Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing.

Human dipeptidyl peptidase I was expressed in the insect cell/baculovirus system and purified in its active (rhDPPI) and precursor (pro-rhDPPI) forms. RhDPPI was very similar to the purified enzyme (hDPPI) with respect to glycosylation, enzymatic processing, oligomeric structure, CD spectra, and catalytic activity. The precursor, which was a dimer, could be activated approximately 2000-fold with papain. Cathepsin L efficiently activated pro-rhDPPI in vitro at pH 4.5 (k(app) approximately 2 x 10(3) min(-)(1) M(-)(1)), and two cleavage pathways were characterized. The initial cleavage was within the pro region between the residual pro part and the activation peptide. Subsequently, the activation peptide was cleaved from the catalytic region, and the latter was cleaved into the heavy and light chains. Alternatively, the pro region was first separated from the catalytic region. Cathepsin S was a less efficient activating enzyme. Cathepsin B and rhDPPI did not activate pro-rhDPPI, and the proenzyme was incapable of autoactivation. Incubation of both pro-rhDPPI and rhDPPI with cathepsin D resulted in degradation. Cystatin C and stefins A and B inhibited rhDPPI with K(i) values in the nanomolar range (K(i) = 0.5-1.1 nM). The results suggest that cathepsin L could be an important activator of DPPI in vivo and that cathepsin D and possibly the cystatins may contribute to DPPI downregulation.

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease.

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.

Biochemical analysis of recombinant fungal mutanases. A new family of alpha1,3-glucanases with novel carbohydrate-binding domains.

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum alpha1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH(2)-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong alpha-amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 degrees C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with K(D) around 0.11 and 0.13 microM at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mutanase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.

Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1.

A laccase from Coprinus cinereus is active at alkaline pH, an essential property for some potential applications. We cloned and sequenced three laccase genes (lcc1, lcc2, and lcc3) from the ink cap basidiomycete C. cinereus. The lcc1 gene contained 7 introns, while both lcc2 and lcc3 contained 13 introns. The predicted mature proteins (Lcc1 to Lcc3) are 58 to 80% identical at the amino acid level. The predicted Lcc1 contains a 23-amino-acid C-terminal extension rich in arginine and lysine, suggesting that C-terminal processing may occur during its biosynthesis. We expressed the Lcc1 protein in Aspergillus oryzae and purified it. The Lcc1 protein as expressed in A. oryzae has an apparent molecular mass of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and absorption maxima at 278 and 614 nm. Based on the N-terminal protein sequence of the laccase, a 4-residue propeptide was processed during the maturation of the enzyme. The dioxygen specificity of the laccase showed an apparent K(m) of 21 +/- 2 microM and a catalytic constant of 200 +/- 10 min(-1) for O(2) with 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the reducing substrate at pH 5.5. Lcc1 from A. oryzae may be useful in industrial applications. This is the first report of a basidiomycete laccase whose biosynthesis involves both N-terminal and C-terminal processing.

Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae).

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.

Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae.

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.

Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109. A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall.

Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.

Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENCI) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENCI amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 degrees C.

Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes.

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.

Bovine histidine-rich glycoprotein is a substrate for bovine plasma factor XIIIa.

Histidine-rich glycoprotein was purified from bovine plasma and the identity of the protein confirmed through amino acid sequencing. Activated bovine factor XIIIa catalyzed the incorporation of 1 nmol of 1,4-[14C]putrescine into 1 nmol of bovine histidine-rich glycoprotein, showing that histidine-rich glycoprotein has the ability to participate in transglutaminase-catalyzed reactions in vivo.

Bovine factor XIIa inhibitor.

Bovine factor XIIa inhibitor was purified by an improved method employing affinity for heparin. N-terminal amino acid sequencing revealed a unique sequence without homology to any other known protein sequences. Peptide sequencing, however, showed that a part of the bovine factor XIIa inhibitor was homologous to human C1-inhibitor with a fraction of identical amino acid residues around 70%. Deglycosylation studies and carbohydrate analysis showed the presence of N- and O-linked carbohydrate. Bovine factor XIIa inhibitor did not inhibit plasma kallikrein and trypsin. The reactive site comprised an Arg-Asn bond, and represents the first example of asparagine as a P1' residue in Serpins with well documented inhibitory activity.

Complete primary structure of bovine beta 2-glycoprotein I: localization of the disulfide bridges.

The complete primary structure of bovine beta 2-glycoprotein I was determined by a combination of cDNA and peptide sequencing. Bovine beta 2-glycoprotein I was purified from citrated plasma, and by sequencing selected peptides, the complete disulfide bridge patterns of the 11 disulfide bridges were established as well as the positions of the five asparagine-linked carbohydrate groups. Bovine beta 2-glycoprotein I comprises five mutually homologous domains or Short Consensus Repeats, each containing two disulfide bridges, except for the fifth most C-terminal domain which diverges from the Short Consensus Repeat consensus by containing an additional disulfide bridge. In the four N-terminal domains, the first and third and the second and fourth half-cystines are disulfide-linked, while in the fifth domain the first and fourth, the second and fifth, and the third and sixth half-cystines are disulfide-linked.

Purification and characterization of an astrocyte GABA-carrier inducing protein (GABA-CIP) released from cerebellar granule cells in culture.

A glycoprotein that induces gamma-aminobutyric acid (GABA) carriers in cultured cerebellar astrocytes was isolated and purified from conditioned media from cultured cerebellar granule cells by anion exchange chromatography, affinity chromatography, and gel filtration. Following gel filtration three fractions corresponding to M(r) 30,000, 60,000, and 240,000 exhibited GABA carrier inducing activity. SDS-PAGE of the M(r) 30,000 fraction revealed under non-reducing conditions three bands corresponding to M(r) 30,000, 60,000, and 120,000. Under reducing conditions only the band corresponding to an M(r) of 30,000 was visible. An identical N-terminal amino acid sequence and amino acid composition was found in the M(r) 30,000 and the M(r) 60,000 fraction from the gel filtration. These results suggest that the protein polymerizes into di- and tetramers. Computer base analysis of the N-terminal amino acid sequence revealed no obvious homology with previously reported N-terminal amino acid sequences. Application of the glycoprotein to cerebellar astrocytes led time and dose dependently to an increased GABA uptake. The effect became maximal after 24 h exposure of the cells. Kinetic analysis of the GABA uptake showed that exposure of the astrocytes to the glycoprotein led to an increase in Vmax for GABA uptake without affecting Km, suggesting an increase in the number of GABA carrier molecules. Addition of actinomycin D together with the glycoprotein abolished this effect suggesting that the glycoprotein acts by stimulating de novo synthesis of GABA carriers. Hence, the newly purified protein secreted from neurons is named GABA-carrier inducing protein (GABA-CIP).

Zinc ions promote the binding of factor XII/factor XIIA to acidic phospholipids but have no effect on the binding of high-Mr kininogen.

Binding of high-Mr kininogen and factor XII/factor XIIa to phospholipids coated on to polystyrene microtiter plates was investigated by ELISA. Both high-Mr kininogen and factor XII/factor XIIa bound specifically to the phospholipid surface. Binding was observed to negatively charged phospholipids only. The binding of high-Mr kininogen was not affected by the presence of zinc ions. At a surface concentration of 20% phosphatidylinositol phosphate in phosphatidylcholine a dissociation constant (kD) of 10 nM for the binding of high-Mr kininogen was calculated. The amount of bound purified alpha-factor XIIa could be increased 4-5-fold in the presence of zinc ions. The lowest zinc ion concentration giving maximal binding was 0.1 mM. The binding of alpha-factor XIIa was inhibited by high-Mr kininogen. Independent of the presence of zinc ions or high-Mr kininogen, a kD of 7.9 nM was calculated for alpha-factor XIIa binding. The binding of prekallikrein was dependent upon the presence and the concentration of high-Mr kininogen. In plasma containing aprotinin, the binding of high-Mr kininogen was apparently inhibited in the presence of zinc ions, which was a prerequisite for the binding of factor XII. This apparently inhibitory effect of zinc ions on the binding of high-Mr kininogen was probably due to the increased binding of factor XII, which displaced high-Mr kininogen.