Signal Peptide Cleavage Research Articles

Expression pattern of GASA, downstream genes of DELLA, in Arabidopsis

Separation and functional research of related components involved in gibberellins (GAs) signaling are important to clarify the mechanism of GA functioning. Research on the downstream components of DELLA, the key factor of the GA signaling pathway, is limited at present. GASA (GA-Stimulated in Arabidopsis) family contains 15 genes usually regulated by GA in Arabidopsis thaliana. All GASA proteins have a cleavable signal peptide in N terminus and a conserved GASA domain including 12 cysteines in C terminus. RT-PCR analysis revealed that the expression of GASA4 and GASA6 were down-regulated, but GASA1 and GASA9 were up-regulated in the DELLA mutants, gai-t6 and rga-24, as well as the double mutant, consisting with the results that GASA4 and GASA6 were induced, but GASA1 and GASA9 were inhibited by exogenous GA3. In addition, the expression patterns of other GASA genes were regulated by GA and ABA, separately or cooperatively. Most of GASA genes were expressed in roots, stems, leaves, flowers and developing siliques. GUS gene driven by the promoters of GASA6, GASA7, GASA8, GASA9, GASA10, GASA11 and GASA12 were used as reporters and it was found that all GASA genes expressed in the growing and differentiating organs and abscission zones, suggesting the role of these genes in cell growth and differentiation. This study provided an important basis for functional study of the GASA gene family in the GA and ABA signaling pathway.

Genetic Toggling of Alkaline Phosphatase Folding Reveals Signal Peptides for All Major Modes of Transport across the Inner Membrane of Bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.

Discovery and Validation of Novel Peptide Agonists for G-protein-coupled Receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

Flanking signal and mature peptide residues influence signal peptide cleavage

BackgroundSignal peptides (SPs) mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I), and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i) eukaryotes (Euk) (ii) Gram-positive (Gram+) bacteria, and (iii) Gram-negative (Gram-) bacteria.ResultsIn this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups.ConclusionWe conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

Role of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein‐specific signal peptidase II (LspA) in localization and physiological function of lipoprotein MsmE in Streptococcus mutans

To clarify the role that prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) play in the physiological function of MsmE, we constructed lgt-deficient and lspA-deficient mutants of Streptococcus mutans 109c and examined the potential role of Lgt and LspA in membrane anchoring and growth in a melibiose medium of S. mutans. The lgt-, lspA-, and msmE-deficient mutants of S. mutans 109c were constructed by double-crossover recombination of their respective genes. Localization of MsmE was demonstrated by Western blot analysis with an MsmE antiserum. The growth of S. mutans cells was examined in a Trypton medium containing melibiose or glucose. In the S. mutans lgt mutant, localization of the surface lipoprotein MsmE changed with the culture supernatant. The growth of the S. mutans lgt and lspA mutants was remarkably reduced in the melibiose medium; however, growth was recovered in the strains complemented with the lgt or the lspA gene. Therefore, lipid-modification by Lgt and subsequent signal peptide cleavage by LspA were crucial for membrane anchoring and the physiological function of MsmE in S. mutans. These results demonstrate that MsmE is required for melibiose metabolism in S. mutans and that modification by Lgt and LspA are important processes for the physiological function of MsmE.

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn(18) and Asn(33)) of the human delta-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn(33) was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the delta-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

A Dentin Sialophosphoprotein Mutation That Partially Disrupts a Splice Acceptor Site Causes Type II Dentin Dysplasia

The dentin sialophosphoprotein ( DSPP) gene on chromosome 4q21.3 encodes the major noncollagenous protein in tooth dentin. DSPP mutations are the principal cause of dentin dysplasia type II, dentinogenesis imperfecta type II, and dentinogenesis imperfecta type III. We have identified a DSPP splice junction mutation (IVS2-6T>G) in a family with dentin dysplasia type II. The primary dentition is discolored brown with severe attrition. The mildly discolored permanent dentition has thistle-shaped pulp chambers, pulp stones, and eventual pulp obliteration. The mutation is in the sixth nucleotide from the end of intron 2, perfectly segregates with the disease phenotype, and is absent in 200 normal control chromosomes. An in vitro splicing assay shows that pre-mRNA splicing of the mutant allele generates wild-type mRNA and mRNA lacking exon 3 in approximately equal amounts. Skipping exon 3 might interfere with signal peptide cleavage, causing endoplasmic reticulum stress, and also reduce DSPP secretion, leading to haploinsufficiency.

Conserved and Variant Molecular and Functional Features of Multiple Egg Yolk Precursor Proteins (Vitellogenins) in White Perch (Morone americana) and other Teleosts

Three complete cDNAs encoding different forms of vitellogenin (Vtg) were isolated from a white perch (Morone americana) liver cDNA library and characterized with respect to immunobiochemical and functional features of the three Vtgs and their product yolk proteins (YPs) in this species and in the congeneric striped bass (Morone saxatilis). The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb using the current nomenclature for multiple teleost Vtgs. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Mapping of peptide sequences from the purified Vtgs and their derived YPs to Vtg sequences deduced from the cDNAs definitively identified the white perch VtgAa, VtgAb, and VtgC proteins. Detailed comparisons of the primary structures of each Vtg with partial or complete sequences of Morone yolk proteins or of Vtgs from other fishes revealed conserved and variant structural elements of teleost Vtgs with functional significance, including, as examples, signal peptide cleavage sites, dimerization sites, cathepsin D protease recognition sites, and receptor-binding domains. These comparisons also yielded an interim revision of the classification scheme for multiple teleost Vtgs.

ERp16, an Endoplasmic Reticulum-resident Thiol-disulfide Oxidoreductase: BIOCHEMICAL PROPERTIES AND ROLE IN APOPTOSIS INDUCED BY ENDOPLASMIC RETICULUM STRESS

We have characterized the properties and putative role of a mammalian thioredoxin-like protein, ERp16 (previously designated ERp18, ERp19, or hTLP19). The predicted amino acid sequence of the 172-residue human protein contains an NH(2)-terminal signal peptide, a thioredoxin-like domain with an active site motif (CGAC), and a COOH-terminal endoplasmic reticulum (ER) retention sequence (EDEL). Analyses indicated that the mature protein (comprising 146 residues) is generated by cleavage of the 26-residue signal peptide and is localized in the lumen of the ER. Biochemical experiments with the recombinant mature protein revealed it to be a thioldisulfide oxidoreductase. Its redox potential was about -165 mV; its active site cysteine residue Cys(66) was nucleophilic with a pK(a) value of approximately 6.6; it catalyzed the formation, reduction, and isomerization of disulfide bonds, with the unusual CGAC active site motif being responsible for these activities; and it existed as a dimer and underwent a redox-dependent conformational change. The observations that the redox potential of ERp16 (-165 mV) was within the range of that of the ER (-135 to -185 mV) and that ERp16 catalyzed disulfide isomerization of scrambled ribonuclease A suggest a role for ERp16 in protein disulfide isomerization in the ER. Expression of ERp16 in HeLa cells inhibited the induction of apoptosis by agents that elicit ER stress, including brefeldin A, tunicamycin, and dithiothreitol. In contrast, expression of a catalytically inactive mutant of ERp16 potentiated such apoptosis, as did depletion of ERp16 by RNA interference. Our results suggest that ERp16 mediates disulfide bond formation in the ER and plays an important role in cellular defense against prolonged ER stress.

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.

Characterization of Recombinant Human Prolyl 3-Hydroxylase Isoenzyme 2, an Enzyme Modifying the Basement Membrane Collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.

Functional analysis of an ADAMTS10 signal peptide mutation in Weill-Marchesani syndrome demonstrates a long-range effect on secretion of the full-length enzyme

We report the identification and functional analysis of the first missense ADAMTS10 mutation (c.73G>A; p.Ala25Thr) causing recessive Weill-Marchesani syndrome (WMS). The Ala25 residue affected by the missense mutation is at the -1 position relative to the ADAMTS10 signal peptidase cleavage site. p.Ala25Thr substituted full-length ADAMTS10 showed consistent and significantly diminished secretion in both HEK293F and Cos-1 cells. However, a C-terminally truncated construct lacking the ancillary domain and containing only the signal peptide, the propeptide and the catalytic domain (p.Ala25Thr Pro-Cat) was efficiently secreted in both HEK293F cells and Cos-1 cells. Edman degradation of purified p.Ala25Thr Pro-Cat and p.Ala25Thr substituted full-length ADAMTS10 from HEK293F cells demonstrated correct signal peptide processing. Thus, the p.Ala25Thr substitution hinders secretion of full-length ADAMTS10, but not Pro-Cat from cells, yet permits signal peptide removal. We infer that folding of the complex C-terminal ancillary domain is the rate-limiting step in biosynthesis of ADAMTS10, and that it (but not Pro-Cat) is sensitive to subtle changes in efficiency of signal peptide cleavage. These observations represent an unprecedented effect of a signal peptide mutation and support a model in which the initial cotranslational processing events during protein biosynthesis can have long-range effects on protein folding and secretion.

Expression of Bioactive Single-Chain Murine IL-12 in Transgenic Plants

Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic. However, its clinical application is limited in part by lack of an effective bioproduction system for this complex heterodimeric glycoprotein. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. To test the potential of plants to effectively produce bioactive IL-12, we developed transgenic tobacco plant lines and derived root cultures yielding high levels of mouse IL-12 (MuIL-12). Functional IL-12 is a heterodimer consisting of two disulfide-linked subunits, p35 and p40. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of MuIL-12. Plant-derived single-chain MuIL-12 was characterized and purified for in vitro bioactivity assays. Our results demonstrated precise cleavage of the endogenous mouse p40 signal peptide in plants as well as addition of N-linked glycans. Plant-derived MuIL-12 triggered induction of interferon-gamma (IFN-gamma) secretion from mouse splenocytes and stimulated splenocyte proliferation with comparable activities to those observed for commercially available animal cell-derived MuIL-12. These studies indicate that plants produce fully functional MuIL-12 at levels compatible with commercial production and may serve as an effective bioproduction platform for bioactive IL-12s from other species for human or veterinary vaccine and therapeutic applications.

Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization

Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for beta-GTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for c-di-GMP [bis-(3',5')-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.

Escherichia coli Signal Peptide Peptidase A Is a Serine-Lysine Protease with a Lysine Recruited to the Nonconserved Amino-Terminal Domain in the S49 Protease Family

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe‐antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression. Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy. HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.

Effects of neighboring sequence environment in predicting cleavage sites of signal peptides

Signal peptide has a pivotal role in the translocation of secretory protein. Some models have been designed to predict its cleavage site. It is reported that the cleavage site has relationship with the neighboring sequence environment, i.e., hydrophobic core h-region, and the specific patterns in c-region. In some studies, this finding does facilitate the prediction of cleavage site. However, in these models, sequence environment information is merely taken account of as model inputs and no detailed investigation into its effect on the prediction of cleavage site has been made. In this work, we analyze the constraint on cleave site placed by the hydrophobic core of signal peptide and then use it to improve the performance of the signal peptide cleavage site prediction. Our model is designed as follows: firstly, a sliding window is used to scan sample and artificial neural network (ANN) is employed to give cleavage site/non-cleavage site scores. Then, based on an estimated hydrophobic h-region a correcting function is proposed to improve the prediction result, in which the sequence environment is taken into account. A trend of cleavage site is indicated by our analysis for each position, which is consistent with experimental findings. Through this correcting step, the improvement of prediction accuracy is over 7%. It therefore demonstrates the neighboring sequence environment is helpful for determination of cleavage site. Program written in Matlab can be downloaded from http://www.scucic.cn/combined model/source code.html.

The Non-catalytic N-terminal Extension of Formylglycine-generating Enzyme Is Required for Its Biological Activity and Retention in the Endoplasmic Reticulum

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.