Comparative Proteomic Analysis Of Buccal Gland Secretion From Fasting And Feeding Lampreys (Lampetra Morii)

Lampreys are one of the most ancient jawless vertebrates still lived today,who possess the semi-parasitic life cycles.Usually,adult lampreys feed on the flesh and blood of fishes by the buccal funnels in their heads.Thus,lampreys are also known as “blood suckers”.Recently,a great deal of studies has shown that the buccal gland secretion(also called lamphredin)could play important roles during their feeding time.Although previous studies have reported that lamphredin contains various regulators including anticoagulants,ion channel blockers,and immune suppressors.But the proteins of lamphredin with low abundance are still not identified yet.In addition,the biological functions of these proteins remain unknown.Furthermore,previous studies might have shortcomings since they reported the characteristics and functions of buccal gland proteins from lampreys at spawning migration stage.Therefore,previous studies might fail to provide information on the expression of total proteins from the buccal glands of lampreys during feeding time in a real-time manner.In order to further clarify the relationship between lampreys lamphredin and their feeding habits,NanoLC–MS/MS with higher resolution,as well as lamprey Ensembl and NCBI databases were used to compare the proteomics of buccal gland secretion from China northeast lampreys(Lampetra morii)at the different feeding stages in the present study.Based on the analysis of proteomic data,the feeding habits of lampreys are closely related to the numbers and categories of proteins present in their buccal gland secretion.The data are summarized as follows: firstly,the number of proteins and peptides identified from buccal glands of lampreys which have been fed for 60 min was found increased significantly,nearly ten times of that in fasting groups.Secondly,the molecular weights of buccal gland proteins were distributed more extensively in the 60 min feeding group than that in the fasting group.When the feeding time was extended,more macromolecules(proteins)and micromolecules(peptides)were generated.Thirdly,GO analysis suggested that the buccal glands of lampreys could secrete more proteins which are related to biology progress,molecular function and cellular components to help them blocking the adverse reactions encountered during their feeding time.Finally,according to the function predictions,lampreys could secrete a variety of proteins to block the blood coagulation,nociceptive,oxidative stress and immune responses during their feeding stages.During the evolution of hundreds of millions years,theemergence of these proteins provides a solid foundation for the unique semi-parasitic life cycles of lampreys.In addition,a novel cystatin F was indentified in the buccal glands of feeding lampreys(60 min).In recent years,cystatin from snake venom or mosquito's salivary glands,etc were shown to play important roles in antibacterial and anti-tumor studies.Therefore,Lampetra morii cystatin F(Lm-CSTF)was also cloned from the buccal glands of lampreys by molecular cloning methods in the present study.Furthermore,Lm-CSTF was further analyzed by bioinformaticis.Due to the rare codons,the cDNA sequence of Lm-CSTF was optimized without changing its amino acid sequence.Subsequently,Lm-CSTF was subcloned into pCold I prokaryotic vector.When pCold I-Lm-CSTF plasmid was transformed into E.coli Chaperone Competent BL21 and optimized the induced conditions,recombinant Lm-CSTF was expressed as a soluble protein,which provided theoretical and material basis for further studying the biology functions of Lm-CSTF.In conclusion,the protein components in the buccal gland secretion of lampreys during different feeding stages were analyzed systematically by comparative proteomics in the present study,which could not only allow us to clarify the unique feeding mechanisms of lampreys,but also provides clues for the identification of functional proteins in the buccal glands of lampreys.Furthermore,the present study would promote the protection of medicinal gene resources in lamprey buccal glands,screening novel drugs with intellectual property rights and the development of marine creatures in China.