来自生物信息学分析的燕麦磷脂酶的新见解外文文献New insights into phospholipases in oat (Avena sativa) from bioinformatic analysis

New insights into phospholipases in oat Avena sativa from bioinformatic analysis Nihed Ben Halima Faculty of Medicine of Sfax University of Sfax Sfax Tunisia a b s t r a c ta r t i c l ei n f o Article history Received 9 January 2019 Received in revised form 22 April 2019 Accepted 22 April 2019 Available online 23 April 2019 Phospholipases from plants in particular from oat Avena sativa could not be purifi ed to homogeneity due to their association with other proteins Interestingly bioinformatics is a useful tool for the identifi cation of such new sequences of enzymes The Avena sativa phospholipases could be identifi ed by functional proteomics and bioinformaticsanalysiswiththeaidofdatabasesearches BaseduponTranscriptomeShotgunAssembly TSA se quences predicted genes were identifi ed for Avena sativa PLD PLA and PLC and assigned as AsPLD1 AsPLA2 1 and AsPiPLC1 respectively Insights into the structural characterization of the oat predicted enzymes were ana lyzed using in silico approaches Our results on sequence analysis of the oat phospholipasesprovide a detail viewof the main residues character istics of such biocatalysts 2019 Published by Elsevier B V Keywords Avena sativa Bioinformatic tools Phospholipases 1 Introduction The ability of any organism e g plant to make any particular prod uct e g lipids is generally predicated on its capability for the secretion of a particular set of enzymes e g lipolytic enzymes such as phospholi pasesthatplayimportantrolesinplantgrowth developmentandstress response Many reports have focalized in studying the mechanism of action of lipolytic enzymes and their diverse functions 1 6 The mechanism of action of major lipolytic enzymes and their roles in lipid signaling in particular in plants remain to be elucidated High performance analytical techniques as well as bioinformatics analysis would be very interesting research tools to accelerate discovering of novelproteins enzymes aswellaslipidsusingthedenovosequencing approaches Plant phospholipases are groups of lipolytic enzymes including different classes of enzymes with diverse interesting characteristics In fact they can be classifi ed into different biocatalysts that cleave either acyl groups of phospholipids e g phospholipases A PLAs or their head group e g phospholipases C PLCs and D PLDs Phospholipases in particular D C and A are among the major lipid hydrolyzingenzymes implicated inlipid mediatedsignaling whichare ubiquitousenzymespresentinbacteria mammalsandplants Theyplay critical roles in multiple plant growth and developmental processes as well as in hormone and stress responses and possible multivalent functionality could be assigned for these enzymes 3 5 7 10 Phospholipases D PLDs hydrolyze the phosphodiester bond of phospholipids PLs to produce phosphatidic acid PA and an alcohol moiety PLDs could also catalyze the interconversion of polar head groups of PLs by the transphosphatidylation reaction This latter reaction leads to syntheses naturally less abundant PLs such as phosphatidylglycerol PG phosphatidylserine PS or phosphatidyl ethanolamine PE from highly abundant ones such as lecithin or phosphatidylcholine PC 11 Phospholipases A PLAs catalyze the hydrolysis of acyl groups of phospholipids generating lysophospholipids and free fatty acids PLAs could be specifi c for the sn 1 position PLA1 or be specifi c to the sn 2 position PLA2 In fact PLAs from plants could be classifi ed into four families namely the PC hydrolyzing PLA1 PLA1 the phosphatidic acid PA preferring PLA1 PA PLA1 the secretory low molecular weight PLA2 sPLA2 and the patatin like PLA pPLA based on biolog ical properties and sequence data 12 Phospholipases C PLCs hydrolyze the phosphodiester bond on the glycerol side of phospholipids to produce diacylglycerol DAG and a phosphorylated head group Plant PLCs could be divided into three groups according to substrate specifi city and cellular functions Non specifi c PLC NPC that acts on the common phospholipids e g PC and PE Phosphoinositide specifi c PLC PI PLC that hydrolyzes phosphoinositides and the Glycosylphosphatidylinositol GPI PLC that hydrolyzes GPI anchored proteins 5 6 The current report would focus especially on PLD PLA2 and PI PLC from oats Avena sativa L which are among the most cultivated crops thatbelongtothePoaceaefamily Gramineae A sativawouldbepromis ing crop plant worldwide thanks to its benefi cial components that could International Journal of Biological Macromolecules 133 2019 804 810 E mail address nihed benhalima medecinesfax org https doi org 10 1016 j ijbiomac 2019 04 161 0141 8130 2019 Published by Elsevier B V Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage be valorized in industrial applications 13 Oats are unique among the common cereal grains since oats have a high lipid content and their lipolytic enzymes are 10 15 times more active than those of wheat 14 Wild oats are diploid but those cultivated Avena sativa are hexa ploid with an estimated 1C genome size of 13 23 pg corresponding to about 13 000 Mbp 15 In fact the common oat Avena sativa L is an allohexaploid 2n 6x 42 crop species and thus it has a large ge nome that remains to be fully assessed Complex polyploidy genomes such as oats need substantial studies and contributions of the bioinfor matics pipeline No cultivar of oat has been reported to have a completed genome sequence hence no sequence of phospholipase from oat for instance Avena sativa PLC has been fully characterized Indeed functional proteomics could one of the useful tools to identify de novo protein sequences from those of uncompleted sequence genome such that of oat Since no amino acid sequence of oat phospholipase is available in the protein data bank an attempt has been made to more characterization and identifi cation of the catalytic amino acid residues by bioinformatics tools to defi ne predict biochemical properties of soluble oat seedling extracts Therefore the primary aim of this work is to identify new insights into oat PLD PLA and PI PLC based on bioinformatic tools and so far to get an insight about the oat genome 2 Materials and methods 2 1 Plant material Seeds of oat Avenasativa L were usedin this studyasstarted plant material in which during seedling extract hydrolases in particular phospholipases were the subject of this study 2 2 Extraction of hydrolases from oat seedling Avenasativaseedswereplacedtogerminateonwettissuepaperina plasticbox Theyweregrowninthedarkjustfor5daysatroomtemper ature On day 10 after planting A sativa seedlings were ground using mortar and pestle with 0 02 M sodium acetate buffer pH 5 6 fi ltered through two layers of cheesecloth to remove large particles and the supernatant obtained was centrifuged at 15 000 g for 20 min The supernatant was used as an oat crude extract of hydrolases Then ace tone was added to the oat crude extract 2 1 v v sample centrifuged at 14 000 g for 15 min and the supernatant discarded The partially delipidated acetone powder was resuspended in water The mixture wasstirredfor20minat4 C sonicatedfor5minandfi nallycentrifuged at 14 000 g for 5 min before collecting the supernatant fraction which was used as oat fraction enriched in hydrolase activity 2 3 Determination of soluble protein concentration The concentration of soluble protein in oat extract was determined by the method of Bradford 16 aswell asby the BCA method following the manufacturer s instructions 17 using bovine serum albumin as a standard 2 4 SDS PAGE Proteinelectrophoresiswasperformedusingsodiumdodecylsulfate SDS polyacrylamide gel PAGE NuPAGE Novex 4 12 w v Bis Tris Gel 1 0 mm 12well Invitrogen and NuPAGE MOPS SDS for Running Buffer Invitrogen according to the method described by Laemmli 18 The apparent molecular weights of proteins were esti mated by co electrophoresis of marker proteins See Blue Invitrogen with weights ranging from 14 to 97 kDa The protein in the sample buffer 0 9 g glycerol 0 1 mL 1 bromophenol blue 1 mL 10 w v SDS and 0 5 L tris 2 carboxyethyl phosphine reducing conditions with NuPAGE LDS 4 LDS Sample Buffer Invitrogen was heated for 5 minin boilingwaterand appliedto the gel Theproteinsseparated on the SDS PAGE were stained with Coomassie Brilliant Blue R 250 Simply Blue Safe Stain Invitrogen 2 5 In geltryptic digestionandproteinidentifi cationbymassspectrometry Bands of interest were manually excised from gels and automated tryptic digestion was conducted as previously described 19 21 or manually treated as follows Gel bands were manually excised in a sterilelaminar fl ow hood transferred individually to 1 5 mL microtubes and cut into cubes of roughly 1 mm3 Gel cubes were destained for 1 h and 30 min at 4 C using a solution of 45 acetonitrile and 55 mM ammonium bicarbonate After gel cubes washing and in gel trypsin proteolysis of proteins the peptides produced were extracted onto Poros beads and purifi ed with ZipTips Millipore France as previously described 22 Extracted proteolytic peptides were analyzed by nanoUltraHPLC nanoESI UHR QTOF MS Experiments were performed using an UltiMate 3000 NanoRSLC System Dionex Sunnyvale CA connected to a Bruker MaXis UHR QTOF 2 GHz mass spectrometer equipped with an online nano ESI ion source The LC MS setup was controlled by Bruker Hystar software version 3 2 Acquired MS MS spectra were searched against the UniProtKB Swiss Prot TrEMBL database version 51 6 257 964 sequence entries non redundantNCBI http www ncbi nlm nih gov andthe Expressed sequence tags ESTs A sativa L database containing 25 400 entries AM071411 CN180783 using the Mascot identifi cation engine version 2 3 Matrix Science France The search was conducted allowing for a maximum of two missed cleavages 5 ppm tolerance for precursor ions and 0 04 Da for fragment ions respectively Since contaminations from human mainly keratins origin could be present in the samples analyzed the search in databases was restricted to plant species using UniProtKB Swiss Prot TrEMBL 49 887 sequence entries NCBI nr 551 056 sequence entries In the case of peptides matching to multiple members of a protein family the presented pro tein was selected based on both the highest score and the highest number of matching peptides 2 6 Retrieval of protein sequences The amino acid sequences from plant phospholipases serving to comparison with the predicted oat proteins were retrieved from the protein database of the National Center for Biotechnology Information NCBI http www ncbi nlm nih gov protein The sequences were saved in FASTA format An outline of the in silico approach steps followed in this study has been portrayed in Fig 1 2 7 Sequence analysis Bioinformatic analysisoftheA sativapeptidesequences ESTs geno mic sequences and deduced protein sequences was performed using the following tools Multiple sequence alignment was performed using the ClustalW algorithm 23 The peptide sequences could be compared with the NCBI National Center for Biotechnology Information USA non redundant sequence databases the Transcriptome Shotgun Assembly TSA A sativa database and the ESTs A sativa database using BLAST 24 Primary structure analysis using predicted amino acid sequences was performed using the ExPASy Proteomics tools The Translate tool web expasy org translate was used to translate DNA sequences to protein sequences whereas the Compute pI Mw tool web expasy org compute pi was used to compute the theoretical isoelectric point pI and molecular mass 25 26 The BioEdit software package 805N Ben Halima International Journal of Biological Macromolecules 133 2019 804 810 27 was used to manipulate edit and compare DNA and amino acid sequences The prediction of the signal peptide sequence was performed using the signalP 4 1 application 28 To predict N and O glycosylation sites the servers NetNGlyc 1 0 www cbs dtu dk services NetNGlyc and NetOGlyc 4 0 www cbs dtu dk services NetOGlyc 29 were used Phylogenetic analyses were performed using Molecular Evolution ary Genetics Analysis MEGA package version 7 30 The program MUSCLE 31 implemented in MEGA7 package was used to perform multiple alignments of amino acid sequences of oat phospholipases and their homologs for phylogenetic analysis The evolutionary history wasinferred using theUPGMA method 32 Theevolutionary distances werecomputedusingtheJTTmatrix basedmethod 33 andwereinthe units of the number of amino acid substitutions per site All positions containing gaps and missing data were eliminated The robustness of the inferred tree was evaluated by bootstrap 1000 replications 34 2 8 Conserved protein motifs analysis and subcellular location prediction Conserved protein motifs of the protein sequences from oat were analyzed using Multiple Expectation Maximization for Motif Elicitation MEME v 4 11 4 35 37 http meme suite org with the number of different motifs as 30 motif sites distribution as zero or one occurrence per sequence and motifs width as 6 minimum and 50 maximum The functional annotations of these motifs were analyzed by InterProScan http www ebi ac uk Tools pfa iprscan 38 The map ping between Pfam http pfam xfam org analysis and Gene Ontology GO is provided by InterPro 39 The prediction on subcellular localization of oat protein was carried out using the CELLO v 2 5 server http cello life nctu edu tw 40 3 Results and discussion Previous studies have reported PLC activity in oat Avena sativa tis sues in particular PI PLC in oat roots 41 43 Avena sativa PLD activity has also been discussed in the report of Kabachevskaya et al 44 with its relation to photosensitivity and photosynthesis in oat seedlings The lipid degradation data in Avena sativa seedling reported by Tjellstr m et al 45 refl ect a signifi cant involvement of PLD rather than PLC In plasma membranes from oat a PLD type activity and a phosphatidic acid phosphatase were the dominant lipase activities induced by phos phate defi ciency 46 In almost all cases no plant phospholipases were ever purifi ed due to their association with other proteins such as actin co purifi ed with an oat root PI PLC 42 Taken together these fi ndings we have intended in this study to the identifi cation of phospholipases in particular PLD PLA and PLC from oat by functional proteomics and bioinformatics analysis By the mean of LC MS MS technique and bioinformatics tools novel amino acid sequences of oat PLD PLA2 and PI PLC were reconstructed and characterized 3 1 Extraction and identifi cation of A sativa seedling proteins of hydrolyt ically catalytic activities Oat AvenasativaL seedlingsfrom10 dayoldwereusedasstarting material for hydrolases extraction In fact although this extract was enrichedinamylolyticactivitiessuchas amylasesasdescribedbypre viousreports 47 49 phospholipaseswerealsointendedinthisextract because of their relevant existence in oat An aliquot of this extract was analyzed by SDS PAGE followed a Coomassie blue staining step and a number of protein bands were excised from the preparative gel Fig 2 Multiple amino acid sequences alignment of oat PLD AsPLD1 with homologous PLDs identifi ed from NCBI databases http www ncbi nlm nih gov We have chosen homologs frommonocotsandeudicotsgroups TheMonocotsPLDshomologsfromPoaceaefamilyareasfollowing Triticumurartu EMS45543 1 Aegilopstauschii XP 020160059 1 Brachypodium distachyon XP 010234931 2 Oryza sativa CAD11899 1 Zea mays ONL99359 1 and Sorghum bicolor XP 021306705 1 While the monocots non Poaceae used are Elaeis guineensis XP 010933911 1 from Arecaceae family and Phalaenopsis equestris XP 020572394 1 from Orchidaceae family The eudicots used are Glycine max XP 003520025 1 and Medicago truncatula XP 003623811 1 from the Fabaceae family The predicted residues implicated in conserved plant PLD domains are highlighted by rectangles The numbers of the residues relative to the fi rst residue of each protein are shown on the right side of each sequence Retrieval of protein sequences with phospholipase activity PLA PLC and PLD Potential de novo sequencing of phospholipases from oat Avena sativa seedling extract Sequence and structure analyses of the deduced potential oat phospholipases Potential analysis of proteins from oat Avena sativa seedling extract by LC MS MS MASCOT Search Swiss Prot DNA EST Databases Search against databases EST and TSA Avena sativa Fig 1 Flow chart depicting the in silico steps used in the study for the prediction and analysis of oat phospholipases 806N Ben Halima International Journal of Biological Macromolecules 133 2019 804 810 Fig S1 The majority of the protein bands excised from SDS PAGE Fig S1 were identifi ed as glycoside hydrolases Table S1 some of these bands corresponding to several proteins Signifi cant scores could be obtained for the match of protein band 1 with PLD and protein band 2 3 and 4 with PI PLC as either full or truncated sequences in the DNA EST Avena sativa databases Fig S1 Therefore de novo sequence peptides could be identifi ed for band 1 correspondingtoAvenasativaPLD AsPLD1 Fig S2 or band 2 3 and 4 corresponding to Avena sativa PI PLC AsPiPLC1 Fig S4 Indeed the peptide sequences obtained could be used to screen for A sativa EST genomic sequences dataset AM071411 CN180783 GAJE01000001 GAJE01050182 Interestingly three genomic se quences TSA A sativa GAJE01023621 1 GAJE01023623 1 and one EST A sativa CN815254 1 could be identifi ed and corresponded to the peptide sequences of bands 2 3 or 4 using Basic Local Alignment SearchTool BLAST http blast ncbi nlm nih gov 50 Thesegenomic sequences are useful tools for the identifi cation of at least two oat PI PLCs sequences Moreover the following genomic sequences TSA A sativa GAJE01030597 GAJE01030603 as well as GFHU01003495 and GFHU01003496 GFHU01000532andGFHU01000533 and ESTs A sativa GO594938 and GO591438 that could be corresponding to the peptide sequences of protein band 1 were identifi ed using BLAST http blast ncbi nlm nih gov 50 These genomic sequences are useful tools for the id