Signal peptidase I processed secretory signal sequences: Selection for and against specific amino acids at the second position of mature protein

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

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Evaluation of signal peptide prediction algorithms for identification of mycobacterial signal peptides using sequence data from proteomic methods

Microbiology ◽

10.1099/mic.0.025270-0 ◽

2009 ◽

Vol 155 (7) ◽

pp. 2375-2383 ◽

Cited By ~ 29

Author(s):

Nils Anders Leversen ◽

Gustavo A. de Souza ◽

Hiwa Målen ◽

Swati Prasad ◽

Inge Jonassen ◽

...

Keyword(s):

Markov Model ◽

Signal Peptide ◽

Cleavage Site ◽

Hidden Markov ◽

Signal Peptidase ◽

Secreted Proteins ◽

Signal Peptides ◽

Sanger Institute ◽

Signal Peptidase I ◽

Peptide Prediction

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

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Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.1873-1881.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 1873-1881

Author(s):

S M Glaser ◽

B R Miller ◽

M G Cumsky

Keyword(s):

Amino Acids ◽

Mitochondrial Matrix ◽

Mature Protein ◽

Wild Type ◽

Inner Membrane ◽

Hydrophobic Domain ◽

Mitochondrial Inner Membrane ◽

The Matrix ◽

Inner Membrane Protein ◽

Cognate Protein

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.

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Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate

Biochemical Journal ◽

10.1042/bj2860761 ◽

1992 ◽

Vol 286 (3) ◽

pp. 761-769 ◽

Cited By ~ 31

Author(s):

F P Barry ◽

J U Gaw ◽

C N Young ◽

P J Neame

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Signal Peptidase ◽

Amino Acid Residues ◽

Terminal Sequence ◽

Binding Region ◽

Keratan Sulphate ◽

Laryngeal Cartilage ◽

Hyaluronan Binding

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

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Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans

Infection and Immunity ◽

10.1128/iai.68.12.6777-6784.2000 ◽

2000 ◽

Vol 68 (12) ◽

pp. 6777-6784 ◽

Cited By ~ 41

Author(s):

Roberto La Valle ◽

Silvia Sandini ◽

Maria Jesus Gomez ◽

Francesca Mondello ◽

Giulia Romagnoli ◽

...

Keyword(s):

Amino Acids ◽

Mononuclear Cells ◽

Opportunistic Pathogen ◽

Inducible Promoter ◽

Protein Band ◽

Signal Peptidase ◽

Pulse Field ◽

Expression Library ◽

Peripheral Blood Mononuclear ◽

Glycosylation Sites

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

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3.4.21.89 Signal Peptidase I

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.tb20774.x_3 ◽

1995 ◽

Vol 232 (1) ◽

pp. 1-2

Keyword(s):

Signal Peptidase ◽

Signal Peptidase I

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Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity

Infection and Immunity ◽

10.1128/iai.00050-20 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

G. J. Staton ◽

S. D. Carter ◽

S. Ainsworth ◽

J. Mullin ◽

R. F. Smith ◽

...

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Functional Characterization ◽

Signal Peptidase ◽

Effective Vaccine ◽

Digital Dermatitis ◽

Diverse Range ◽

Content Type ◽

Signal Peptidase I

ABSTRACT Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

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Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen

The Journal of Cell Biology ◽

10.1083/jcb.200701158 ◽

2007 ◽

Vol 177 (2) ◽

pp. 211-218 ◽

Cited By ~ 11

Author(s):

Thomas B. Nicholson ◽

Clifford P. Stanners

Keyword(s):

Amino Acids ◽

Carcinoembryonic Antigen ◽

Cell Adhesion Molecule ◽

Signal Sequence ◽

Mature Protein ◽

Functional Specificity ◽

Gpi Anchor ◽

Neural Cell Adhesion ◽

Tumorigenic Activity ◽

Necessary And Sufficient

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6–10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.

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