SPase Activity Research Articles

2-O-α-D-glucosyl glycerol production by whole-cell biocatalyst of lactobacilli encapsulating sucrose phosphorylase with improved glycerol affinity and conversion rate

Background2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing.ResultsHere, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01% in the total product. However, the LrSP exhibited an initial forward reaction rate of 11.83/s and reached equilibrium of 56.90% at 110 h, indicating low glycerol affinity and conversion rate. To improve catalytic efficiency, the LrSP was overexpressed in Lb. paracasei BL-SP, of which the intracellular SPase activity increased by 6.67-fold compared with Lb. reuteri SDMCC050455. After chemically permeabilized with Triton X-100, the whole-cell biocatalysis of Lb. paracasei BL-SP was prepared and showed the highest activity, with the initial forward reaction rate improved to 50.17/s and conversion rate risen to 80.79% within 17 h. Using the whole-cell biocatalyst, the final yield of 2-αGG was 203.21 g/L from 1 M sucrose and 1 M glycerol.ConclusionThe food grade strain Lb. paracasei was used for the first time as cell factory to recombinantly express the LrSP and construct a whole-cell biocatalyst for the production of 2-αGG. After condition optimization and cell permeabilization, the whole-cell biocatalyst exhibited 23.89% higher equilibrium conversion and 9.10-fold of productivity compared with the pure enzyme catalytic system. This work would provide a reference for large-scale bioprocess of 2-αGG.

TRANSGLYCOSYLATION ACTIVITY AND CHARACTERIZATION OF RECOMBINANT SUCROSE PHOSPHORYLASE FROM LEUCONOSTOC MESENTEROIDES MBFWRS-3(1) EXPRESSED IN ESCHERICHIA COLI

Objective: Sucrose phosphorylase (SPase) is an enzyme that catalyzes the transfer of glucosyl to various acceptor molecules. Distinct types of SPaseshave been reported, and their transglycosylase activities have been shown to differ. In general, glycosylation is a process that is used to modifybioactive compounds. As such, glycosylation can increase the chemical stability of compounds and improve their characteristics such as reduce strongsmell and sour taste. We previously cloned recombinant SPase (SPaseWRS-3[1]) from Leuconostoc mesenteroides MBFWRS-3[1] in Escherichia coli.In the current study, we aimed to characterize SPaseWRS-3 and determine its transglycosylation activity using benzoic acid (BA), ascorbic acid, andkojic acid (KA).Methods: Expression analyses were conducted in lysogeny broth (LB) medium supplemented with tetracycline and expression was induced usingisopropyl-β-d-thiogalactopyranoside. The characteristics of the 56 kDa recombinant SPase (rec-SPase) were confirmed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Rec-SPase activity was determined spectrophotometrically using sucrose as the substrate and NADPHas the end-product at 340 nm. Transglycosylation activity was evaluated using thin-layer chromatography (TLC) on silica gel plates.Results: Our results demonstrated that the rec-SPase had an activity of 98.52% relative to the reference SPase (ref-SPase). BA and KA were determinedto undergo glucosyl transfer by rec-SPase using ref-SPase, as observed with TLC. Our findings are consistent with those reported previously for theSPase isolated from L. mesenteroides.Conclusion: Recombinant SPase activity is comparable to reference SPase activity. Our study could be the initial study to deeply observe SPase activityin other substrates as well.

Stable Signal Peptides and the Response to Secretion Stress in Staphylococcus aureus.

ABSTRACTProtein secretion is essential, but how it is managed is poorly understood. In bacteria, most secreted proteins require release from the outer surface of the cytoplasmic membrane by type I signal peptidase (SPase), which cleaves the mature protein from its membrane-bound N-terminal signal peptide. As the first step that occurs outside the protected cytoplasmic environment and because insufficient activity can rapidly result in the toxic accumulation of preproteins, the activity of SPase is expected to be closely monitored and perhaps supplemented when insufficient. Indeed, we previously demonstrated that inhibition of SPase in Staphylococcus aureus results in derepression of the ayrRABC operon, which encodes an alternate mechanism to release proteins. However, in this case, the proteins are released with partially intact signal peptides, with the exception of IsaA, which is released with a virtually intact signal peptide. Here we show that mutation of AyrA [ayrA(R233K)] results in constitutive derepression of ayrRABC and that mutation of IsaA’s signal peptide [isaA(K2Q)] results in hyperderepression upon SPase inhibition, which also requires AyrA. Further studies demonstrate that the inducing signal for ayrRABC derepression is accumulation of a subset of preproteins with signal peptides that are stable toward further processing and that the signal is critically amplified by the K2Q mutation and relayed to AyrR by AyrA. These results elucidate the mechanism by which S. aureus monitors and responds to secretion stress. The presence of ayrRA in other bacteria suggests that it may represent a general strategy linking membrane stress to appropriate transcriptional responses.

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.

Origins of Yersinia pestis sensitivity to the arylomycin antibiotics and the inhibition of type I signal peptidase.

Yersinia pestis is the etiologic agent of the plague. Reports of Y. pestis strains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported that Y. pestis strain KIM6+, unlike most Enterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range of Y. pestis strains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of the Y. pestis SPase for the antibiotic and that alterations to each component of the Y. pestis lipopolysaccharide-O antigen, core, and lipid A-make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity against Y. pestis and potentially other Gram-negative pathogens.

Defective maintenance of pH of stratum corneum is correlated with preferential emergence and exacerbation of atopic-dermatitis-like dermatitis in flaky-tail mice

BackgroundNeutralization of stratum corneum (SC) pH, which is induced by a variety of stimuli, such as scratching, use of soap and inflammation, can stimulate activity of serine protease (SPase). Activation of SPase induces production of thymic stromal lymphopoietin (TSLP) through protease-activated receptor-2. Both reduced expression of natural moisturizing factors, which are required for maintenance of SC pH, and the preferential development of atopic dermatitis (AD)-like dermatitis are found in flaky-tail mice (FTM) with a loss-of-function mutation in filaggrin. ObjectiveWe examined possible correlations between disturbance of responses to an exogenous stimulus of SC neutralization and the preferential emergence of AD-like dermatitis in FTM. MethodsFTM and wild-type mice (C57BL/6) were subjected to an SC-neutralization stimulus via application of 1,1,3,3-tetramethylguanidine (TMG). TMG was applied to young mice at a time when FTM had not yet developed significant dermatitis, and we examined their ability to maintain SC acidity and several parameters associated with AD-like dermatitis. ResultsThe recovery of SC pH after the application of TMG was delayed in FTM, presumably because of unchanged expression of Na+/H+ antiporter 1, which is involved in maintenance of SC acidity. Cutaneous inflammation with elevated SPase activity and serum levels of TSLP, thymus and activation-regulated chemokine and IgE were induced only in TMG-treated FTM. ConclusionOur results suggest that defective maintenance of pH of SC is correlated with emergence and exacerbation of AD-like dermatitis in FTM.

Pseudomonas aeruginosa Possesses Two Putative Type I Signal Peptidases, LepB and PA1303, Each with Distinct Roles in Physiology and Virulence

Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P. aeruginosa possesses two noncontiguous SPase homologues, LepB (PA0768) and PA1303, which share 43% amino acid identity. Reverse transcription (RT)-PCR showed that both proteases were expressed, while a FRET-based assay using a peptide based on the signal sequence cleavage region of the secreted LasB elastase showed that recombinant LepB and PA1303 enzymes were both active. LepB is positioned within a genetic locus that resembles the locus containing the extensively characterized SPase of E. coli and is of similar size and topology. It was also shown to be essential for viability and to have high sequence identity with SPases from other pseudomonads (≥ 78%). In contrast, PA1303, which is small for a Gram-negative SPase (20 kDa), was found to be dispensable. Mutation of PA1303 resulted in an altered protein secretion profile and increased N-butanoyl homoserine lactone production and influenced several quorum-sensing-controlled phenotypic traits, including swarming motility and the production of rhamnolipid and elastinolytic activity. The data indicate different cellular roles for these P. aeruginosa SPase paralogues; the role of PA1303 is integrated with the quorum-sensing cascade and includes the suppression of virulence factor secretion and virulence-associated phenotypes, while LepB is the primary SPase.

Type I Signal Peptidase and Protein Secretion in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection.

Type I Signal Peptidase and Protein Secretion inStaphylococcus epidermidis

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the β-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

Molecular and functional characterization of type I signal peptidase from Legionella pneumophila.

Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.

A retinoblastoma susceptibility gene product, RB, targeting protease is regulated through the cell cycle

Degradation of cyclin B and cyclin-dependent kinase inhibitor, p27, at a specific time has been shown to play a critical role in regulating the cell cycle. SPase, a nuclear and cytosol protease with cathepsin B- and L-like proteolytic activity, has been identified in several cell lines. This proteolytic enzyme selectively degraded nuclear proteins such as retinoblastoma susceptibility gene product, RB, and transcription factor, SP-1. High levels of SPase activity were detected at the G 1/S, moderate levels at the G 1 and S phases, and undetectable activity at the M phase of synchronized CV-1 cells, suggesting that SPase activity is regulated through the cell cycle. Degradation of RB correlated with SPase activity throughout the cell cycle, suggesting that SPase regulates RB, which has a functional role in regulating cell cycle. These results demonstrated that SPase plays an integral role in regulating the nuclear regulator, RB, in controlling cell cycle progression.

Subcellular distribution of two enzymes with neuropeptide converting activity in rat spinal cord

The subcellular distribution of a dynorphin converting enzyme (DYNase) and a substance P endopeptidase (SPase) has been examined in rat spinal cord using differential and density gradient centrifugation. The DYNase activity was highest in a 10,000 g supernatant after differential centrifugation. In the step gradient, the highest activity was found in synaptosomal fractions. In the continuous density gradient using a synaptosomal lysate as starting material, peak activities were found in the top fractions and at 0.8–1.05 M sucrose. This distribution is compatible with a localization in a soluble compartment and to synaptosomes. The SPase activity was highest in the P1 (nuclear) and P2 (mitochondrial) fractions, suggesting it is mainly membrane bound during these conditions. In a step gradient centrifugation (0.32/0.8/1.4 M sucrose), most of the activity was found in synaptosomal (0.8 M) fractions. After partial osmotic lysis of the synaptosomal fraction and centrifugation on a continuous density gradient (0.1–1.4 M sucrose), peaks of SPase activity were found around 0.55 M, possibly representing material associated with neuronal or plasma membrane vesicles, and at 1.0–1.25 M, representing synaptosomes, and/or mitochondria.

High performance liquid chromatography for the detection of homozygotes and heterozygotes of Niemann-Pick disease.

A method is developed for the detection of homozygotes and heterozygote carriers of Niemann-Pick disease type A by employing a high performance liquid chromatography (HPLC) technique for the measurement of sphingomyelin (SP) as well as for the assay of the enzyme sphingomyelinase (SPase) in skin fibroblasts and amniotic fluid cell cultures. A simple, less than 10-min, HPLC of the lipid extract of fibroblasts (about 2 x 10(6) cells) and measurement of the ratio of the area of SP peak to that of other phospholipids present in the cell-lipid extract show accumulation of SP in Niemann-Pick type A fibroblasts compared to normal. The SPase assay was performed by HPLC measurement of the substrate SP before and after hydrolysis by the enzyme. By employing this technique, it was shown that cells from patients with Niemann-Pick disease type A have less than 10% of the normal SPase activity, whereas the heterozygote carriers of this disease have about 50% of the normal activity.