Presence Of Signal Peptide Research Articles

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SignificanceThe comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

In Silico Analysis of the Dextransucrase Obtained From Leuconostoc mesenteroides Strain IBUN 91.2.98.

The DSR-IBUN dextransucrase produced by Leuconostoc mesenteroides strain IBUN 91.2.98 has a short production time (4.5 hours), an enzymatic activity of 24.8 U/mL, and a specific activity of purified enzyme 2 times higher (331.6 U/mg) than that reported for similar enzymes. The aim of this study was to generate a structural model that, from an in silico approach, allows a better understanding, from the structural point of view, of the activity obtained by the enzyme of interest, which is key to continue with its study and industry application. For this, we translated the nucleotide sequence of the dsr_IBUN gene. With the primary structure of DSR-IBUN, the in silico prediction of physicochemical parameters, the possible subcellular localization, the presence of signal peptide, and the location of domains and functional and structural motifs of the protein were established. Subsequently, its secondary and tertiary structure were predicted and a homology model of the dextransucrase under study was constructed using Swiss-Model, performing careful template selection. The values obtained for the model, Global Model Quality Estimation (0.63), Quality Mean (-1.49), and root-mean-square deviation (0.09), allow us to affirm that the model for the enzyme dextransucrase DSR-IBUN is of adequate quality and can be used as a source of information for this protein.

Computational Clues of Immunogenic Hotspots in Plasmodium falciparum Erythrocytic Stage Vaccine Candidate Antigens: In Silico Approach.

Malaria is the most pernicious parasitic infection, and Plasmodium falciparum is the most virulent species with substantial morbidity and mortality worldwide. The present in silico investigation was performed to reveal the biophysical characteristics and immunogenic epitopes of the 14 blood-stage proteins of the P. falciparum using comprehensive immunoinformatics approaches. For this aim, various web servers were employed to predict subcellular localization, antigenicity, allergenicity, solubility, physicochemical properties, posttranslational modification sites (PTMs), the presence of signal peptide, and transmembrane domains. Moreover, structural analysis for secondary and 3D model predictions were performed for all and stable proteins, respectively. Finally, human helper T lymphocyte (HTL) epitopes were predicted using HLA reference set of IEDB server and screened in terms of antigenicity, allergenicity, and IFN-γ induction as well as population coverage. Also, a multiserver B-cell epitope prediction was done with subsequent screening for antigenicity, allergenicity, and solubility. Altogether, these proteins showed appropriate antigenicity, abundant PTMs, and many B-cell and HTL epitopes, which could be directed for future vaccination studies in the context of multiepitope vaccine design.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

Genome and Transcriptome Sequence Resources and Effector Repertoire of Pythium myriotylum Drechsler.

In silico analysis and prediction of immunogenic epitopes for pre-erythrocytic proteins of the deadly Plasmodium falciparum.

Malaria is the deadliest parasitic disease in tropical and subtropical areas around the world, with considerable morbidity and mortality, particularly due to the life-threatening Plasmodium falciparum. The present in silico investigation was performed to reveal the biophysical characteristics and immunogenic epitopes of the six pre-erythrocytic proteins of the P. falciparum using comprehensive immunoinformatics approaches. For this aim, different web servers were employed to predict subcellular localization, antigenicity, allergenicity, solubility, physico-chemical properties, post-translational modification sites (PTMs), the presence of signal peptide and transmembrane domains. Moreover, the secondary and tertiary structures of the proteins were revealed followed by refinement and validations. Finally, NetCTL server was used to predict cytotoxic T-lymphocyte (CTL) epitopes, followed by subsequent screening in terms of antigenicity and immunogenicity. Also, IEDB server was utilized to predict helper T-lymphocyte (HTL) epitopes, followed by screening regarding interferon gamma induction and population coverage. These proteins showed appropriate antigenicity, abundant PTMs as well as many CTL and HTL epitopes, which could be directed for future vaccination studies in the context of multi-epitope vaccine design.

Immunoinformatics based design and prediction of proteome-wide killer cell epitopes of Leishmania donovani: Potential application in vaccine development

Despite several extensive and exhaustive efforts, search for potential therapy against leishmaniasis has not made much progress. In the present work, we have employed mining strategy to screen Leishmania donovani proteome for identification of promising vaccine candidate. We have screened 21 potential antigenic proteins from 7960 total protein of L. donovani, based on the presence of signal peptide, GPI anchor, antigenicity prediction and substractive proteomic approach. Secondly, we have also performed comprehensive immunogenic epitope prediction from the screened 21 proteins, using IEDB-AR tools. Out of the 21 antigenic proteins, we obtained 11 immunogenic epitopes from 9 proteins. The final results revealed that four predicted epitopes namely; YPAFAALVF, VAVAATVAY, AAAPTEAAL and MYPLVAVVF, have significantly better binding potential with respective alleles and could elicits immune responses. Docking analysis using PATCHDOCK server and molecular dynamic simulation using GROMACS revealed the potential of the sequences as immunogenic epitopes. In silico studies also suggested that the epitopes occupied almost same binding cleft with the respective alleles, when compared with the reference peptides. It is also suggested from the molecular dynamic simulation data that the peptides were intact in the pocket for longer periods of time. Our study was designed to select MHC class I restricted epitopes for the activation of CD8 T cells using immunoinformatics for the prediction of probable vaccine candidate against L. donovani parasites. Communicated by Ramaswamy H. Sarma

In silico analysis of κ-theraphotoxin-Cg2a from Chilobrachys guangxiensis

κ-theraphotoxin-Cg2a is a 29- residue polypeptide extracted from the venomous glands of the Chinese earth tiger tarantula Chilobrachys guangxiensis. Plethoras of cancers are being associated with irregular functions of potassium ion channels. An extensive understanding of the toxin’s interaction with the voltage-gated potassium channels is of utmost necessity for it to be screened as a potential pharmacological molecule which may perhaps serve as toxin-based therapy to manage various cancer channelopathies. Physicochemical properties were studied, the evolutionary analysis was done to visualize the conserved domain among different toxins of tarantula family, docking studies between κ-theraphotoxin-Cg2a and a voltage-gated potassium ion channel was done by ClusPro 2.0. The presence of signal peptide was observed using PSIPRED. Cysteine – disulfide bonds present in the amino acid sequence was predicted by DiANNA server. Multiple sequence alignment illustrated conserved residues with other families of tarantula’s toxin. The docking of κ-theraphotoxin-Cg2a with the voltage-gated potassium channel was found to be interactive. The presence of cysteine –disulfide bonds were observed potentially playing a crucial role in the docking process. The interaction between the receptor and the ligand was found to be interactive which could turn out to help develop strategies to assist in creating potential pharmacological drug-based therapies.

Recombinant polypeptide of Mycobacterium leprae as a potential tool for serological detection of leprosy

Current prevention methods for the transmission of Mycobacterium leprae, the causative agent of leprosy, are inadequate as suggested by the rate of new leprosy cases reported. Simple large-scale detection methods for M. leprae infection are crucial for early detection of leprosy and disease control. The present study investigates the production and seroreactivity of a recombinant polypeptide composed of various M. leprae protein epitopes. The structural and physicochemical parameters of this construction were assessed using in silico tools. Parameters like subcellular localization, presence of signal peptide, primary, secondary, and tertiary structures, and 3D model were ascertained using several bioinformatics tools. The resultant purified recombinant polypeptide, designated rMLP15, is composed of 15 peptides from six selected M. leprae proteins (ML1358, ML2055, ML0885, ML1811, ML1812, and ML1214) that induce T cell reactivity in leprosy patients from different hyperendemic regions. Using rMLP15 as the antigen, sera from 24 positive patients and 14 healthy controls were evaluated for reactivity via ELISA. ELISA-rMLP15 was able to diagnose 79.17% of leprosy patients with a specificity of 92.86%. rMLP15 was also able to detect the multibacillary and paucibacillary patients in the same proportions, a desirable addition in the leprosy diagnosis. These results summarily indicate the utility of the recombinant protein rMLP15 in the diagnosis of leprosy and the future development of a viable screening test.

A new collagenase enzyme of the marine sponge pathogen Pseudoalteromonas agarivorans NW4327 is uniquely linked with a TonB dependent receptor

The primary pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile, recently identified as a novel strain (NW4327) of Pseudoalteromonas agarivorans, produced collagenase which degraded R. odorabile skeletal fibers. We now report the collagenase of P. agarivorans as a metalloprotease which required Ca2+ and Zn2+ as cofactors. The collagenase was a TonB dependent receptor (TBDR) having a carboxypeptidase regulatory like domain (CRLD) in the N-terminal along with an outer membrane (OM) channel superfamily domain. The genes for TBDR sub-components and collagenase formed one unified entity in the genome of P. agarivorans NW4327. This association of a collagenase with a TBDR distinguished it from all known functional collagenases till date and for the first time, established the enzymatic capability of TBDRs. Predicted TBDR model demonstrated only 15% identity with ferripyoverdin receptor and the CRLD displayed merely 24% identity with carboxypeptidase catalytic chain. Presence of signal peptide, lack of transmembrane helices, absence of N-terminal in the cytoplasmic side, extracellular localization and recovery from the culture supernatant implicated that the TBDR was secreted. Stronger binding of the collagenase with marine sponge type IV collagen than type I collagen, revealed through molecular docking, indicated higher specificity of the enzyme towards type IV collagen.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

De novo transcriptome sequencing and analysis of the cereal cyst nematode, Heterodera avenae.

The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction.

Fluorescence spectroscopy of solubleE. coliSPase I Δ2-75 reveals conformational changes in response to ligand binding

The bacterial Sec pathway is responsible for the translocation of secretory preproteins. During the later stages of transport, the membrane-embedded signal peptidase I (SPase I) cleaves the signal peptide from a preprotein. We used tryptophan fluorescence spectroscopy of a soluble, catalytically active E. coli SPase I Δ2-75 enzyme to study its dynamic conformational changes while in solution and when interacting with lipids and signal peptides. We generated four single Trp SPase I Δ2-75 mutants, W261, W284, W300, and W310. Based on fluorescence quenching experiments, W300 and W310 were found to be more solvent accessible than W261 and W284 in the absence of ligands. W300 and W310 inserted into lipids, consistent with their location at the enzyme's proposed membrane-interface region, while the solvent accessibilities of W261, W284, and W300 were modified in the presence of signal peptide, suggesting propagation of structural changes beyond the active site in response to peptide binding. The signal peptide binding affinity for the enzyme was measured via FRET experiments and the Kd determined to be 4.4 μM. The location of the peptide with respect to the enzyme was also established; this positioning is crucial for the peptide to gain access to the enzyme active site as it emerges from the translocon into the membrane bilayer. These studies reveal enzymatic structural changes required for preprotein proteolysis as it interacts with its two key partners, the signal peptide and membrane phospholipids.

Alternatively spliced variants of gamma-subunit of muscle-type acetylcholine receptor in fetal and adult skeletal muscle of mouse.

Gamma-subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse. This gene is located on chromosome 1, spans 6.5kb, and contains 12 exons and 11 introns. Previous studies have reported three transcript variants (C1-3) produced by alternative splicing; C1 contains all the 12 reported exons, C2 uses an in-frame alternate splice site in exon-2, and C3 produced by exon-5 skipping. These variants differ in their channel kinetics and opening times. In our study, we report the presence of two new transcript variants (T1 and T2) of chrng expressed in mouse postnatal day 3 and adult skeletal muscles. These transcripts contain novel first coding exon either N1 or N2. N1 is located in the 5' UTR, while N2 is an extended exon-2. 5' extension of exon-2 contains an initiation codon which produces a novel transcript variant. Either of the two exons can splice with the internal exons to produce mature transcripts making different 5' ends of the transcripts. Consequently, the proteins encoded by these two transcripts differ at N-termini. The presence of N2 exon containing transcript was further supported by the availability of EST from the database. These new variants display heterogeneous properties. They differ in the presence of signal peptide, phosphorylation, and acetylation of their amino acid residues of the new N-termini of the gamma subunit.

Alternatively Spliced Three Novel Transcripts of gria1 in the Cerebellum and Cortex of Mouse Brain

Glutamate receptor type 1 (GluR1) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors plays an important role in the expression of long-term potentiation and memory formation. GluR1 is encoded by gria1 gene containing 16 exons and 15 introns in mouse. Previous studies have reported two alternatively spliced variants of this subunit. These flip and flop variants differ enormously in their properties as well as expression. In our studies, we report the presence of three new transcripts of this gene present in the cerebellum and cortex of mouse brain produced by alternative splicing at 5' end. Four new exons are reported; N1 is located in 5' untranslated region, N2 is located in the 1st intronic region while N3 and N4 are located in the 2nd intronic region. The properties of these new exons encoding N-terminal variants are highly diverse. N1, N3 and N4 are coding while N2 is a non-coding exon and results in a truncated transcript. The existence of N2 exon containing transcript is further supported by the presence of an Expressed Sequence Tag from the database. The translated amino acid sequences of these transcripts differ in the presence of signal peptide as well as in their phosphorylation and acetylation pattern. The differences in their properties might be involved in receptor modulation.

Expression, purification and identification of gibberellin-induced cysteine-rich protein ofGymanadenia conopsea

AbstractPrimers bearing restriction enzyme sites forEcoR I andHind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of thegcgasagene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation intoEscherichia coliBL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana

Proteins entering the secretory pathway of eukaryotic cells are folded into their native structures in the endoplasmic reticulum (ER). Disruption of protein folding causes ER stress and activates signaling cascades, designated the unfolded protein response (UPR), that restore folding capacity. In mammals and yeast, the protein disulfide isomerases (PDIs) are key protein folding catalysts activated during UPR. However, little is known about the response of PDI genes to UPR in plants. In Arabidopsis thaliana, we identified 12 PDI genes that differed in polypeptide length, presence of signal peptide and ER retention signal, and the number and positions of thioredoxin and transmembrane domains. AtPDI gene expression was investigated in different tissues, in response to chemically induced UPR, and in null mutants of UPR signaling mediators (AtIRE1-2 and AtbZIP60). The expression of six AtPDI genes was significantly up-regulated by UPR and sharply attenuated by the transcription inhibitor, actinomycin D, indicating UPR induced AtPDI gene transcription. AtPDI and BIP2 (Binding protein) gene expression was not affected in the Atire1-2 mutant exposed to UPR, however, the expression of four AtPDI genes was decreased in the Atbzip60 mutant. We proposed that additional UPR signaling factors complement AtbZIP60 in the activation of AtPDI gene expression during ER stress in plants.

A New Type of Bacterial Sulfatase Reveals a Novel Maturation Pathway in Prokaryotes

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine ("Cys-type" sulfatases) or a serine ("Ser-type" sulfatases) into a Calpha-formylglycine (FGly). This conversion depends on a strictly conserved sequence called "sulfatase signature" (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.

FXYD3 (Mat-8), a New Regulator of Na,K-ATPase

Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K- as well as H,K-ATPases when expressed in Xenopus oocytes. However, in situ (stomach), FXYD3 is associated only with Na,K-ATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopus oocytes, FXYD3 modulates the glycosylation processing of the beta subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na+ and K+ of Na,K-ATPase.

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein.

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

ABSTRACTA complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte‐specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte‐specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N‐terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin‐like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.