Mutations in the NH2-terminal domain of the signal peptide of preproparathyroid hormone inhibit translocation without affecting interaction with signal recognition particle

The amino-terminal domain of a eukaryotic signal peptide, from bovine parathyroid hormone, was altered by in vitro mutagenesis of the cDNA. The function of "internalized" signal sequence mutants and of deletion mutants was assayed using an in vitro translation-translocation system. The addition of 11 amino acids to the NH2 terminus of the signal peptide did not prevent normal processing of the precursor protein, whereas a 23-amino acid extension blocked processing. These data suggest that the NH2-terminal sequences of internal signal peptides must be permissive of the signal function. Deletion of 6 NH2-terminal amino acids from the signal peptide had no effect on its cleavage by microsomal membranes, but removal of 10 or 13 amino acids, including all charged residues prior to the hydrophobic core, prevented processing. For both the extension and deletion mutations, processed proteins were protected from proteolytic digestion, whereas unprocessed forms were not, which indicated that the unprocessed mutant proteins were not translocated across the microsomal membrane. Translation of both the extension and deletion translocation-deficient mutants was arrested by signal recognition particle, and salt-washed microsomal membranes reversed the translational arrest. These data demonstrate that the NH2-terminal domain is not required for the interaction of signal recognition particle with the signal peptide or with signal recognition particle receptor, but is required for formation of a maximally translocation-competent complex with the microsomal membrane.     

Characterization of the secretion efficiency of a plant signal peptide in Bacillus subtilis.

Summary The ability of the Bacillus subtilis secretion machinery to interact with a heterologous signal peptide was studied using a plant (wheat -amylase) signal peptide. The plant signal peptide was capable of mediating secretion of Escherichia coli alkaline phosphatase and B. amyloliquefaciens levansucrase from B. subtilis. This secretion was dependent on the plant signal peptide, as deletion of five amino acids from the hydrophobic core resulted in a block of secretion. Attempts to improve the efficiency of the plant signal peptide in B. subtilis were made by increasing the length of the hydrophobic core from 10 to 16 residues by insertion of 2, 4, 5 or 6 amino acids. None of the alterations improved the secretion efficiency relative to the wild-type plant signal peptide.     

Regions in the transit peptide of SSU essential for transport into chloroplasts

Summary Deletion mutations, 3–19 amino acids in size, were introduced into the transit peptide (57 amino acids) of a small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea. Transport of the authentic small subunit precursor (pSSU) and of the mutant pSSUs by isolated chloroplasts of pea was examined. We show that the transit peptide contains two different, separated functional regions. A deletion mutation in the central region of the transit peptide, a region purported to be important for function, barely affected transport. Changes in the amino-terminal region of the transit peptide drastically reduced transport. Processing of mutants affected in either the amino-terminal or central portion of the transit peptide appeared normal. A deletion mutation at the carboxy-terminus of the transit peptide interfered with both transport and processing. From the aberrant processing we suggest that pSSU is matured in more than one step, and that the maturation signal is located within the carboxy-terminal 16 amino acids. The methionine residue at the evolutionarily conserved cleavage site (cysteine-methionine) between the transit peptide and the mature protein is not essential for processing.     

Optimal secretion of alkali-tolerant xylanase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by signal peptide screening

Xylanases are industrially important enzymes for xylan digestion. We experimentally screened over 114 Sec and 24 Tat pathway signal peptides, with two different promoters, for optimal production of an alkaline active xylanase (XynBYG) from Bacillus pumilus BYG in a Bacillus subtilis host. Though both promoters yielded highly consistent secretion levels (0.97 Pearson correlation coefficient), the Sec pathway was found to be more efficient than the Tat pathway for XynBYG secretion. Furthermore, the optimal signal peptide (phoB) for XynBYG secretion was found to be different from the optimal peptides for cutinase and esterase reported in previous studies. A partial least squares regression analysis further identified several statistically important variables: helical properties, amino acid composition bias, and the discrimination score in Signal P. These variables explain the observed 23 % variance in the secretion yield of XynBYG by the different signal peptides. The results also suggest that the helical propensity of a signal peptide plays a significant role in the beta-rich xylanase, but not in the helix-rich cutinase, suggesting a coupling of the conformations between the signal peptide and its cargo protein for optimal secretion.     

Modifications of a signal sequence for antibody secretion from insect cells

Monoclonal antibodies and antibody fragments have recently been developed for use in diverse diagnostic and therapeutic applications. Insect cells can efficiently secrete recombinant proteins such as antibody molecules through post-translational processing and modifications that are similar to those performed in mammalian cells. In eukaryotic cells, the signal sequence in a nascent polypeptide is recognized by the signal recognition particle, and the polypeptide is then folded and modified in the endoplasmic reticulum. The signal sequence consists of three regions, a positively charged N-terminus, a hydrophobic core, and a polar C-terminus. In the present study, we examined the substitutions of the characteristic amino acids of a Drosophila immunoglobulin heavy chain binding protein signal sequence, and investigated the effect on the secretory production of an antibody Fab fragment from lepidopteran insect cells in transient expression. A modification of the signal sequence for the heavy chain resulted in a twofold increase in the secreted Fab fragment, while the modification for the light chain led to a more than 3.6-fold increase.     

Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis.

A series of alterations in the Bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in Bacillus subtilis was studied. Some of the alterations resulted in a completely defective signal peptide. These included the removal of positively charged residues from the N-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. Analysis of the signal peptide processing-site alterations revealed that small residues are preferred at the -1 and -3 positions. However, a wide variety of amino acids are tolerated at the +1 position.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5′ end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis

Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation.     

A mutant <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS–PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.     

Identification of residues important for cleavage of the extracellular signaling peptide CSF of Bacillus subtilis from its precursor protein

Extracellular Phr pentapeptides produced by gram-positive, spore-forming bacteria regulate processes during the transition from exponential- to stationary-phase growth. Phr pentapeptides are produced by cleavage of their precursor proteins. We determined the residues that direct this cleavage for the Bacillus subtilis Phr peptide, CSF, which is derived from the C terminus of PhrC. Strains expressing PhrC with substitutions in residues -1 to -5 relative to the cleavage site had a defect in CSF production. The mutant PhrC proteins retained a functional signal sequence for secretion, as assessed by secretion of PhrC-PhoA fusions. To determine whether the substitutions directly affected cleavage of PhrC to CSF, we tested cleavage of synthetic pro-CSF peptides that corresponded to the C terminus of PhrC and had an amino acid substitution at the -2, -3, or -4 position. The mutant pro-CSF peptides were cleaved less efficiently to CSF than the wild-type pro-CSF peptide whether they were incubated with whole cells, cell wall material, or the processing protease subtilisin or Vpr. To further define the range of amino acids that support CSF production, the amino acid at the -4 position of PhrC was replaced by the 19 canonical amino acids. Only four substitutions resulted in a >2-fold defect in CSF production, indicating that this position is relatively immune to mutational perturbations. These data revealed residues that direct cleavage of CSF and laid the groundwork for testing whether other Phr peptides are processed in a similar manner.     

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 °C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze β-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.     

Positively charged amino acids placed next to a signal sequence block protein translocation more efficiently in Escherichia coli than in mammalian microsomes

Positively charged amino acids are known efficiently to block protein secretion in Escherichia coli, when placed within a short distance downstream of a signal sequence. It is not known whether the same applies to protein secretion in eukaryotic cells, though statistical studies of signal sequences of prokaryotic and eukaryotic secretory proteins have suggested that the situation may be different in this case. Here, we show that identical charge mutations in a model protein have different effects on membrane translocation in E. coli and in mammalian microsomes, and that the ‘charge block’ effect is much more pronounced in the prokaryotic system. This finding has implications not only for our understanding of the mechanisms of protein secretion, but also points to a potential problem in the expression of eukaryotic secretory proteins in bacteria.     

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.     

Secretion of thermophilic bacterial cellobiohydrolase in Saccharomyces cerevisiae

The partial DNA sequence corresponding to the N-terminal amino acid sequence of cellobiohydrolase derived from a thermophilic anaerobe NA10 was determined. The cellobiohydrolase gene fused to the secretion signal (signal peptide and T-S region) from Saccharomyces diastaticus was expressed in an ethanologenic yeast, S. cerevisiae YIY345, under control of the glucoamylase promoter. The recombinant yeast produced cellobiohydrolase: approximately 40% of the total cellobiohydrolase activity was detected in the medium, and the remaining cellobiohydrolase was localized in the intracellular fraction. An analysis of the N-terminal amino acid sequence of the main intracellular cellobiohydrolase revealed that the signal peptide and T-S region were removed proteolytically. Alteration of the amino acid residues at the cleavage site by insertion of a Bgl II linker led to an approximately 3.5-fold increase in the total cellobiohydrolase production, but did not affect the efficiency of secretion into the medium. Cellobiohydrolase production was not repressed in the presence of glucose. The recombinant yeast hydrolyzed carboxymethyl cellulose in the medium. The results suggest the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.     

Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.     

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.     

Functional analysis of the signal-sequence processing site of yeast acid phosphatase

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.     

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.