酿酒酵母中表达神经肉毒素A及其功能研究
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摘要
肉毒神经毒素(BoNT)是肉毒梭菌产生的神经毒素,是最强大的毒素之一。为了研究肉毒神经毒素A在酵母中的活性及功能,在酿酒酵母细胞中表达BoNT/A LC,观察其对酵母生长影响以及通过蛋白免疫印迹方法研究其在酵母中蛋白表达水平。结果表明:BoNT/A抑制野生型酵母的生长;在酿酒酵母细胞中BoNT/A具有水解酶活性,当其活性位点突变后,BoNT/A突变体的表达不会抑制酿酒酵母的生长;过量表达酵母的SNAP25同源基因SEC9可以回补BoNT/A轻链过表达的酵母的生长。在BoNT/A轻链表达的酵母细胞中,其催化活性可以通过酵母的生长来检测。因此,这类特殊的酵母细胞可以用来分析BoNT/A。
Botulinum toxin(BoNT) is a neurotoxin produced by Clostridium botulinum,which is one of the most powerful toxin. To study the activity and function of BoNT/A in yeast,BoNT/A LC in yeast is expressed,observing the yeast growth and BoNT/A protein expressing level by western blot. Expression of LC of BoNT cerotype A caused a growth defect in yeast cells,and the toxicity is due to the hydrolytic activity. It is because catalytically inactive BoNT/A LC does not cause a growth defect in yeast cells. Overexpression of a yeast SNAP-25 homologue gene,SEC9,could rescue the growth defect in BoNT/A LC expressing cells. The results demonstrate that,in BoNT/A LC expressing yeast cells,the hydrolytic activity can be monitored through their growth. Thus,such cells can be used to analyze BoNT/A.
Botulinum toxin(BoNT) is a neurotoxin produced by Clostridium botulinum,which is one of the most powerful toxin. To study the activity and function of BoNT/A in yeast,BoNT/A LC in yeast is expressed,observing the yeast growth and BoNT/A protein expressing level by western blot. Expression of LC of BoNT cerotype A caused a growth defect in yeast cells,and the toxicity is due to the hydrolytic activity. It is because catalytically inactive BoNT/A LC does not cause a growth defect in yeast cells. Overexpression of a yeast SNAP-25 homologue gene,SEC9,could rescue the growth defect in BoNT/A LC expressing cells. The results demonstrate that,in BoNT/A LC expressing yeast cells,the hydrolytic activity can be monitored through their growth. Thus,such cells can be used to analyze BoNT/A.
引文
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[16]RUMMEL A,KARNATH T,HENKE T,et al. Synaptotagmins I and II act as nerve cell receptors for botulinum neurotoxin G[J].Journal of Biological Chemistry,2004,279(29):30865-30870.
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[19]SCOTT A B. Botulinum toxin injection of eye muscles to correct strabismus[J]. Transactions of the American Ophthalmological Society,1981,79:734-770.
[20]JANKOVIC J,BRIN M F. Botulinum toxin:historical perspective and potential new indications[J]. Muscle&Nerve Supplement,1997,6:129-145.
[21]ERBGUTH F J. Historical notes on botulism,Clostridium botulinum,botulinum toxin,and the idea of the therapeutic use of the toxin[J]. Journal of the Movement Disorder Society,2004,19(8):2-6.
[22]LUVISETTO S,GAZERANI P,CIANCHETTI C,et al. Botulinum toxin type a as a therapeutic agent against headache and related disorders[J]. Toxins,2015,7(9):3818-3844.
[23]KUKREJA R V,SHARMA S,CAI S,et al. Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase[J]. Biochimica et Biophysica Acta,2007,1774(2):213-222.
[2]WANG Liping,SHAO Kankai,GAO Xiaodong,et al. Functional analysis of the SNARE domain of syntaxin family proteins using yeast sporulation[J]. Journal of Food Science and Biotechnolagy,2015,30(5):530-535.(in Chinese)
[3]STROP P,KAISER S E,VRLJIC M,et al. The structure of the yeast plasma membrane SNARE complex reveals destabilizing water-filled cavities[J]. The Journal of Biological Chemistry,2008,283(2):1113-1119.
[4]SIMPSON L. The life history of a botulinum toxin molecule[J]. Journal of the International Society on Toxinology,2013,68:40-59.
[5]SMITH T J,LOU J,GEREN I N,et al. Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization[J]. Infection and Immunity,2005,73(9):5450-5457.
[6]ROSSETTO O,PIRAZZINI M,MONTECUCCO C. Botulinum neurotoxins:genetic,structural and mechanistic insights[J].Nature Reviews Microbiology,2014,12(8):535-549.
[7]SHAPIRO R L,HATHEWAY C,BECHER J,et al. Botulism surveillance and emergency response. A public health strategy for a global challenge[J]. Jama,1997,278(5):433-435.
[8]DASGUPTA B R,DEKLEVA M L. Botulinum neurotoxin type A:sequence of amino acids at the N-terminus and around the nicking site[J]. Biochimie,1990,72(9):661-664.
[9]BLASI J,CHAPMAN E R,LINK E,et al. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25[J]. Nature,1993,365(6442):160-163.
[10]BLASI J,CHAPMAN E R,YAMASAKI S,et al. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin[J]. The EMBO Journal,1993,12(12):4821-4828.
[11]SCHIAVO G,SHONE C C,ROSSETTO O,et al. Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin[J]. The Journal of Biological Chemistry,1993,268(16):11516-11519.
[12]SCHIAVO G,MALIZIO C,TRIMBLE W S,et al. Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala-Ala peptide bond[J]. The Journal of Biological Chemistry,1994,269(32):20213-20216.
[13]SCHIAVO G,BENFENATI F,POULAIN B,et al. Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin[J]. Nature,1992,359(6398):832-835.
[14]LOMNETH R,GIMENEZ J,MARTIN T F,et al. Calcium-dependent release of norepinephrine from permeabilized PC12 cells is inhibited by approximately 48 and approximately 112 kDa fragments of botulinum neurotoxin type E[J]. Neuropharmacology,1993,32(3):285-289.
[15]LALLI G,HERREROS J,OSBORNE S L,et al. Functional characterisation of tetanus and botulinum neurotoxins binding domains[J]. Journal of Cell Science,1999,112(16):2715-2724.
[16]RUMMEL A,KARNATH T,HENKE T,et al. Synaptotagmins I and II act as nerve cell receptors for botulinum neurotoxin G[J].Journal of Biological Chemistry,2004,279(29):30865-30870.
[17]LACY D B,TEPP W,COHEN A C,et al. Crystal structure of botulinum neurotoxin type A and implications for toxicity[J].Nature Structural Biology,1998,5(10):898-902.
[18]BROOKS V B. The action of botulinum toxin on motor-nerve filaments[J]. Journal of Physiology,1954,123(3):501-515.
[19]SCOTT A B. Botulinum toxin injection of eye muscles to correct strabismus[J]. Transactions of the American Ophthalmological Society,1981,79:734-770.
[20]JANKOVIC J,BRIN M F. Botulinum toxin:historical perspective and potential new indications[J]. Muscle&Nerve Supplement,1997,6:129-145.
[21]ERBGUTH F J. Historical notes on botulism,Clostridium botulinum,botulinum toxin,and the idea of the therapeutic use of the toxin[J]. Journal of the Movement Disorder Society,2004,19(8):2-6.
[22]LUVISETTO S,GAZERANI P,CIANCHETTI C,et al. Botulinum toxin type a as a therapeutic agent against headache and related disorders[J]. Toxins,2015,7(9):3818-3844.
[23]KUKREJA R V,SHARMA S,CAI S,et al. Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase[J]. Biochimica et Biophysica Acta,2007,1774(2):213-222.