C1A cysteine-proteases and their inhibitors in plants.

Plant cysteine-proteases (CysProt) represent a well-characterized type of proteolytic enzymes that fulfill tightly regulated physiological functions (senescence and seed germination among others) and defense roles. This article is focused on the group of papain-proteases C1A (family C1, clan CA) and their inhibitors, phytocystatins (PhyCys). In particular, the protease-inhibitor interaction and their mutual participation in specific pathways throughout the plant's life are reviewed. C1A CysProt and PhyCys have been molecularly characterized, and comparative sequence analyses have identified consensus functional motifs. A correlation can be established between the number of identified CysProt and PhyCys in angiosperms. Thus, evolutionary forces may have determined a control role of cystatins on both endogenous and pest-exogenous proteases in these species. Tagging the proteases and inhibitors with fluorescence proteins revealed common patterns of subcellular localization in the endoplasmic reticulum-Golgi network in transiently transformed onion epidermal cells. Further in vivo interactions were demonstrated by bimolecular fluorescent complementation, suggesting their participation in the same physiological processes.

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.

Salivary (SD-type) cystatins: over one billion years in the making--but to what purpose?

Human saliva contains relatively abundant proteins that are related ancestrally in sequence to the cystatin superfamily. Most, although not all, members of this superfamily are potent inhibitors of cysteine peptidases. Four related genes have been identified, CST1, 2, 4 and 5, encoding cystatins SN, SA, S, and D, respectively. CST1, 4, and probably CST5 are now known to be expressed in a limited number of other tissues in the body, primarily in exocrine epithelia, and the term SD-type cystatin is more appropriate than 'salivary cystatin'. These genes are co-ordinately regulated in the submandibular gland during post-natal development. The organization of these tissue-specifically-expressed genes in the genome, and their phylogeny, indicate that they evolved from an ancestral housekeeping gene encoding the ubiquitously expressed cystatin C, and are members of a larger protein family. Their relationship to rat cystatin S, a developmentally regulated rodent submandibular gland protein, remains to be established. In this review, the evolution of the SD-type cystatins in the cystatin superfamily, their genomics, expression, and structure-function relationships are examined and compared with known cystatin functions, with the goal of providing clues to their biological roles.

Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism.

In the tropical species Carica papaya, the articulated and anastomosing laticifers form a dense network of vessels displayed in all aerial parts of the plant. Damaging the papaya tree inevitably severs its laticifers, eliciting an abrupt release of latex. Besides the well-known cysteine proteinases, papain, chymopapain, caricain and glycyl endopeptidase, papaya latex is also a rich source of other enzymes. Together, these enzymes could provide an important contribution to plant defence mechanisms by sanitising and sealing the wounded areas on the tree.

Structural and functional roles of asparagine 175 in the cysteine protease papain.

The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175-->Gln and Asn175-->Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.

Identification of the murine coronavirus p28 cleavage site.

Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p28. Single-amino-acid substitutions of other residues between P7 and P2' were generally permissive for cleavage, although a few changes did greatly reduce proteolysis. The relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.

Could proteolytic enzyme modulate the interaction platelets/vessel wall in presence of ASA at ultra low doses?

Acetylsalicylic acid (ASA) is known to act on platelets and vessel walls. At ultra low doses it reverses the inhibitory effects produced by a vascular fragment. Use of papain on normal platelets in vitro led to the appearance of platelet aggregation without collagen induction with a range of 20.25 +/- 28.91%. In the presence of vascular fragments (without ASA), this "spontaneous" aggregation remained but was reduced (13.26 +/- 27.73%). This effect was reversed by ASA treatment (29.41 +/- 24.17%). Reversion of vascular inhibition by ASA was not modified by papain.