Signal Peptide Cleavage Research Articles

A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

Primary Effusion Lymphoma: Secretome Analysis Reveals Novel Candidate Biomarkers with Potential Pathogenetic Significance

A signal peptide missense mutation associated with nicotine dependence alters α2*-nicotinic acetylcholine receptor function

A cytosine to thymidine (C → T) missense mutation in the signal peptide (SP) sequence (rs2472553) of the nicotinic acetylcholine receptor (nAChR) α2 subunit produces a threonine-to-isoleucine substitution (T22I) often associated with nicotine dependence (ND). We assessed effects on function of α2*-nAChR (‘*’indicates presence of additional subunits) of this mutation, which could alter SP cleavage, RNA/protein secondary structure, and/or efficiency of transcription, translation, subunit assembly, receptor trafficking or cell surface expression. Two-electrode voltage clamp analyses indicate peak current responses to ACh or nicotine are decreased 2.8–5.8-fold for putative low sensitivity (LS; 10:1 ratio of α:β subunit cRNAs injected) α2β2- or α2β4-nAChR and increased for putative high sensitivity (HS; 1:10 α:β subunit ratio) α2β2- (5.7–15-fold) or α2β4- (1.9–2.2-fold) nAChR as a result of the mutation. Agonist potencies are decreased 1.6–4-fold for putative LS or HS α2(T22I)β2-nAChR or for either α2*-nAChR subtype formed in the presence of equal amounts of subunit cRNA, slightly decreased for LS α2(T22I)β4-nAChR, but increased 1.4–2.4-fold for HS α2(T22I)β4-nAChR relative to receptors containing wild-type α2 subunits. These effects suggest that the α2 subunit SP mutation generally favors formation of LS receptor isoforms. We hypothesize that lower sensitivity of human α2*-nAChR to nicotine could contribute to increased susceptibility to ND. To our knowledge this is the first report of a SP mutation having a functional effect in a member of cys-loop family of ligand-gated ion channels.

Characterization of Riemerella anatipestifer CH-1 gldJ gene and GldJ protein

Riemerella anatipestifer (RA) CH-1, a highly virulent field strain, was isolated and identified by our laboratory. The gldJ gene was conserved in RA, and it had a typical TATA promoter region and AU-rich sequence within the 5' untranslated region. The GldJ protein was an outer-membrane lipoprotein with a signal peptide cleavage site between amino acids 20 and 21. GldJ was also a member of proteins involved in gliding motility. The RA GldJ protein had 16 phosphorylation sites and 4 N-glycosylation sites. These functional sites played an important role in the posttranslational modification of the GldJ protein. In addition, the GldJ protein of RA CH-1 had strong immunogenicity. Fifteen B-cell epitopes were identified in the GldJ protein, which might be a good biological material for the development of a subunit vaccine and diagnostic reagents. The GldJ protein also had the activity of formylglycine-generating sulfatase enzyme. The 3-dimensional structure models of GldJ were constructed based on 2 templates using the SwissModel automatic modeling mode in the SWISS-MODEL workplace. Here, we characterized the RA gldJ gene and GldJ protein to contribute to the functional annotation for the GldJ protein.

Characterization of α-type carbonic anhydrase (CA) gene and subcellular localization of α-CA in the gametophytes of Saccharina japonica

To understand the CO2 concentration mechanism in the phaeophyte kelp Saccharina japonica, a full-length complementary DNA (cDNA) encoding carbonic anhydrase (CA) was cloned from the gametophytes based on the two screened clones from a suppressive subtracted cDNA library. The cDNA sequence was composed of 2,804 bp in length, including a 166-bp 5′-untranslated region (UTR), a 1,765-bp 3′-UTR, and an 873-bp open read frame. No intron separating this gene was found after comparing its cDNA and DNA sequences. The deduced precursor protein of S. japonica CA consisted of 290 amino acids with a typical signal peptide cleavage site between Gly 20 and Val 21 from the N terminus. The mature protein contained three conserved His residues chelated with a zinc ion constituting a catalytic active site. This cloned CA gene from S. japonica could be grouped into the α-type as shown by a constructed phylogenetic tree and most identity within the conserved domains characteristic of the α-CA. Quantitative real-time PCR results demonstrated that the diurnal transcription of this CA gene in the gametophytes cultured under the addition of CO2 or HCO3− were not significantly different from those cultured only with filtered air supply at any sampling time, suggesting that this CA might not be periplasmic. After preparation of polyclonal antibody with this recombinant CA in Escherichia coli, the gold immunolocalization showed that this α-CA was associated with the chloroplast envelopes and thylakoid membranes suggesting that this α-CA could provide the chloroplasts with sufficient CO2 for carbon assimilation.

Co-translational Processing of Glycoprotein 3 from Equine Arteritis Virus: N-GLYCOSYLATION ADJACENT TO THE SIGNAL PEPTIDE PREVENTS CLEAVAGE

Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes.

Signal peptide discrimination and cleavage site identification using SVM and NN

About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification.The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification.The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model.

Phylogenetic and sequence analysis of the growth hormone gene of two sturgeons, Huso huso and Acipenser Gueldenstaedtii

In this study, the cDNA Growth Hormone (cGH) of the Belugasturgeon (Husohuso) and Russian sturgeon (Acipensergueldenstaedtii) were cloned and sequenced, and phylogenetic relationships were examined using nucleic acid and amino acid sequences. The nucleotide sequence of the Beluga GH has an open reading frame of 645 nucleotides encoding a protein 214 amino acid residues. The signal peptide cleavage site was predicted to be at position 72, yeilding a signal peptide of 24aminoacid residues and a mature peptide of 190 amino acids. The cDNAsequence of the Russian sturgeon was similar to that of the Beluga cGH. The phylogenetic analysis was performed based on amino acid and DNA sequences using the neighbor oining(NJ) and Maximum parsimony (MP) method. Phylogenic trees by the two methods wereidentical in most of the clades with the high bootstrap support, and the topology of amino acid and DNA sequences showed highest similarity with mammalian sequences.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Topology and Membrane Anchoring of the Lysosomal Storage Disease-Related Protein CLN5

One late infantile variant of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL) is caused by a mutation in the CLN5 gene. CLN5 encodes a lysosomal glycoprotein whose structure and function have not yet been clearly defined. In the present study, we used epitope-tagged CLN5 to determine the topology and solubility of the CLN5 protein. Our results indicated that CLN5 is synthesized as a type II transmembrane (TM) glycoprotein with a cytoplasmic N-terminus, one TM segment, and a large luminal C-terminal domain containing an amphipathic helix (AH). The cytoplasmic and TM domains were rapidly removed following signal-peptide cleavage, and the resulting mature CLN5 was tightly associated with the lumen of the membrane through the AH. CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded by the proteasomal machinery. We experimentally define the topology of CLN5 and demonstrate the existence of an AH that anchors the protein to the membrane. Our work sheds light on the basic properties of CLN5 required to better understand its biological functions and involvement in NCL pathogenesis.

Chaperonin-mediated Protein Folding

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.

A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies.

Exclusively membrane-inserted state of an uncleavable Tat precursor protein suggests lateral transfer into the bilayer from the translocon

In bacteria, the export of proteins by the twin-arginine translocase (Tat) pathway is directed by cleavable N-terminal signal peptides. We studied the relationship between transport and maturation using a substrate, YedY, that contains an Ala > Leu substitution at the -1 position of the signal peptide. This blocks maturation and leads to the accumulation of a membrane-bound precursor form with the mature domain exposed to the periplasm. Its accumulation does not block transport of other Tat substrates, indicating that exit from the translocation channel has taken place, and the precursor protein is fir mLy integrated into the membrane bilayer. The membrane-integrated nature of the precursor, and complete absence of precursor protein in the periplasm, strongly suggest that the precursor has undergone lateral transfer into the bilayer during translocation. We propose that subsequent proteolytic processing releases the mature protein into the periplasm. A delay in processing results in an inhibition of cell growth, emphasizing a requirement for efficient maturation of Tat substrates.

Signal Peptide Cleavage from GP5 of PRRSV: A Minor Fraction of Molecules Retains the Decoy Epitope, a Presumed Molecular Cause for Viral Persistence

Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a “decoy epitope” located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the “decoy epitope” is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the “decoy epitope” sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the “decoy epitope”.

Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells

Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

Production of the Cannibalism Toxin SDP Is a Multistep Process That Requires SdpA and SdpB

During the early stages of sporulation, a subpopulation of Bacillus subtilis cells secrete toxins that kill their genetically identical siblings in a process termed cannibalism. One of these toxins is encoded by the sdpC gene of the sdpABC operon. The active form of the SDP toxin is a 42-amino-acid peptide with a disulfide bond which is processed from an internal fragment of pro-SdpC. The factors required for the processing of pro-SdpC into mature SDP are not known. We provide evidence that pro-SdpC is secreted via the general secretory pathway and that signal peptide cleavage is a required step in the production of SDP. We also demonstrate that SdpAB are essential to produce mature SDP, which has toxin activity. Our data indicate that SdpAB are not required for secretion, translation, or stability of SdpC. Thus, SdpAB may participate in a posttranslation step in the production of SDP. The mature form of the SDP toxin contains a disulfide bond. Our data indicate that while the disulfide bond does increase activity of SDP, it is not essential for SDP activity. We demonstrate that the disulfide bond is formed independently of SdpAB. Taken together, our data suggest that SDP production is a multistep process and that SdpAB are required for SDP production likely by controlling, directly or indirectly, cleavage of SDP from the pro-SdpC precursor.

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics

Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combined database of the NCBI annotations and a six-frame translation of the genome. Database searches with Mascot and X! Tandem revealed 1115 proteins identified by at least two peptides with a peptide false discovery rate below 1%. This represents 71% of the predicted proteome. So far this is the most extensive proteome study of Helicobacter pylori. Our proteogenomic approach unambiguously identified four previously missed annotations and furthermore allowed us to correct sequences of six annotated proteins. Since secreted proteins are often involved in pathogenic processes we further investigated signal peptidase cleavage sites. By applying a database search that accommodates the identification of semi-specific cleaved peptides, 63 previously unknown signal peptides were detected. The motif LXA showed to be the predominant recognition sequence for signal peptidases. The results of MS-based proteomic studies highly rely on correct annotation of protein coding genes which is the basis of conventional data analysis. However, the annotation of protein coding sequences in genomic data is usually based on gene finding software. These tools are limited in their prediction accuracy such as the problematic determination of exact gene boundaries. Thus, protein databases own partly erroneous or incomplete sequences. Additionally, some protein sequences might also be missing in the databases. Proteogenomics, a combination of proteomic and genomic data analyses, is well suited to detect previously not annotated proteins and to correct erroneous sequences. For this purpose, the existing database of the investigated species is typically supplemented with a six-frame translation of the genome. Here, we studied the proteome of the major human pathogen Helicobacter pylori that is responsible for many gastric diseases such as duodenal ulcers and gastric cancer. Our in-depth proteomic study highly reliably identified 1115 proteins (FDR<0.01%) by at least two peptides (FDR<1%) which represent 71% of the predicted proteome deposited at NCBI. The proteogenomic data analysis of our data set resulted in the unambiguous identification of four previously missed annotations, the correction of six annotated proteins as well as the detection of 63 previously unknown signal peptides. We have annotated proteins of particular biological interest like the ferrous iron transport protein A, the coiled-coil-rich protein HP0058 and the lipopolysaccharide biosynthesis protein HP0619. For instance, the protein HP0619 could be a drug target for the inhibition of the LPS synthesis pathway. Furthermore it has been proven that the motif "LXA" is the predominant recognition sequence for the signal peptidase I of H. pylori. Signal peptidases are essential enzymes for the viability of bacterial cells and are involved in pathogenesis. Therefore signal peptidases could be novel targets for antibiotics. The inclusion of the corrected and new annotated proteins as well as the information of signal peptide cleavage sites will help in the study of biological pathways involved in pathogenesis or drug response of H. pylori.

Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100°C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.

Interferon-Lambda3 (IFN-λ3) and Its Cognate Receptor Subunits in Tree Shrews (Tupaia belangeri): Genomic Sequence Retrieval, Molecular Identification and Expression Analysis

Type III IFNs (IFN-λs) constitute a new subfamily with antiviral activities by signaling through a unique receptor complex composed of IFN-λs receptor 1 (IFNλR1) and interleukin-10 receptor 2 (IL10R2). As tree shrews (Tupaia belangeri) have shown susceptiblility to several human viruses, they are a potentially important model for analyzing viral infection. However, little is known about their IFN-λs system. We used the tree shrew genome to retrieve IFN-λs and their receptor contig sequences by BLASTN and BLASTZ algorithms, and GenScan was used to scan transcripts from the putative contig sequences. RT-PCR and bioinformatic methods were then used to clone and characterize the IFN-λs system. Due to its highest identity with human IFN-λ3, we opted to define one intact IFN-λ gene, tsIFN-λ3, as well as its two receptor subunits, tsIFNλR1 and tsIL10R2. Additionally, our results showed that tsIFN-λ3 contained many features conserved in IFN-λ3 genes from other mammals, including conserved signal peptide cleavage and glycosylation sites, and several residues responsible for binding to the type III IFNR. We also found six transcript variants in the receptors: three in tsIFNλR1, wherein different extracellular regions exist in three transmembrane proteins, resulting in different affinities with IFN-λs; and three more variants in tsIL10R2, encoding one transmembrane and two soluble proteins. Based on tissue distribution in the liver, heart, brain, lung, intestine, kidney, spleen, and stomach, we found that IFN-λs receptor complex was expressed in a variety of organs although the expression level differed markedly between them. As the first study to find transcript variants in IL-10R2, our study offers novel insights that may have important implications for the role of IFN-λs in tree shrews’ susceptibility with a variety of human viruses, bolstering the arguments for using tree shrews as an animal model in the study of human viral infections.

Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to optimize an integrated experimental/informatics approach to more confidently characterize the range of post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of complementary proteolytic digestion/peptide fragmentation processes allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.