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PubMed1984

Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin.

Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine.

Journal Article↗
PubMed1999

Isolation and primary structure of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis hook.

The dried latex of the mountain papaya, Carica candamarcensis, was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-alpha-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCIa and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88-89% similarity with the three subvariants (G90/R212, E90/R212, E90/K212) of CCIa. 139-140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys25, His159, Asn175) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed.

Amino Acid Sequence↗
PubMed1995

Application of the proteolytic enzyme papain in routine platelet serology.

The use of proteolytic enzymes is well established in red cell serology. These enzymes modify some antigen structures and remove sialic acid from the red cell membrane. Enzyme-sensitive structures have also been identified on the platelet membrane. The effect of papain, a proteolytic enzyme used widely in red cell serology, on the detection of various platelet alloantibodies was examined to determine its usefulness in platelet serology. Antisera with the specificities anti-HPA-la, -2b, -3a, -4a, -5a, -5b, and -Naka were examined. HLA antibodies were also included. All sera were tested by a solid-phase red cell adherence technique in parallel with untreated platelets (UP) and platelets treated with papain (PP) for 15 minutes at 37 degrees C. The reactivity of anti-HPA-2b was eliminated and that of anti-HPA-3a was either eliminated or almost eliminated with PP. Antisera specific for the other alloantigens tested reacted similarly or more strongly with PP compared with UP. These findings were confirmed by flow cytometry. The reactivity of HLA antibodies with PP was generally enhanced. Inactivation by papain of platelet alloantigens in the HPA-2 and HPA-3 systems, but not in other systems, may assist in resolving mixtures of platelet alloantibodies. Also, detection of weak antibodies of other specificities may be enhanced. The use of PP may be a simple and useful serologic tool for investigating platelet alloantibodies.

Journal Article↗
PubMed2005

Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition.

Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry. This protease is especially selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S(-)/(His)-Im(+)H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.

Amino Acid Sequence↗
PubMed1999

A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .

A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.

Amino Acid Sequence↗
PubMed1962

An investigation into the use of activated and non-activated papain in routine rhesus blood grouping.

The activities of three commercial products of papain were compared as supplied and after activation, and when used in the enzyme-serum method for rhesus (D) grouping. Certain dangers in activation became apparent, and accurate determination of the proportions of papain to anti-D appeared more important than indiscriminate activation without reference to the initial enzyme activity. A technique for assessing these proportions is described.

Blood Group Antigens↗
PubMed1986

A naturally occurring, warm-reactive macroglobulin specific for papain-treated human platelets: preliminary characterization.

Sera from 28 of the 113 normal children and adults (25%) studied were found to contain an immunoglobulin capable of causing complement-dependent lysis of normal platelets treated with small quantities of papain. This factor reacts equally well at 4 degrees C and at 37 degrees C with a determinant induced on platelets from normal subjects by treatment with papain or bromelain, but not by trypsin, chymotrypsin, or neuraminidase. It does not bind to red cells treated with any of these enzymes. The site(s) for which the factor was specific could not be induced on platelets from six patients with type I Glanzmann's thrombasthenia (lacking glycoproteins IIb and IIIa), in contrast to platelets from each of 20 normal donors. Isolation and characterization of the factor has been difficult because of its intolerance to chemical and physical manipulation. In 11 of the 20 individuals studied, however, it was found to have the properties of an IgM immunoglobulin. The factor appears to be different from any previously described, naturally occurring human immunoglobulin. It has not yet been shown to be associated with any disease state, but in the presence of complement, it is capable of causing profound damage to platelets previously subjected to minimal proteolysis, and the possibility that it can provoke platelet destruction in some conditions deserves further study.

Adolescent↗
PubMed2006

Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases.

Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing a novel O-acyl hydroxyurea warhead was found to function as covalent inhibitors of papain-like proteases. These inhibitors can be easily synthesized on solid support, which allows rapid optimization of compounds with improved selectivity and potency for a given target enzyme. We present here one such optimized inhibitor that showed selective inhibition of falcipain 1, a protease of the malaria-causing parasite, Plasmodium falciparum.

Animals↗
PubMed1976

Immune response region-associated antigens of the mouse: biochemical properties of detergent and papain solubilized molecules.

Immune response region-associated (Ia) antigens and classical H-2 antigens are glycoproteins which differ in their molecular weight and tissue distribution. Ia and H-2 molecules also show differences in overall charge, apparent isoelectric point and susceptibility to proteolytic degradation, acid treatment and exposure to 56 degrees C. In the present paper, Ia.11d antigens were shown to have a molecular weight of about 35000 after solubilization with nonionic detergents. Purified Iad antigens, solubilized by controlled papain digestion, display a molecular weight of about 26000 after final purification by indirect immune coprecipitation. This figure corresponds to the molecular weight of a highly purified H-2 polypeptide fragment obtained after papain solubilization. The remarkable size similarity of proteolytic fragments from Ia and H-2 antigens could suggest that these genetically and functionally related molecules possess some structural features in common.

Animals↗
PubMed2006

Binding site-based classification of coronaviral papain-like proteases.

The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.

Amino Acid Sequence↗
PubMed2000

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes.

The structures of the two leader protease (Lpro) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 A. This singular, pseudo-polymeric organization is present in a new Lpro crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this Lpro structure, refined at 1.9 A resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of Lpro activity on a fluorescent hexapeptide substrate showed that Lpro, in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the Lpro structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties.

Amino Acid Substitution↗
PubMed1995

Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase.

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants.

Amino Acid Sequence↗
PubMed1998

Characterization of a papain-like cysteine-proteinase encoded by gene 1 of the human coronavirus HCV 229E.

Expression of the coronaviral gene 1 polyproteins, pp 1a and pp 1ab, involves a series of proteolytic events that are mediated by virus-encoded proteinases similar to cellular papain-like cysteine-proteinases and the 3C-like proteinases of picornaviruses. In this study, we have characterized, in vitro, the human coronavirus HCV 229E papain-like cysteine-proteinase PCP 1. We show that PCP 1 is able to mediate cleavage of an aminoterminal polypeptide, p9, from in vitro translation products representing the aminoproximal region of pp 1a/pp 1ab. Mutagenesis studies support the prediction of Cys1054 and His1278 as the catalytic amino acids of the HCV 229E PCP 1, since mutation of these residues abolishes the proteolytic activity of the enzyme.

Coronavirus↗
PubMed1992

Clinical relevance of soluble HLA and interaction of papain derived class I molecules with alloreactive CTL.

MHC class I and class II molecules are expressed in soluble form in the serum of both healthy and diseased individuals. Our aim was to investigate whether soluble class I (sHLA) levels in allograft patients correlate with their clinical status. Altogether, 20 renal and 30 cardiac graft recipients were examined. High levels of sHLA were measured at least 5 days preceding acute rejection episodes. Immune complexes between anti-HLA antibodies and sHLA were detected in a patient who died of a severe vascular rejection. In another study the interaction of papain-derived sHLA on alloreactive CTL in vitro was investigated. In a chromium-51-release cytotoxicity assay, 1.25 microg/ml of papain-digested class I molecules reduced CTL cytotoxicity to background levels. On the contrary, immobilized molecules triggered the release of serine esterase allospecifically. These data showed that the MHC molecule alone was a sufficient ligand for the interaction with alloreactive CTL.

Biomarkers↗
PubMed1989

Single breath N2 washout in papain-induced pulmonary emphysema.

Single breath nitrogen washout tests were analyzed in dogs (n = 8) with healthy lungs and after development of emphysema. The animals were in the supine position and studied during anaesthesia and mechanical ventilation (FiO2 = 0.4, FiN2 = 0.6). During controlled expiration with constant flow (VE = 0.15 l/s) onset of phase IV of the alveolar plateau was related to airway closure of dependent lung regions (closing volume CV). In the control state, CV accounted for 6.2 +/- 1.5% VC, and closing capacity (CC) was lower than functional residual capacity (FRC). Likewise, gas exchange was normal in all animals (PaO2 = 24.7 +/- 3.32 kPa, PaCO2 = 5.18 +/- 0.53 kPa, PA-aO2 = 2.6 +/- 0.3 kPa). Panlobular emphysema (PLE) was induced by inhalation of papain (100 mg/kg). After three weeks development of PLE was documented by measurements of lung volumes (functional residual capacity (FRC), expired vital capacity (EVC), total lung capacity (TLC), residual volume (RV], pulmonary mechanics (dynamic and static compliance (Cdyn, Cstat), mean airway resistance (Raw], gas exchange (PaO2, PaCO2, PA-aO2), and by radiomorphological analysis. In the PLE-group, FRC and RV (p less than or equal to 0.05), and Cstat (p less than or equal to 0.01) were significantly elevated. CV increased to 16.2 +/- 2.7% VC (p less than or equal to 0.01) and CC exceeded FRC by 80 ml, indicating that tidal volume breathing took place within the range of closing volume. Oxygenation was significantly impaired (PaO2 = 18.6 +/- 3.72 kPa, PA-aO2 = 6.5 +/- 1.1 kPa, p less than or equal to 0.05), but not CO2-elimination. Pathological analysis by radiomorphological means showed dissiminate parenchymal lesions compatible with emphysema of grade II severity located predominantly in subpleural areas. In dogs with papain-induced PLE, premature closure of dependent airways is enhanced, which is due to structural changes and a loss of elastic recoil in the lungs.

Animals↗
PubMed1979

Fresh immature articular cartilage allografts. A study on the integration of chondral and osteochondral grafts both in normal and in papain-treated knee joints of rabbits.

The integration of immature cartilage allografts to knee joints of both normal rabbits and those submitted to arthritis through papain was studied for a period of up to 9 months. Two types of grafts were compared: chondral--constituted only by cartilage, and osteochondral--cartilage plus a thin layer of subchondral bone. The integration of the grafts was analyzed through inspection of the articular surface and histologic sections. The viability of the grafts was checked through the incorporation of 35SO4 on the chondral matrix. The following conclusions were drawn: (1) A large member of the grafted cartilages were well accepted without histological evidence of immunological rejection, and metabolically active 9 months after transplantation. (2) The chondral graft was found to be superior to the osteochondral one regarding the integration, but both showed tendency to degeneration with time. (3) The presence of arthritis previously induced by papain affected the integration of the grafts causing a higher precentage of degeneration on the grafts causing a higher precentage of degeneration on the grafted cartilage.

Animals↗
PubMed1992

Characterization of cell-bound papain-soluble beta-lactamases in BRO-1 and BRO-2 producing strains of Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens.

In Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens strains isolated from clinical specimens in the south of Sweden two variants of beta-lactamase were distinguished by isoelectric focusing (IEF). The BRO-1 (Ravasio type) enzyme was the most common in Branhamella catarrhalis, constituting about 90% of the beta-lactamase found in this species, while the BRO-2 enzyme (1908 type) was as common as BRO-1 in Moraxella nonliquefaciens. The determinants mediating the production of BRO-1 and BRO-2 were both transferable by conjugation. Cell-bound beta-lactamase from reference strains producing BRO-1 and BRO-2 could be solubilized by papain digestion. The isoelectric point of the solubilized enzymes differed distinctly between BRO-1 (pI 6.5) and BRO-2 (pI 6.9). The molecular species of BRO-1 and BRO-2 released by papain digestion were purified by affinity chromatography with phenylboronic acid agarose gel. They had identical molecular weights of approximately 28,000. Their kinetic constants were indistinguishable for a number of substrates and beta-lactamase inhibitors.

Anti-Bacterial Agents↗