Enzymatic Synthesis Of Ganglioside GM1 Derivatives And Their Neurite Outgrowth Activities

Ganglioside GM1 is one kind of glycosphingolipids?GSLs?on the mammalian cell membrane that shows important physiological functions and medicinal activity.GM1 has been widely used in the treatment of various neurodegenerative diseases such as Parkinson's disease,Alzheimer's disease,and other central nervous system injury.The structure of GM1 are composed of three modules including a oligosaccharide chain,a sphingosine base and a fatty acid,in which the structure of the fatty acid module significantly affects the membrane fluidity,their distribution on the plasma membrane and biological activitives.Therefore,modification of fatty acid module of GM1 may be a feasible way to obtain GM1 derivatives with novel biological activities.Sphingolipid ceramide N-deacylase?SCDase?is a dule function enzyme:it can not only catalyze GSLs hydrolysis to generate lyso-GSLs and fatty acids,but also catalyze the reverse reaction to synthesize GSLs.Based on the catalytic properties of the enzyme,we developed an efficient reaction system for enzymatic substitution of the fatty acid module of GM1 in this study,and synthesized a series of GM1 derivatives with different unsaturated fatty acids.Some of the derivatives were found to show higher neurite outgrowth activities than GM1,and the possible mechanism of the enhanced activity was explored by RNA-seq analysis.1)Efficient enzymatic hydrolysis of GM1 for preparation of lyso-GM1.The heterologous expression of SCDase?SA_SCD?derived from Shewanella alga G8 in Escherichia coli was optimized by culture medium selection and expression condition optimization.As a result,the yield of SA_SCD was improved from 4-5 mg/L to more than 40 mg/L,which paved the way for the large-scale enzymatic reaction.Furthermore,to solve the problem of low conversion rate caused by the competition between the hydrolytic activity and synthetic activity of the enzyme,the effects of different reaction conditions on the GM1 hydrolysis were systematically studied.We found that the stearic acid generated from hydrolyzed GSLs can be precipitated in the presence of both Ca²⁺and taurodeoxycholate hydrate?TDC?,which pushed the reaction equilibrium toward hydrolysis direction.In the optimized condition(pH 5.8,100 mM Ca²⁺,and at least 0.28%TDC),the yield of lyso-GM1 was improved from51%to 96%.Importantly,similar improvement also can be observed in other GSLs such as GM3,GlcCer,GalCer,and sphingomyelin.Finally,lyso-GM1 was prepared by using the optimized hydrolysis reaction system in large-scale,and the yield of lyso-GM1 was up to 90%?20 ml reaction system,100 mg GM1,12 h reaction time?,which is significantly higher than the best yield reported previously?24 ml reaction system,10 mg GM1,2 weeks reaction time,a yield of 72%?.2)Enzymatic synthesis of libray of GM1 derivatives.Based on our understanding of various factors that affected the reaction equilibrium,we found that when the pH value was adjusted to 7.5 and the detergent and metal ion was absent in the reaction system,the hydrolytic activity of SCDase can be inhibited,which pushed the equilibrium toward synthesis direction.Using the established synthetic reaction system?pH 7.5,0.75 mM lyso-GM1,1.5 mM fatty acid,10%dimethoxyethane?,we effectively synthsize ten GM1 derivatives with different unsaturated fatty acid modules such as palmitoleic acid,oleic acid,and docosahexenoic acid,and the yields were 16-67%after purification.Furthermore,the structure of the products were identified by high resolution mass spectra?HRMS?and H¹ NMR.3)The neurite outgrowth activities of GM1 derivatives.The neurite outgrowth activities of the synthesized GM1 derivatives were assayed by using Neuro2a cells as a model.It was found that some GM1 derivatives such as C16:1-GM1 and C18:1-GM1 showed a higher neurite outgrowth activities than GM1.Furthermore,through isolating the two species of GM1,C16:1-GM1 and C18:1-GM1 which contain C18-and C20-sphingosine bases,we further revealed that both of the fatty acid module and the sphingosine base can profoundly affect their biological activities.Among them,the derivative C16:1-GM1?d18?had the highest neuritogenic activity while it also had the highest cytotoxicity.The derivative C18:1-GM1?d20?,on the other hand,had higher neuritogenic activity and comparable low cytotoxicity compared to GM1?d18?,suggesting its potential therapeutic value in nerve regeneration.The cells treated by C16:1-GM1?d18?were further analyzed by RNA-seq to study the possible molecular mechanism of its enhanced activity.It was found that C16:1-GM1?d18?can more effectively upregulate expression of neurite outgrowth-asociated genes such as Egr1,Klf7,Epha7 and Dock10,and downregulate expression of Id2,Id2 and Hes5 which negatively regulate neurite outgrowth.This study provided new perspective for further studying and optimizing the functions of medicinal GSLs.