results indicated that pro- teinase B is capable to catalyze the correct post-translational cleavage of V fubu pro-legumin B4 in vitro (not shown). The proteinase cleaves the peptide bond between Asn and Gly which links the C-terminal end of the acidic a-chain to the N-terminus of the basic P-chain of legumin as confirmed by microsequenc- ing.

N-terminal peptide sequencing. To determine the N-terminal amino acids of the purified components B1 and B2, aliquots of

8 h 1 2 3 4 5 6 7 8 1 2 1 5

days after imbibition (DAI)

Fig.4. Northern-blot analysis of proteinase B mRNA during seed germination. Total RNA from cotyledons of different germination days were prepared. 5 pg were denatured by glyoxal and electrophoresed on an 1.2% agarose gel [24]. The numbers indicate the different days after imbibition (DAI).

the enzymes were subjected to SDSPAGE and blotted onto a nitrocellulose membrane. After staining, the 37-kDa and the 39- kDa bands were excised from the blot and used for automated amino acid sequencing. The N-terminal amino acid of mature

460 Becker et al. (Eul: J. Biochem. 228)

GGATTCCCTTCGATCATGGGCTCTTCTCAACTCTCCACTCTTCTCTTTTTCACCATCGTCGTCACCTTCCTCACCGTCGTCTCCAGCGGC M G S S Q L S T L L F F T I V V T F L T V V S S A G CGTGATCTCCCCGGAGACTATCTCCGATTGCCTTCTGAAACCTCCAGATTCTTCCGTGAACCTAAAAACGATGACGACTTTGAGGGAACT

R D L P G D Y L R L P S E T S R F F R E P K N D D D F E G T A A-ATTTTACTAGCTGGTTCTAATGGTTACTGGAATTATAGACATCAGTCTGATGTTTGTCATGCGTATCAATTACTGAGGAAA R.AI. A G S N G Y W N Y R H Q S D V C H A Y Q L L R K

GGTGGTTCGAAGGAAGAAAATATTATTGTTTTCATGTATGATGATATTGCTTCCAATGAAGAGAATCCAAGGCCTGGTGTCATAATTAAC G G S i l K k E E N I I V F M Y D D I A S N E E N P R P G V I I N

AAACCTGATGGGGATGATGTTTATGCAGGAGTTCCAAAGGATTATACTGGTGCAGAAGTACATGCGGACAATTTCTATGCTGCTTTACTT K P D G D D V Y A G V P K D Y T G A E V H A D N F Y A A L L

GGAAATAAATCAGCTCTTACAGGTGGGAGTGGGAAAGTTGTGGATAGTGGTCCCAATGATCATATTTTTGTATACTACACTGATCATGGT G N K S A L T G G S G K V V D S G P N G H I F V Y Y T D H G

GGTCCAGGGGTTCTTGGTATGCCTGTTGGTCCTTACTTGTATGCATCTGATCTGAATGAAGTCTTGAAGAAAAAGCATGCTTCTGGAACA G P G V L G M P V G P Y L Y A S D L N E V L K K K H A S G T

TATAAGAGCCTAGTATTTTATCTAGAGGCATGTGAATCTGGGAGTATCTTTGAAGGTCTTCTTCCAGATGATCTCAATATCTACGCGACA Y K S L V F Y L E A C E S G S I F E G L L P D @ D L N l Y A T

ACGGCTTCAAATGCAGAAGAAAGCAGTTGGGGATACTATTGCCCTGGGGATAAACCTCCCCCACCCCCAGAGTACTCAACCTGTTTGGGT T A S N A E E S S W G Y Y C P G D K P ’ P P P P E Y S T C L G

GACCTATACAGTATTGCTTGGATGGAAGACAGTGAAGTACACAATTTGCAAACTGAAAGTTTGCAACAACAATATAAATTGGTTAAGAAT D L Y S I A W M E D S E V H N L Q T E S L Q Q Q Y K L V K N

AGGACTATTAGTGAACCATATGGATCTCATGTGATGGAATATGGTGATATAGGTCTTAGCAAAAATGATCTCTACCAATATTTGGGTACA R T I S E P Y G S H V M E Y G D I G L S K N D L Y Q Y L G T

AATCCTGCCAATGATAATAATTCCTTTGTGGACGAAACTGAAAACTCCTTGAAATTGAGAACACCTTCAGCCGCAGTCAATCAAAGGGAT N P A N G N N S F V D E T E N S L K L R T P S A A V N Q R D

GCTGATCTCATCCATTTCTGGGAAAAGTTCCGCAAAGCACCTGAGGGTTCTTCGCAGAAAAACGAAGCTGAGAAACAAGTTTTGGAAGCA

T G

A

T

A

A D L I H F W E K F R ~ K A P E G S S Q K N E A E K Q V L E A A ATGTCTCACAGGAAGCATATAGACAACAGTGTGAAACTGATTGGGCAGCTCTTATTTGGCATTGAAAAGGGTACTGAACTGCTCGACGTT

M S H R K H I D N S V K L I G O L L F G I E K G T E L L D V

GTTAGACCTGCTGGATCACCCCTTGTTGATAACTGGGACTGCCTCAAAACCATGGTAAAGACTTTTGAGACACACTGTGGATCCCTATCT V R P A G S P L V D N W D C L K T M V K T F E T H C G S L S

CAGTATGGTATGAAACATATGAGGTCTTTTGCGAACATCTGCAATGCAGGAATACCGAATGAGCCAATGGCCGAGGCCTCAGCACAAGCT

A

Q Y G M K H M R S F A N I C N A G I P N E P M A E A S A O A

TGTGCCAGTATTCCTGCCAACCCCTGGAGTTCTCTGCAAGGAGGTTTCAGTGCATAGTCTCTAAAATGCGCACTTTGTATAAACCATGTP C A S I P A N P W S S L Q G G F S A

CAACTGCTGAACATTGGTCATGATCTCATGTCACGCTTCTGTAAAAATATAATTGGGACACCACTAAGACATGGAGGAATAAGATTTCTC TCCATTTATGTATTATTTCAAGAAAAATAATTTTTAGATGGGAAAAATACAAATATGGCAGGAAAAGTTGTATAGAGTCAAAGGGCAATl GGCAGATAGGAGCTACCCTAGACACTTTGCAAAATACATAATTGGCAACTCTTTGTATAATTATTTTAGTTGGATATGCTATTCATACA~ CTATTGTTTAATATTAAAAAAAAAAAAAAAAAAA-

90 2 5

180 55

270 8 5

360 115

450 145

540 175

630 205

720 235

810 265

900 295

990 325

1080 355

1170 385

1260 415

1350 445

1440 475

1530 493

1620 1710 1800 1834

Fig. 5. Nucleotide and the deduced amino acid sequences of the cDNA clone pPB14 encoding proteinase B. Nucleotide mismatches found in other cDNA clones are: C-T at position 278 in pSK54 and pPB33 results in Ser+Leu; A-G at position 281 in pPB33 results in Lys-Arg; T-A at position 699 results in Asp-Glu; C-+T at position 775 results in P r w S e r and G-A at position 1112 results in Arg-His. The last three mismatches were all found in pSK54 and pPR25. The three mismatches in the coding region which do not cause an amino acid substitution are T-C at position 531 and C-T at position 528 in pPR25, and C-A at position 1281 in pSK54 and pPR25. The long horizontal arrows indicate oligonucleotide primer positions. Arrows indicate putative sites of co-translational cleavage of the signal peptide (A) and post-translational cleavage of pro-peptides (A). The amino acid sequence obtained from Edman degradation of the purified enzyme as well as a potential polyadenylation signal are underlined. The potential glycosylated sites are labeled by dots.

proteinase B is Asp. The N-terminal sequence of both compo- nents is DDDFEGTRWAILLAGS. This sequence failed to dis- play any sequence identity to common papain-like cysteine pro- teinases but is highly similar to the N-terminal amino acid se- quences of the reported asparaginyl-specific endopeptidases from jack bean [ll] and castor bean [12] seeds.

Immunoblot analysis. Antibodies raised against both compo- nents of the proteinase B recognized two proteins with molecu- lar masses of 39 kDa and 37 kDa (Fig. 3), indicating that they are really present in total extracts of cotyledons from different germination stages and might be isoforms. As the proteinase B antibodies were raised against the purified enzyme from plant material, some faintly cross-reacting bands with plant proteins of higher molecular masses were obtained. Antibodies raised against recombinantly formed proteinase B should eliminate these cross reactions. This work is now in progress.

Proteinase B first became detectable at day 4 of germination. Its amount increased up to day 10. Appearance of the enzyme directly corresponds to the synthesis of proteinase B mRNA (Fig. 4).

Proteinase B antibodies strongly cross-reacted with a 35-kDa band which is detectable at middle to late stages of vetch seed development and remains detectable in cotyledon extracts up to day 4 of germination (Fig. 3).

Isolation and characterization of cDNA clones. To analyze the primary structure of proteinase B, we performed PCR cloning of its cDNA. Five full-length cDNA clones (pPB14, pPB22, pPR25, pPB33 and pSK54) were sequenced on both strands. They all contained the complete sequence coding for the peptide DDDFEGTRWAILLAGS known from the N-terminal sequenc- ing of the purified enzyme. 13 mismatches were found in the nucleotide sequences of the five clones; eight are located in the

Becker et al. ( E m J. Biochem. 228) 46 1

We suppose that at least some of them reflect different alleles. To examine the number of proteinase-B-specific sequences, ge- nomic DNA was isolated from young roots and shoots of vetch seedlings and digested with different restriction enzymes. The fragments were analyzed by Southern-blot hybridization with proteinase-B-specific cDNA. Four or five different hybridizing bands appeared in each restriction digest (Fig. 6). Most of them are much larger than the full-length cDNA, suggesting that there are at least two genes for proteinase B.

kb

-21.2

-5.2 -4.9 -3.5

-2.0

BamHI EcoFU EcoRV

Fig.6. Southern-blot analysis of K safiva genomic DNA. Genomic DNA was isolated from roots and shoots of seedlings. 10 pg were di- gested with either BarnHI, EcoRV or EcoRI. Autoradiography was per- formed for one week at -70°C.

coding region and five in the 3' untranslated region. Five mis- matches result in the amino acid substitutions highlighted in Fig. 5 , which shows the nucleotide and derived amino acid se- quences of clone pPB14. Proteinase B cDNA consists of an open reading frame of 1479 bp encoding a precursor polypeptide of 493 amino acids (M, = 54480). There exists a putative signal peptide, the cleavage site of which is predicted to be at the C- terminal site of Ser24 according to (-3, -1) rule (PC/GENE, subprogram PSIGNAL). 24 amino acid residues which seem to correspond to a propeptide of still unknown function are located between the cleavage site of the signal peptide and the N-termi- nal amino acid (Asp49) of mature proteinase B. The derived molecular mass from the N-terminus of the mature enzyme to the stop codon is 49089 Da. This is 10 kDa and 12 kDa larger than the 39 kDa and 37 kDa found for the purified enzyme. Therefore, a second post-translational modification has most likely to occur in the C-terminal region of the proteinase B pre- cursor. There are two potential glycosylation sites at Asn29.5 and Asn331. Until now we have no indication that the purified pro- teins are glycosylated.

Northern-blot analysis. To investigate expression of the pro- teinase-B-specific gene(s), Northem-blot analysis was per- formed with total RNA from cotyledons of germinating seeds at different stages. First transcripts appeared at day 3 of germina- tion, indicating that proteinase B is de novo synthesized (Fig. 4). Expression of the gene(s) reached a maximum at day 8. Under less stringent hybridization conditions, e.g. lower temperature, transcripts of similar size were detected in developing seeds and, in the seed coat at middle to late stages of seed development, in axes, leaves and roots (not shown). This finding supports the idea that Asn-specific enzymes with similiar structure occur widely in the plant.

Southern-blot analysis. Sequence analyses of different protein- ase B cDNA clones revealed single nucleotide mismatches or substitutions, some of which result in amino acid substitutions.

DISCUSSION

The results describe the purification and molecular charac- terization of proteinase B from germinating vetch seeds. The purification resulted in two proteins with molecular masses of 39 kDa (Bl) and 37 kDa (B2), both of which had the same N- terminal amino acid sequence. The two components displayed enzymic activity against gelatin, as shown by activity staining in SDS/polyacrylamide gels. Since the proteins were both detec- table in cotyledon extracts, isoforms are apparantly present in the germinating seed. We suppose that the hypothesis that these isoforms might represent either differences in the electric charge of the proteins or in the glycosylation patterns.

The cleavage specificity of proteinase B was compared with that observed with a high molecular-mass protease purified from developing soybeans [ 81. Both enzyme preparations correctly and efficiently cleaved the octapeptide ETRNGVEE, which con- tained the four amino acids on either side of the conserved post- translational cleavage site in G2 proglycinin. The two enzyme preparations also cut octapeptides in which the Asn residue was replaced by Asp, although reactions with this substrate did not proceed as efficiently as in the case of the octapeptide that con- tained the conserved Asn-Gly bond. Substitution of Gln for Asn on the N-terminal side of the cleavage site eliminated activity for both types of enzymes. Differences between the two prepara- tions became visible when analyzing cleavage of ETRNIGEE. Proteinase B cleaved this substrate at the C-terminal side of Asn, whereas no cleavage took place by the high molecular-mass pro- tease from soybean. In addition, both enzyme preparations were able to carry out the correct cleavage of subunits of pro-legumin B trimers into acidic and basic chains.

cDNA sequence analyses revealed that proteinase B must be synthesized as a precursor molecule comprising a putative signal peptide of 24 amino acids, an interspaced propeptide of 24 amino acids and the sequence for a 49-kDa product which is larger than the purified proteinases B1 and B2. The cDNA-de- rived amino acid sequence of an Asn-specific proteinase from castor bean also displayed synthesis of a larger pre-propolypep- tide. Autocatalytic processing in the C-terminal region was pro- posed to convert the inactive precursor molecule into the active proteinase [12]. We found, in the C-terminal part of the protein- ase B sequence, two Asn residues (Asn394 and Asn376) which might determine cleavage sites to remove C-terminal peptides. The presumptive processing products (38564 Da and 36447 Da) would display molecular masses similar to those of the purified proteinase B1 (39 kDa) and B2 (37 m a ) .

The presence of a signal peptide indicates that the proteinase B precursor is synthesized at the rough endoplasmic reticulum and enters the endomembrane system. In addition, it can be an- ticipated that the enzyme acts on its substrate inside the protein storage vacuoles where it has presumably to be transported in an inactive state. Therefore, one might speculate that the 24 amino acids N-terminal to the mature protein might stabilize the proteinase B precursor against premature activiation by the assumed autolysis in the C-terminal region. Since the N-terminal

462 Becker et al. (Eul: J. Biochem. 228)

amino acid of the purified proteinases B1 and B2 was shown to be Asp49, autocatalytic cleavage at Am48 could take place to remove this interspaced sequence. Preliminary results on heter- ologous expression of the proteinase B1-specific cDNA in dif- ferent E. coli strains indicated that mature proteinase B (Mr = 38.5 j is very unstable. Expression products of different sizes, presumably due to autolysis, were only detectable by imrnu- noblotting (results not shown).

The primary structure analysis of proteinase B displayed 59 % sequence identity to the vacuolar Asn-dependent proteinase from developing castor bean endosperm [12] which is assumed to also be involved in the maturation of 11s globulins. We have used our proteinase B antibodies to investigate whether there is a corresponding proteinase in developing vetch seed. The cross- reacting 35-kDa protein appeared during the middle and late stages of seed development and remained detectable up to day 4 of germination. As this synthesis profil is very similar to those described for the enzymes in developing castor bean [12] and jack bean [13], we suppose that the 35-kDa protein might be an Asn-specific proteinase from developing vetch seeds. Whether these Asn-specific proteinases in developing and germinating seeds with molecular masses of 35-40 kDa are isoenzymes with different functions but encoded by a small gene family whose expression is differentially regulated remains to be further inves- tigated. DNA probes encoding proteinase B revealed the pres- ence of a small gene family. The developmentally regulated ex- pression of proteinase B during germination has already been shown by Northern-blot analysis. Purification of the Asn-spe- cific proteinase from developing vetch seeds, cloning of the cor- responding gene(s) and promoter analyses are now under way to investigate in more detail expression and regulation of these similar enzymes involved in different physiological stages of plant development.

We are grateful to S. Konig for performing sequence analyses on the automated DNA-sequencer and R.-M. Gillandt for synthesis of oligonu- cleotides. This work was supported, in part, by grant Mu925/5-I from the Deutsche Fu~schungsgerneinschuft (DFG). A. D. Shutov (grant 436 Mol-17/4/93) and V. I. Senyuk (grant 436 Mol-17/3/93) wish to thank the DFG for support during their research stays in Gatersleben, Germany.

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35, 713-718.

1651 - 1659.

191-196.

26, 1207-1212.