The Effect Of N-glycosylation Design On The Structure And Properties Of ?-glucuronidase

Glycosylation plays a critical role in the regulation of multi-function of protein such as the correct structure,biological activity,solubility,antigenicity and stability.However,the regulation mechanism of glycosylation towards protein is still unclear,and it has limited its application in the rational modification of protein.In this thesis,?-glucuronidase was selected as a model enzyme,and the glycosylation modification was manually introduced based on the conformation.The enzymatic characteristics of glycoprotein were investigated.Furthermore,the kinetic and thermodynamic characteristics of glycoprotein were studied by combining the experimental data and molecular dynamics simulation.Based on this,the conformation properties and interaction between sugar chain and protein were elucidated which provides insight into the oriented modification of protein by glycosylation.The main results are as follows:Four glycosylation sites were identified in different domains of the recombinant ?-glucuronidase expressed in Pichia pastoris,and their effect on the enzymatic properties was studied.N28 glycosylation could significantly improve the enzyme activity,N383 glycosylation could improve the enzyme thermal stability,and N594 could stabilize the conformation of the other three glycosylation modifications.Then,22 new glycosylation sites were introduced to the 12 loop/turn of ?-glucuronidase The glycosylation at N26,N150 and N543 significantly improved the activity,and the glycosylation at N115 and N418 improved the affinity between enzyme and the substrate,while the glycosylation at N26 and N150 enhanced the catalytic efficiency of the enzyme.In addition,the introduced glycosylation at different sites had not apparent influence on the pH and temperature profiles of the enzyme.Notably,glycosylation at N40 significantly improved the thermal stability of the enzyme,while glycosylation at N418 dramatically decreased the thermal stability of the enzyme.Thermodynamic analysis indicated that N40 mutant had the highest midpoint transition temperature(T_m3)and denaturation enthalpy(?H),and showed one more conformational transition than other mutants.The T_m3 of N418 mutant was at least 5? lower than other mutant along with the highest ?H.Fluorescence spectroscopy discovered sugar chain modification changed the protein tertiary structure.Circular dichroism spectroscopy indicated that only N40 mutant after incubation at 70 ? retained integral secondary structure,and the glycosylation at this site significantly increased the middle state free energy change(? G).Molecular dynamics simulation displayed sugar chain at N40 formed a "glycan hairpin" with the lowest energy between the two subunits to enhance the stability of the overall structure.The sugar chain at N208 was located at the junction of three interacting domains forming a "glycan gasket " structure.The sugar chain at N418,adjacent to the catalytically active sites,was likely to swing with the changes in the environment.At the same time,the glycosylation has superimposed effect,and the thermal stability enhancement effect of "glycan hairpin" was not affected by other sugar chains.And "glycan hairpin" will not affect the type of enzyme catalyzed reactions and specificity.