Papaya proteinase IV amino acid sequence

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.     

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.     

The thiol proteinases from the latex of Carica papaya L. II. The primary structure of proteinase omega

The complete primary structure of the proteinase omega isolated from the latex of the Carica papaya fruits is given. The polypeptide chain contains 216 amino-acid residues, the alignment of which was deduced from sequence analyses of the native enzyme, the tryptic, chymotryptic, peptic and thermolysinolytic peptides and facilitated due to the considerable degree of homology with papain and actinidin. The location of the three disulfide bridges could be established with the help of peptic and thermolysinolytic fragments. Proteinase omega shares 148 identical amino-acid residues (68.5%) with papain and 108 ones (50%) with actinidin, including the three disulfide bridges and the free cysteine residue required for activity, as well as most of the other amino-acid residues involved in the catalytic mechanism and two thirds of the glycine residues which are of structural significance. The homology with other cysteine proteinases of different origin is discussed.     

A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.

1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.     

Molecular cloning of cDNA for rat cathepsin C. Cathepsin C, a cysteine proteinase with an extremely long propeptide

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The deduced amino acid sequence of cathepsin C comprises 462 amino acid residues: 28 NH2-terminal residues corresponding to the signal peptide, 201 residues corresponding to the propeptide, and 233 COOH-terminal residues corresponding to the mature enzyme region. Four potential glycosylation sites were found, three located in the propeptide region, and one in the mature enzyme region. The amino acid sequence of mature cathepsin C has 39.5% identity to that of cathepsin H, 35.1% to that of cathepsin L, 30.1% to that of cathepsin B, and 33.3% to that of papain. Cathepsin C, therefore, is a member of the papain family, although its propeptide region is much longer than those of other cysteine proteinases and shows no significant amino acid sequence similarity to any other cysteine proteinase.     

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.     

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.     

The Arabidopsis Xylem Peptidase XCP1 Is a Tracheary Element Vacuolar Protein That May Be a Papain Ortholog

XCP1 is a xylem-specific papain-like cysteine peptidase in Arabidopsis. To determine whether XCP1 could be involved in tracheary element autolysis, promoter activity and localization of XCP1 were investigated using XCP1 promoter-beta-glucuronidase fusions and immunofluorescence confocal microscopy. A tracheary element expression pattern was detected for XCP1. Results from confocal microscopy and biochemical subcellular fractionation indicated that XCP1 was localized in the vacuole. Ectopic expression of XCP1 resulted in a reduction in plant size in some lines and early leaf senescence, as indicated by early loss of leaf chlorophyll. Reduced plant size was correlated with higher levels of XCP1, as shown by immunoblot and peptidase activity gel analyses. The XCP1 prodomain exhibits exceptionally high similarity (greater than 80%) to the prodomains of papain and other papain-like enzymes isolated from papaya (Carica papaya) laticifers when compared with all other reported papain-like enzymes. The potential for XCP1 and papain to perform common functions as catalysts of autolytic processing following cell death due to programmed suicide or to wounding is discussed.     

Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex

Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.     

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.     

Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana.

A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Chymopapain. Chromatographic purification and immunological characterization.

Chymopapain (EC 3.4.22.6) was purified from commercially available spray-dried latex of papaya (Carica papaya) fruit by (NH4)2SO4 fractionation and fast protein chromatography on the Mono S cation-exchange column. Multiple forms of chymopapain separated chromatographically were shown to be immunologically identical. A major form was isolated and found to be homogeneous by several criteria, and fully active, and its N-terminal amino acid was identified as tyrosine. Latex from fresh unripe papaya fruit contained predominantly one form of chymopapain, and it is concluded that chymopapain is a single enzyme distinct from the other cysteine proteinases of C. papaya latex.     

Papaya (Carica papaya) Lysozyme Is a Member of the Family 19 (Basic, Class II) Chitinases

The most comprehensive studies on a plant lysozyme (EC 3.2.1.17) are those on the enzyme from papaya (Carica papaya) latex, published in 1967 and 1969. However, the N-terminal amino acid sequence of five amino acid sequence of this enzyme, determined by manual Edman degradation, did not allow assignment to any of the much later-classified families of glycosyl hydrolases. N-Terminal sequence analysis of 22 residues of papaya lysozyme now shows unambiguously that the enzyme belongs to the family 19 chitinases. It has properties similar to those of basic class I chitinases with lysozyme activity, such as cleavage specificity at the C-1 of N-acetylmuramic acid with inversion of configuration, but as it lacks an N-terminal hevein domain, it should be classified as a class II chitinase. Received: 3 February 1999 / Accepted 25 July 1999     

Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2''-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely 'essential' for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

Purification and in situ immobilization of papain with aqueous two-phase system

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33-17.65% (w/w) PEG 6000, 14.27-14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain.     

Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin

The comparison of the amino acid sequences of 5 cysteine proteinases: papain, actinidin, rat cathepsins B and H and chicken cathepsin L, demonstrates a striking homology among their sequences. The N-terminal region (residues 1-70 in papain) and C-terminal region (residues 118-212 in papain) display the highest sequence homologies, whereas the lowest sequence homologies are observed in the middle region (residues 71-117 in papain); a segment where most insertions/deletions are observed. The highest sequence homology is observed between rat cathepsin H and chicken cathepsin L. As shown by X-ray studies, papain and actinidin have a clearly defined double domain structure. Each domain contains a core of non-polar side chains, which are retained in cathepsins B, H and L, except for the non-polar residue 203 of the core which is replaced by glutamic acid in cathepsin B. The percentage and the location of alpha-helix and beta-sheets of cathepsins B, H and L, assessed using the methods of Garnier et al. (1978, J. Mol. Biol. 120, 97-120) and Chou and Fasman (1974, Biochemistry 13, 222-245), show that the main ordered structures in papain and actinidin are probably retained in cathepsins B, H and L. The differences observed occur essentially in the middle region, a place where sequences display the lowest homologies and which is far removed from the active site.     

The thiol proteinases from the latex of Carica papaya L. I. Fractionation, purification and preliminary characterization

Three thiol proteinases, namely papain, chymopapain and proteinase omega were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as S-sulphenylthiosulfate derivative or after blocking with p-chloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase omega. The active sites of chymopapain and proteinase omega could not be distinguished from that of papain neither by the analysis of the pH dependence of kcat/Km nor by the examination of the pH dependence of the fluorescence emission spectra.