Function of the prosequence for in vivo folding and secretion of active Rhizopus oryzae lipase in Saccharomyces cerevisiae

The role of the prosequence of Rhizopus oryzae lipase (ROL) with a preprosequence was analyzed by an expression system using Saccharomyces cerevisiae. When the mature portion of ROL (mROL) fused to the pre-alpha-factor leader sequence was expressed, secretion of active mROL was not observed. However, when mROL was synthesized together with the prosequence in trans (individually and coincidentally), secretion of active mROL was observed. The results indicate that the prosequence of ROL helped correct folding of mROL and its subsequent secretion from the yeast cells, and that physical linkage (cis) of the prosequence to the mature region was not prerequisite. From the expression of the ROL mutants with deletions at the N-terminal end of the prosequence together with mROL in trans, the residues from 20 to 37 in the prosequence were essential for the secretion, and those from 38 to 57 were essential for the formation of the active ROL and might play a role as an intramolecular chaperone. The results using the fragment of the prosequence confirmed that these residues (20-57) were significant for in vivo folding and secretion of active mROL.     

Mutated intramolecular chaperones generate high-activity isomers of mature enzymes

The propeptide of carboxypeptidase Y precursor (proCPY) acts as an intramolecular chaperone that ensures the correct folding of the mature CPY (mCPY). Here, to further characterize the folding mechanism mediated by the propeptide, folding analysis was performed using a yeast molecular display system. CPYs with mutated propeptides were successfully displayed on yeast cell surface, and the mature enzymes were purified by the selective cleavage of mutated propeptides. Measurement of the activity and kinetics of the displayed CPYs indicated that the propeptide mutation altered the catalytic efficiency of mCPY. Although the mature region of the wild-type and mutant CPYs had identical amino acid sequences, the mCPYs from the mutant proCPYs had higher catalytic efficiency than the wild-type. These results indicate that proteins with identical amino acid sequence can fold into isomeric proteins with conformational microchanges through mutated intramolecular chaperones.     

Creation of Rhizopus oryzae lipase having a unique oxyanion hole by combinatorial mutagenesis in the lid domain

Combinatorial libraries of the lid domain of Rhizopus oryzae lipase (ROL; Phe88Xaa, Ala91Xaa, Ile92Xaa) were displayed on the yeast cell surface using yeast cell-surface engineering. Among the 40,000 transformants in which ROL mutants were displayed on the yeast cell surface, ten clones showed clear halos on soybean oil-containing plates. Among these, some clones exhibited high activities toward fatty acid esters of fluorescein and contained non-polar amino acid residues in the mutated positions. Computer modeling of the mutants revealed that hydrophobic interactions between the substrates and amino acid residues in the open form of the lid might be critical for ROL activity. Based on these results, Thr93 and Asp94 were further combinatorially mutated. Among 6,000 transformants, the Thr93Thr, Asp94Ser and Thr93Ser, Asp94Ser transformants exhibited a significant shift in substrate specificity toward a short-chain substrate. Computer modeling of these mutants suggested that a unique oxyanion hole, which is composed of Thr85 Oγ and Ser94 Oγ, was formed and thus the substrate specificity was changed. Therefore, coupling combinatorial mutagenesis with the cell surface display of ROL could lead to the production of a unique ROL mutant.     

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I^WT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M^WT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-I^WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I^WT released soluble peptides from SSP faster than did M^WT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.     

A pathway for conformational diversity in proteins mediated by intramolecular chaperones.

Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer's disease. The molecular basis of such diversity is unknown. Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed "protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by Ile-48 --> Thr IMC (IMCI-48T) acquires an "altered" enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates. IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin. Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC.     

Total chemical synthesis and oxidative folding of delta-conotoxin PVIA containing an N-terminal propeptide

Small disulfide-rich peptides are translated as larger precursors typically containing an N-terminal prepro sequence. In this study, we investigated the role of a propeptide in the oxidative folding of an extremely hydrophobic delta-conotoxin, PVIA. delta-Conotoxin PVIA (delta-PVIA) is a 29-amino acid neurotoxin stabilized by three disulfide bridges. Previous folding studies on delta-conotoxins revealed that their poor folding properties resulted from their hydrophobicity. However, low folding yields of delta-PVIA could be improved by the presence of a nonionic detergent, which acted as a chemical chaperone. delta-PVIA provided an attractive model to investigate whether the hydrophilic propeptide region could function as an intramolecular chaperone. A 58-amino acid precursor for delta-PVIA (pro-PVIA), containing the N-terminal propeptide covalently attached to the mature conotoxin, was synthesized using native chemical ligation. Oxidative folding of pro-PVIA resulted in a very low accumulation of the correctly folded form, comparable to that for the mature conotoxin delta-PVIA. Our results are in accord with the relevant data previously observed for alpha- and omega-conotoxins, indicating that conotoxin prepro sequences are so-called class II propeptides, which are not directly involved in the oxidative folding. We hypothesize that these propeptide regions may be important for interactions with protein folding catalysts and sorting receptors during the secretory process.     

前导肽介导的蛋白折叠机制及其对脂肪酶影响的研究进展

大多数蛋白质的形成过程主要由合成前体蛋白和合成功能蛋白两个步骤组成.在这个过程中,前导肽能够辅助蛋白质折叠或抑制它的活性.前导肽作为脂肪酶结构中重要的一段多肽链,通常作为分子内分子伴侣来辅助脂肪酶的折叠,同时该序列上包括糖基化位点在内的一些特殊位点,对酶的活性、极端环境稳定性、甲醇耐受性和底物特异性等性质具有重要影响....     

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu^?1-Ser¹ bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu^?12-Lys^?11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys³²-Cys⁴⁸ and Cys¹⁸⁰-Cys¹⁸³ cumulatively contributed to the thermostability of TS-GSE.     

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.     

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an M_r 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000–1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg⁷⁰⁹/Arg¹⁰¹⁸/Arg¹⁵⁴⁵) mutated to glutamine (fV^Q3), a mutant fV molecule with region 1000–1008 deleted (fV^ΔB9), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV^ΔB9/Q3). The recombinant molecules along with wild type fV (fV^WT) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV^Q3 was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, K_D(app) values for fV^ΔB9 (0.9 nm), fVa^ΔB9 (0.4 nm), and fV^ΔB9/Q3 (0.7 nm) were similar to the affinity of fVa^WT for fXa (0.3 nm). Two-stage clotting assays revealed that although fV^Q3 was deficient in its clotting activity, fV^ΔB9/Q3 had clotting activity comparable with fVa^WT. The k_cat value of prothrombinase assembled with fV^ΔB9/Q3 was minimally affected, whereas the K_m value of the reaction was increased 57-fold compared with the K_m value obtained with prothrombinase assembled with fVa^WT. These findings strongly suggest that amino acid region 1000–1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.     

Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.     

Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that the proform, which contains an N-terminal propeptide composed of a core domain (N*) and a linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway is initiated by cis-processing of N* to generate an autoprocessed complex N*-I^WT, and this step is followed by truncation of the linker peptide and degradation of N*. Another autocatalytic pathway is initiated by trans-processing of the linker peptide followed by degradation of N*. Unlike most reported subtilases, the maturation of the WF146 protease occurs not only autocatalytically but also hetero-catalytically whereby heterogeneous proteases accelerate the maturation of the WF146 protease via trans-processing of the proform and N*-I^WT. Although N* acts as an intramolecular chaperone and an inhibitor of the mature enzyme, the linker peptide is susceptible to proteolysis, allowing the trans-processing reaction to occur auto- and hetero-catalytically. These studies also demonstrate that the WF146 protease undergoes subtle structural adjustments during the maturation process and that the binding of Ca²⁺ is required for routing the proform to mature properly at high temperatures. Interestingly, under Ca²⁺-free conditions, the proform is cis-processed into a unique propeptide-intermediate complex (N*-I^E) capable of re-synthesis of the proform. Based on the basic catalytic principle of serine proteases and these experimental results, a mechanism for the cis-processing/re-synthesis equilibrium of the proform and the role of the linker peptide in regulation of this equilibrium has been proposed.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.     

A conserved sequence within the propeptide domain of membrane type 1 matrix metalloproteinase is critical for function as an intramolecular chaperone.

The propeptide domain of secreted matrix metalloproteinases (MMPs) is responsible for maintaining the latency of these proteinases. Recently, the propeptide domain of the prototype membrane type matrix metalloproteinase (MT1-MMP) was demonstrated to act as an intramolecular chaperone (Cao, J., Hymowitz, M., Conner, C., Bahou, W. F., and Zucker, S. (2000) J. Biol. Chem. 275, 29648-29653). In the current study, the role of an unique four-amino acid sequence in the propeptide domain of MT1-MMP was examined. The sequence (42)YGYL(45) is conserved in the propeptide domain of all six members of the MT-MMP subfamily, but not in secreted MMPs. Mutant MT1-MMP cDNAs coding for alanine substitutions (single and double amino acid sequences) in this conserved propeptide region were transfected into COS-1 cells deficient in endogenous MT1-MMP. As demonstrated by immunofluorescence, mutant MT1-MMP protein was synthesized and displayed on the plasma membrane of transfected cells. Alanine substitutions within the (42)YGYL(45) sequence proved to be detrimental for enzyme function in terms of activation of proMMP-2 and binding TIMP-2 to the cell surface (MT1-MMP serves as a cell surface receptor for TIMP-2). In contrast to wild-type MT1-MMP-transfected cells, mutant MT1-MMP-transfected cells were incapable of degrading and migrating on a fibronectin substrate. These data indicate that the conserved (42)YGYL(45) sequence within the propeptide domain of MT-MMPs is required for intramolecular chaperone function of these intrinsic membrane proteinases.     

Characterization of a potent dominant negative mutant variant of Rad51 in Ustilago maydis

Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer–protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51^D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51^WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51^D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog.     

GM2 gangliosidoses in Spain: Analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R²⁰⁰ and A²⁰⁴ inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA^H201L but not rhGAA^WT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA^H201L but not rhGAA^WT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.     

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT^C/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT^C/ala did not affect circadian behavior differently from wild-type DBT (DBT^WT), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT^WT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT^C/ala did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.     

Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli

A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.