| The neuromuscular junction formed between motor neurons and skeletal muscles,is defined as chemical synapse,which dominates the movement of multiple skeletal muscles.The presynaptic membrane proteins in phrenic neuromuscular junction are synthesized in anterior horn motor neurons of the cervical spinal cord,and finally reaches the presynaptic membrane via anterograde transport,which in turn innervate the movement of the diaphragm.Except for their natural functions,many transmembrane proteins at the presynaptic membrane of phrenic NMJ are the targets(receptors)for many presynaptic biotoxins.In order to find the potential receptor of presynaptic neurotoxin Gintexin of Gloudius intermedius,in the present study,the mouse cervical spinal cord and the membrane proteins of phrenic neuromuscular junction were adopted as material,the content of the study was as follows:Part 1:Transcriptome of mouse cervical spinal cord was analyzed by using Illumina HiSeq X Ten sequencing.The raw data were assembled into transcript with Cufflinks software,each FPKM(Fragments Per Kilobase of transcript per Million fragments mapped)which reflects the relative expression level was worked out.Then,the non-redundant(Nr)protein database of mice in NCBI was searched online by Blastx tool,so as to get the protein function clues.Results:A total of 22352 transcripts were obtained.Among them,there were eight major classes of membrane proteins,including synapse related proteins,ion channels,neurotransmitter receptors,neurotrophic factors and cytokine receptors,toxin receptors,viral receptors,other receptors and spinal cord neuron-associated proteins,myosin family members,respectively.The relative expression levels were different from each other.Part 2:With phrenic neuromuscular junction preparations from mice as material,total membrane proteins were prepared by the method of non-continuous sucrose density gradient ultracentrifugation.The membrane proteins were identified by high-resolution LC-MS/MS(Liquid chromatography tandem mass spectrometry),the peptide fingerprints were collected.Mice protein data in NCBI was retrieved by Mascot 2.2 software to search the peptide information.Results:A total of 5530 proteins were identified.After overlapping analysis of transcriptome and proteomic data,at least 1107 proteins from the cervical spinal cord were mapped to phrenic NMJ,and 231 proteins were synaptic membrane related.They were divided into 11 categories:synapse related proteins,ion channel,neurotransmitter receptor,neurotrophic factor receptor,toxin receptor,myosin family member,signal transduction receptor,transporter,other transmembrane proteins,vomeronasal and olfactory receptors and other receptors,respectively.Part 3:With total membrane proteins of mice phrenic neuromuscular junction and presynaptic neurotoxin Gintexin of Gloudius intermedius as initial material,chemical cross-linking was conducted.After separation by SDS-PAGE and electric transfering to PVDF membrane,binding proteins were detected by western blot with anti-Gintexin monoclonal antibody(N16).The positive bands were identified by LC-MS/MS mass spectrometry.The peptide fingerprints were obtained and mice protein database in NCBI was searched by Mascot 2.2 software.Results:A positive band at position about 250 kDa appeared.The identification of cut-off band by LC-MS/MS revealed 22 proteins that bind to Gintexin.Among them,two Ca2+ channel proteins(voltage-dependent L-type calcium channel subunit alpha-1F?voltage-dependent L-type calcium channel subunit alpha-1S iso form 2)may involve in motor neuron peripheral Ca2+ concentration perception,influx and synaptic vesicle fusion with presynaptic membrane;four Na+ channel proteins(sodium channel protein type 1 subunit alpha isoform X1?sodium channel protein type 5 subunit alpha iso form X1?sodium channel protein type 8 subunit alpha isoform 1?sodium channel protein type 10 subunit alpha isoform 1);three cation channels(cation channel sperm-associated protein subunit gamma 1 isoform X1 transient receptor potential cation channel subfamily M member 6?transient receptor potential cation channel subfamily M member 7 iso form 1);four receptor-type tyrosine-protein phosphatases(receptor-type tyrosine-protein phosphatase beta precursor?receptor-type tyrosine-protein phosphatase delta?receptor-type tyrosine-protein phosphatase F precursor?receptor-type tyrosine-protein phosphatase S isoform 1 precursor);four unconventional myosins(unconventional myosin-Va isoform X3?unconventional myosin-??unconventional myosin-????unconventional myosin-XVIIIa isoform 1).Other proteins,including transmembrane protein 131 may play a role in the immune response to viral infection;low-density lipoprotein receptor-related protein 4 isoform 1 precursor plays a key role in the formation and the maintenance of the neuromuscular junction,the synapse between motor neuron and skeletal muscle;thyroid receptor-interacting protein 11 and peripheral-type benzodiazepine receptor-associated protein were also identified.It is worth mentioning that PLA2 receptor for Cobra PLA2 neurotoxin which was found and proven by Lambeau was absent among these 22 proteins.So the specific receptor for Gintexin was not PLA2R1(PLA2 receptor 1),and hidden in the 22 candidate proteins.Conclusion:There are 231 proteins in mouse phrenic NMJ presynaptic membrane protein,which can be classified into 11 categories.Among them,22 proteins may interact with Gintexin.Our work may thus lay the solid foundation for the future investigation of searching for the potential Gintexin receptors. |
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