Keratinolytic proteinase produced by Candida albicans

Candida albicans was cultivated in various media that contained human stratum corneum, human scalp hair or keratin powder (cow's hoof) as a nitrogen source. Production of a keratinolytic proteinase (KPase) was observed when C. albicans was incubated in the medium containing stratum corneum. However, there was no production of a KPase that could digest human stratum corneum in the medium containing hair or keratin powder. alpha-fibrous protein extracted from human stratum corneum was digested by the KPase. The pH optimum of the enzyme was 4.0 and enzyme activity was inhibited by pepstatin A and chymostatin. The KPase, a kind of carboxyl proteinase, may be important for C. albicans to enable it to play a pathogenic role in vivo.     

Induction of secretory acid proteinase in Candida albicans.

Candida albicans and some other pathogenic Candida species, when grown in a medium containing a protein as a sole source of nitrogen, secrete an acid proteinase. Culture supernatants were assayed for proteinase activity, and were also analysed by Western blotting with antibodies raised and affinity-purified against proteinase of C. albicans. Proteinases secreted by C. tropicalis and C. parapsilosis were antigenically related to that of C. albicans, but had different molecular masses. The proteinases secreted by C. lipolytica, C. rugosa and C. lusitaniae were not antigenically related. The kinetics of proteinase secretion by C. albicans were monitored by activity and by Western blotting. With BSA as the nitrogen source, proteinase secretion increased exponentially until about 16 h. Culture supernatants of BSA-grown cultures accumulated proteinase to about a 1000-fold higher level than those of ammonium-sulphate-grown cultures. In vivo labelling experiments showed that proteinase was not detectably accumulated in the cells, but was secreted immediately after synthesis. Immunoprecipitation of in vitro translated poly(A)-containing RNA identified a putative pre-protein of about 54 kDa. As well as BSA, other proteins (haemoglobin, ovalbumin, histone), peptone and tryptone, when used as nitrogen sources, could induce proteinase, but to different levels. When Casamino acids or an amino acid mixture (equivalent to the composition of BSA) was used as nitrogen source, no induction was observed. Ammonium sulphate, or any other ammonium salt, repressed secretion of proteinase.     

A second gene for a secreted aspartate proteinase in Candida albicans.

The gene (PRA11) encoding a secreted aspartate proteinase of Candida albicans has been cloned and sequenced. The nucleotide and deduced amino acid sequences of PRA11 are 77 and 73% identical, respectively, with the reported sequences of PRA10 also cloned from C. albicans. Southern analyses indicated that the genome of each strain examined (ATCC 10231 and ATCC 10261) contains PRA10 and PRA11. Northern (RNA) analyses showed that PRA11 was expressed at a much higher level than was PRA10 when secretion of the proteinase by strain ATCC 10261 was induced with albumin.     

Purification and characterization of secretory proteinase of Candida albicans.

Proteolytic activity of medically important yeasts was tested in both YCB-BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time-course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60-min incubation. The 23 amino acid sequence of the proteinase N-terminus was determined.     

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein

Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion-exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1.     

Acidic cysteine proteinase inhibitor in seminal plasma

A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.     

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.     

Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Detection of intracellular forms of secretory aspartic proteinase in Candida albicans.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Affinity chromatography of a cysteine proteinase inhibitor from chicken egg protein

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

Rapid purification of secretory acid proteinase from Candida albicans and its characterization.

Secretory acid proteinase from C. albicans was purified from culture supernatant to apparent homogeneity by ion-exchange chromatography. Two isozymes of the proteinase were resolved using a novel chromatofocusing method. The enzyme, which appears to be a glycoprotein, consists of a single polypeptide chain with glutamine at the N-terminus. Its molecular weight is about 45,000 and isoelectric point is pH 4.6. At pH 5, the proteinase is stable at 45 degrees C for at least 15 min. It has a broad substrate specificity. With BSA as a substrate, Km was determined to be 1.6 x 10(-4) M. The enzyme is inhibited by pepstatin and thus is a carboxyl proteinase. It undergoes autocatalytic digestion at or below pH 5.0. The kinetics of induction of proteinase by various proteins are also reported.     

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.     

Association of human neuroglobin with cystatin C, a cysteine proteinase inhibitor

Neuroglobin (Ngb) is a newly discovered globin that is expressed in vertebrate brain. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that Ngb protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. In the present study, we identified human cystatin C, a cysteine proteinase inhibitor, as an Ngb-binding protein by using a yeast two-hybrid system. Surface plasmon resonance experiments verified that Ngb binds to cystatin C dimers, not to the monomers. Because both intracellular cystatin C and the amyloidogenic variant of cystatin C form dimers, Ngb may modulate the intracellular transport (or secretion) of cystatin C to protect against neuronal death under conditions of oxidative stress and/or it may have a role in the development of neurodegenerative diseases.