Cloning And Homology Modeling Of A Serine Protease Gene .
Ann Microbiol (2011) 61:511–516DOI 10.1007/s13213-010-0166-5ORIGINAL ARTICLECloning and homology modeling of a serine protease gene(PrC) from the nematophagous fungus Clonostachys roseaLianming Liang & Jinkui Yang & Juan Li &Yuanyuan Mo & Li Li & Xinying Zhao & Ke-Qin ZhangReceived: 7 May 2010 / Accepted: 10 November 2010 / Published online: 25 November 2010# Springer-Verlag and the University of Milan 2010Abstract The nematophagous fungus Clonostachys roseacan parasitize nematodes as well as insects and severalfungi. Proteases play critical roles during infection and areconsidered important virulence factors in these fungi. Inthis study, a cuticle-degrading serine protease gene (PrC)was cloned for the first time from C. rosea. The genecontained three introns and four exons and encodes apolypeptide of 386 amino acid residues. The mature proteinis 277 amino acid residues long and contains a conservedmotif shared by peptidase S8 family members. ItsN-terminal amino acid residues showed a high degree ofsequence similarity with serine proteases from nematophagous and entomopathogenic fungi. Based on the PrCamino acid sequence, the three-dimensional structure hasbeen predicted and compared with that of protease K. Ourresults provide a basis for further understanding themolecular mechanism of C. rosea infection of nematodes.Such knowledge could be explored for improving bygenetic engineering the effectiveness of the use of fungalinfections to control parasitic nematodes.Keywords Clonostachys rosea . Cuticle-degradingprotease . Gene cloning . Homology modeling . SequenceanalysisLianming Liang and Jinkui Yang contributed equally to this work.Electronic supplementary material The online version of this article(doi:10.1007/s13213-010-0166-5) contains supplementary material,which is available to authorized users.L. Liang : J. Yang : J. Li : Y. Mo : L. Li : X. Zhao :K.-Q. Zhang (*)Laboratory for Conservation and Utilization of Bio-Resources,and Key Laboratory for Microbial Resourcesof the Ministry of Education, Yunnan University,Kunming 650091, People’s Republic of Chinae-mail: kqzhang111@yahoo.com.cnIntroductionPlant-parasitic nematodes cause serious damages to crops,worth more than US 100 billion per year globally (Sasserand Freekman 1987). Methods for nematode controlinvolve nematicides, crop rotation and biological agents.As the environment has been polluted by chemicalpesticides, biological control methods using nematophagous fungi, the natural enemy of nematodes, are attractingincreasing attention (Siddiqui and Mahmood 1996;Nordbring-Hertz et al. 2000).The nematode cuticle is a thin and flexible exoskeleton,composed primarily of proteins, including collagens (Coxet al. 1981; Maizels et al. 1993). At the early stage ofnematode infection by nematophagous fungi, penetration ofthe nematode cuticle through combined mechanical activityand hydrolytic enzymes has been proposed to be crucial(Huang et al. 2004). Extracellular serine proteases secretedby fungi can degrade the nematode cuticle and help thepenetration process. During the past several years, severalcuticle-degrading proteases have been purified and characterized from different nematophagous fungi, includingArthrobotrys oligospora (Tunlid et al. 1994; Zhao et al.2004), Metacordyceps chlamydosporia (syn. Verticilliumchlamydosporium) (Segers et al. 1994), and Lecanicilliumpsalliotae (St Leger et al. 1987).Clonostachys rosea (syn. Gliocladium roseum) belongsto the fungal family Bionectriaceae (Blaxter et al. 1992). Itcolonizes living plants as an endophyte, digests materials inthe soil as a saprophyte, and is also known to be a parasiteof other fungi and nematodes (Toledo et al. 2006). Itproduces a wide range of volatile organic compounds thatare toxic to other microbes (Stinson et al. 2003). As aresult, it is of great interest as a biological control agent. C.rosea infects nematodes via conidia that are capable of
512attaching to nematode cuticle, and producing germ tubesthat penetrate the host cuticle and kill the host (Zhang et al.2008). In our previous reports, a protease called PrC wasisolated from C. rosea, and its biochemical properties werecharacterized (Li et al. 2006). PrC was found to be highlysensitive to phenylmethanesulfonyl fluoride (PMSF) andcould degrade a broad range of substrates including casein,gelatin and nematode cuticle (Li et al. 2006). However, itsencoding DNA sequence information was unknown and theputative protein structure was not investigated. In thisstudy, the gene encoding PrC was cloned and compared toother serine proteases from nematophagous and entomopathogenic fungi. Furthermore, we modeled the threedimensional (3D) structure of PrC and compared it withthat of protease K.Materials and methodsStrainsThe isolate (YMF 1.00611) of Clonostachys rosea used inthis study was isolated originally from field soil samples inYunnan Province and has been deposited in the ChinaGeneral Microbiological Culture Collection Center(CGMCC 0806). It is maintained on potato dextrose agar(PDA) medium at 26 C for routine culture.Escherichia coli DH 5α was used in all DNA manipulations and grown in Luria-Bertani (LB) medium.Ann Microbiol (2011) 61:511–516cations were carried out following the manual of thecommercial kit. The primers are listed in Table 1.Cloning and sequencingThe PCR products were purified from a 1% agarose gelusing DNA fragment purification kit ver. 2.0 (Takara,Shiga, Japan) and subcloned into pMD18-T Vector(Takara). The plasmid DNA was sequenced using an ABI3730 automated DNA sequencer (Perkin-Elmer, Fremont,CA) with four fluorescent dyes. Raw sequences wereassembled using seqman of the DNAStar (Lasergene,Madison, WI) software package.Sequence analysisDatabase searches were performed using BlastX (http://www.ncbi.nlm.nih.gov/BLAST). Signal sequence prediction wasperformed using Signal P (http://www.cbs.dtu.dk/services/signalP). Potential post-translational modification sites of theprotease were identified through comparisons with thedatabase PROSITE (http://www.expasy.ch/prosite/). Fifteenreported cuticle-degrading protease sequences were alignedwith PrC using ClustalX2.0. A phylogenetic tree wasconstructed by the neighbor joining (NJ) method using theMEGA program 4.1 (Tamura et al. 2007). Bootstrap analysiswith 1,000 replicates was used to estimate the relativesupport for clades produced by the NJ analysis.Homology modelingAmplification of the nucleotide sequenceThe fungus C. rosea was cultured in PL-4 liquid medium(Yang et al. 2005a) on a rotary shaker (150 rpm) at 28 C for6 days. Genomic DNA was extracted from the myceliumusing an E.Z.N.A. Fungal DNA Mini Kit (Omega BioTek, Norcross, GA) according to the manufacturer’sprotocol.A pair of degenerate primers (p245 and p248) wasdesigned to amplify the conserved sequence of proteasePrC from C. rosea, according to the conserved sequences ofserine proteases from nematophagous and entomopathogenic fungi (Wang et al. 2006). The genomic DNA wasused as template, and PCR conditions followed thosedescribed in a previous report (Yang et al. 2005b). Afterobtaining the conserved sequence of the PrC gene, the 5′and 3′ unknown sequences of the protease gene wereamplified using a DNA walking SpeedUp kit (Seegene,Seoul, Korea). Two groups of three primers were designedaccording to the conserved sequence of PrC: primers 5sp1,5sp2, and 5sp3 were used to amplify the sequence upstreamof the 5′ region and primers 3sp1, 3sp2, and 3sp3 for thesequence downstream of the 3′ region. The PCR amplifi-The sequence of mature PrC was used in homologymodeling. Homology modeling was performed using theSWISS-PROT program (http://swissmodel.expasy.org//SWISS-MODEL.html; (Guex and Peitsch 1997; Schwedeet al. 2003; Arnold et al. 2006) with the 2.5 Å proteinase Kcoordinate (1pfgA) (Saxena et al. 1996) as a template.Pymol Ver. 0.99 (DeLano 2002) was used in the visualization and analysis of the structure. All residues but the lastthree were used in the modeling.Table 1 Primers used for PCR amplificationPrimerSequences AACAA
Ann Microbiol (2011) 61:511–516Results and discussionCloning of the cuticle-degrading serine protease PrCA 1,074-bp PCR product was successfully amplified usingprimers p245 and p248. In silico analysis indicated that thesequence does not contain the full open reading frame (ORF)of the protease when aligned with other serine protease.Consequently, DNA walking was performed based on thesequence obtained. A 710 bp fragment from the 5′ upstreamregion and a 784 bp fragment from the 3′ downstream regionof the conserved sequence were amplified. Finally, the threefragments were assembled into a sequence of 2,568 bpcontaining the complete ORF of the PrC gene. The full lengthnucleotide sequence of PrC has been submitted to GenBank,under accession number GQ149467.Sequence analysisThe sequence of the gene PrC comprises three introns andfour exons (Supplementary Fig. 1). The cDNA sequence(1,161 bp) encodes a polypeptide of 386 amino acid residues,including a conserved signature motif of the peptidase S8family. Alignment with other fungal protease sequencesrevealed that PrC is a typical secreted fungal serine protease,with a pre-pro sequence. The whole polypeptide of PrCcontains a signal peptide of 15 amino acid residues and apro-peptide of 94 amino acid residues, indicating that thisprotease is a secreted protease, similar to several otherreported serine proteases from nematophagous fungi. Themature protease contains 277 amino acids, with a predictedmolecular weight of 27 kDa, and pI of 6.82. The molecularweight of the purified PrC is 33 kDa (Li et al. 2006)—higherFig. 1 Phylogenetic tree showing the relationship betweena cuticle-degrading serineprotease (PrC) and other cuticledegrading proteases fromnematophagous fungi. The twobacterial serine proteases,subtilisin BPN’ from Bacillusamyloliquefaciens and subtilisinCarlsberg from Bacillus licheniformis, were used as outgrouptaxa. Confidence values wereassessed from 1,000 bootstrapreplicates of the originalsequence data513than the predicted value. The molecular weight differencesuggests that PrC is likely modified after translation. The firstten amino acids of PrC are ATQTGAPWGI, differing by twoamino acids from the N-terminal sequence of a previouslyobtained protease PrC (ATQSNAPWGL) (Zhao et al. 2004;Li et al. 2006). This indicates genetic polymorphisms at theN-terminal end of PrC and that more than one PrC genelikely exists in the genome. The PCR conditions usedamplified only one PrC gene. This is consistent with findingswith the proteases from another nematode-trapping fungus,A. oligospora. Disruption of the gene PII in A. oligosporahas only a limited effect on the pathogenicity of A.oligospora. However, mutants containing additional copiesof the PII gene developed more infection structures and had agreater speed of capturing and killing nematodes than wildtype strains (Åhman et al. 2002). These observations suggestthat duplications of protease genes contributing to fungalinfection might be common in fungal pathogens.When scanning the sequence of mature PrC in theprosite database, the three active site motifs of serineproteases were found at positions 35–46 (AFIIDTGIytsH,subtilase asp), 70–80 (HGThVAGtVGG, subtilase his) and223–233 (GTSmAsPhVAG, subtilase ser). Asp39, His69and Ser225 make up the catalytic triad of PrC. An asnglycosylation motif at 131–134 (NMSL) and an alkalinephosphatase active site motif at 96–104 (VlDSSGSGT)were also found in the sequence of PrC.Comparison of PrC with other serine proteases isolatedfrom nematophagous and entomopathogenic fungiPrC showed high amino acid sequence identities (41–52%)with cuticle-degrading proteases from nematophagous and
514entomopathogenic fungi (Supplementary Fig. 2). Thealignment suggests that PrC is a typical serine protease.To standardize the description, all sequences in the alignmentare numbered according to the reference sequence AB120125,with gaps in AB120125 numbered 1, 2 etc. The analysisshows that the catalytic triads (Asp187, His224 and Ser379),as well as the S1 (residues 283–286, 309–313, 377–381) andS4 (residues 255–259, 262, 283–287, 292) substrate-bindingsites are highly conserved.Based on the alignment of only the mature proteasesfrom nematophagous and entomopathogenic fungi, aphylogenetic tree (Fig. 1) was constructed using Mega 4.1(Tamura et al. 2007). Two bacterial serine proteases,subtilisin BPN’ from Bacillus amyloliquefaciens andsubtilisin Carlsberg from B. licheniformis, were used asoutgroup. The 16 proteases were clustered into two clades.Clade I was composed of nine proteases from nematodetrapping fungi, and clade II was composed of six proteasesfrom parasitic fungi. As expected, the aligned sequencesrevealed that the two clusters of proteases were moreconserved within each clade than between the two clades.In clade II, all the signal peptides were 15 amino acids long,and the signal peptides in clade I had 2–6 additional aminoacids. Some conserved indels between the two clades werefound in both the pro-peptide region and in the matureproteins. One common clade I insertion was at position 274Fig. 2 a–c The predictedstructure of PrC. a Ribbonrepresentation of the PrCstructure. The catalytic triadamino acids, D39–H69–S225,are represented as magentasticks, and residues 101–105 and135–139 involved in substratebinding site are represented ascyan sticks. The two disulfidebridges are shown as black linesindicated by black arrows. bSuperimposition of the twoproteases, PrC (green) andproteinase K (red). c The surfacecontour based on electrostaticpotential. The four substratebinding pockets are shownAnn Microbiol (2011) 61:511–516( 1)–274 ( 4). Deletions were observed at positions 57–80,and 200–204. Two disulfide bridges (182–274 [ 4] and329–410) were conserved within clade II (except forU16305 for the second disulfide bridge), but no cysteineexisted in the corresponding position in clade I. Aconserved cysteine was also found in clade I at position365, which was identified as free cysteine and wasconserved in all the proteases except PrC. This is anunusual difference between PrC and other cuticle proteases.Another obvious difference between PrC and other cuticleproteases was an insertion at 103 ( 1)–103 ( 7) in thepropeptide region, although the amino acids near theinsertion (99–105) were quite different (SupplementaryFig. 2). This result is very similar to our previous report(Yang et al. 2007). Because these two groups of fungi havedifferent mechanisms of killing nematodes, these proteaseslikely represent different evolutionary adaptation to facilitate fungal virulence against nematodes.Three-dimensional structure of PrCThe 3D structure of PrC was predicted by homologymodeling using the 3D structure of proteinase K as atemplate sharing 56% sequence identity. The structure ofPrC showed the typical folding of a subtilisin-like serineprotease (Fig. 2). It is composed of seven alpha helices, a
Ann Microbiol (2011) 61:511–516seven-strand parallel beta sheet, and two two-strandantiparallel beta sheets. The substrate binding site of PrCincludes two peptide segments containing residues 101–105and 135–139, respectively. The catalytic triad of PrC iscomposed of residues Asp39, His69 and Ser225. There aretwo disulfide bridges formed by residues Cys34–Cys124and Cys179–Cys250 (Fig. 2a). Figure 2b shows thestructural superimposition of PrC and proteinase K. Theiroverall folds are very similar to each other, the root meansquare deviation (RMSD) of the two proteases is 0.23 Åwhen computed using the backbone atoms. The similaritiesin the overall 3D coordinate, sequence, and structuralidentity of the catalytic triad between PrC and proteinaseK suggest that they have similar catalytic mechanisms. Thepolar residues located at the surface of PrC render theprotease polarized overall. Meanwhile, the catalytic centerof PrC is negatively charged (Fig. 2c). It has been observedthat the anionic character can increase flexibility of anenzyme (Pasternak et al. 1999), and in particular increasethe flexibility around the active site region (Kumar andNussinov 2004). Large parts of PrC surface are positivelycharged, which is similar to VCP1 and Pr1, which weremodeled recently (Liu et al. 2007), suggesting that thepolarized surface may enhance the attraction between theprotease and nematodes.Acknowledgments We are grateful to Prof. Jianping Xu (McMasterUniversity, Canada) for his valuable comments and critical discussions. This work was funded by National Basic Research Program ofChina (approved no. 2007CB411600), by projects from the NationalNatural Science Foundation of China (approved nos. 30630003 and30960229), and the Department of Science and Technology of YunnanProvince (approved no. 2007C007Z and 2009CI052).ReferencesÅhman J, Johansson T, Olsson M, Punt PJ, van den Hondel CA,Tunlid A (2002) Improving the pathogenicity of a nematodetrapping fungus by genetic engineering of a subtilisinwith nematotoxic activity. Appl Environ Microbiol 68:3408–3415Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISSMODEL workspace: a web-based environment for proteinstructure homology modelling. Bioinformatics 22:195–201Blaxter ML, Page AP, Rudin W, Maizels RM (1992) Nematode surfacecoats: actively evading immunity. Parasitol Today 8:243–247Cox G, Kusch M, Edgar R (1981) Cuticle of Caenorhabditiselegans: its isolation and partial characterization. J Cell Biol90:7–17DeLano W (2002) The PyMOL molecular graphics system. DeLanoScientific, Palo AltoGuex N, Peitsch M (1997) SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling.Electrophoresis 18:2714–2723Huang XW, Zhao NH, Zhang KQ (2004) Extracellular enzymesserving as virulence factors in nematophagous fungi involved ininfection of the host. Res Microbiol 155:811–816515Kumar S, Nussinov R (2004) Different roles of electrostatics in heatand in cold: adaptation by citrate synthase. Chembiochem 5:280–290Li J, Yang JK, Huang XW, Zhang K-Q (2006) Purification andcharacterization of an extracellular serine protease from Clonostachysrosea and its potential as a pathogenic factor. Process Biochem41:925–929Liu SQ, Meng ZH, Yang JK, Fu YX, Zhang KQ (2007) Characterizingstructural features of cuticle-degrading proteases from fungi bymolecular modeling. BMC Struct Biol 7:33Maizels RM, Blaxter ML, Selkirk ME (1993) Forms and functions ofnematode surfaces. Exp Parasitol 77:380–384Nordbring-Hertz B, Jansson H, Tunlid A (2000) Nematophagousfungi. In: Encyclopedia of life sciences. Macmillan, BasingstokePasternak A, Ringe D, Hedstrom L (1999) Comparison of anionic andcationic trypsinogens: the anionic activation domain is moreflexible in solution and differs in its mode of BPTI binding in thecrystal structure. Protein Sci 8:253–258Sasser J, Freekman D (1987) A world perspective on nematology:the role of the society. In: Veech JA, Dickerson DW (eds)Vistas on nematology. Society of Nematologists, Hyatsville,pp 7–14Saxena AK, Singh TP, Peters K, Fittkau S, Betzel C (1996)Strategy to design peptide inhibitors: structure of a complex ofproteinase K with a designed octapeptide inhibitor N-Ac-ProAla-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 Å resolution.Protein Sci 5:2453–2458Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL:An automated protein homology-modeling server. Nucleic AcidsRes 31:3381–3385Segers R, Butt T, Kerry B, Peberdy J (1994) The nematophagousfungus Verticillium chlamydosporium produces a chymoelastaselike protease which hydrolyses host nematode proteins in situ.Microbiology 140(Pt 10):2715–2723Siddiqui Z, Mahmood I (1996) Biological control of plant parasiticnematodes by fungi: a review. Bioresour Technol 58:229–239St Leger R, Charnley A, Cooper R (1987) Charac
genetic engineering the effectiveness of the use of fungal infections to control parasitic nematodes. Keywords Clonostachys rosea.Cuticle-degrading protease.Gene cloning.Homology modeling.Sequence analysis Introduction Plant-parasitic nematodes cause serious damages to crops, worth more than US 100 billion per year globally (Sasser and Freekman 1987). Methods for nematode control involve .
Some are established (PCR assembly cloning, end-recession cloning/Gibson cloning) but these are often difficult to control. This RFC adapts the "Phosphorothioate-based ligase-independent gene cloning" PLICing) to BioBrick cloning with restriction sites (e.g. RFC 10) and provides a universal cloning protocol.
As the first step in so many experiments, successful cloning is key to recovering meaningful data. The cloning tools you use can be the difference between wasted time and fast, consistent results. Figure 1. In-Fusion Cloning protocol. The key to In-Fusion Cloning is 15 bp of homology between your insert(s) and linearized vector backbone. Add
Keywords: Homology, evolution, vertebrates, fossils, comparative anatomy Introduction One of the most fundamental concepts underlying student understanding of the theory of evolution is homology, a term coined by Sir Richard Owen in 1848, which discusses the structure of the skeleton in vertebrates (Owen, 1848). Homology may
describing this Rosetta-focused methodology, we note that other groups have also developed excellent tools outside Rosetta for RNA homology modeling, and we advise the reader to try multiple tools and check for Xu&Chen,2018).We also note that current homology modeling methods can be computationally
Homology Modeling Homology Modeling . motif. 5 Measuring protein structure similarity . Sequence Comparison & Searching GenBank NIH genetic sequence database BLAST Psi_BLAST. 9 BLAST (Basic Local Alignment Search Tool) is a popular program for searching biosequences against databases. BLAST was developed and is maintained by a
validation. Homology modeling is known to be one of the best and extensively used computational methods to generate three-dimensional structures when there is more than 35% sequence identity between the known protein structure (template) and the unknown protein structure [10-13]. In silico homology modeling has been successfully ap-
downtime during these events, use the VersaView cloning utility. The VersaView cloning utility provides advanced backup, data protection, and recovery to help you restore your system’s efficiency. The VersaView cloning utility uses the Symantec Ghost cloning program to crea
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