Biochemical Characterization,Structure Analysis And Application Studies Of A Novel Peptide:N-glycosidase(PNGase Dj)from Dyella Japonica

N-glycans play an important role in mammalian cells and plants biological functions,and different carbohydrate structures also have different effects on their biological functions.In order to correlate the glycan structures with their repective functions,N-glycans must first be released from glycoconjugates.Peptide:N-glycosidase(PNGase,EC:3.5.1.52)catalyzes the cleavage and release of N-linked glycan moieties from glycoproteins and/or glycopeptides.PNGases are valuable tools in glycopretin analysis.The currently available commercial N-glycosidase mainly includes two different types:PNGase F and PNGase A.Recombinant PNGase F is the most common choice and permits the release of all types of N-glycans,except the structures bearing a core ?1,3 fucose.Therefore,PNGase A is usually used for the analysis of plant N-glycans.However,this enzyme deglycosylates only proteolytically derived glycopeptides,and is unable to deglycosylate native glycoproteins.The novel PNGases also have some limitations.PNGase F? has lower efficiency towards the structures containing a core al,3 flucose and PNGase H+ only shows high activity under extremely acidic conditions.Considering the continuous development of N-glycoproteome studies,the requirement for the novel types of PNGases that integrates the benifit of PNGase A with PNGase F is becoming more and more urgent.In addition,determing the structures of different types of PNGases is the way to gain a great understanding of these enzymes from the mechanistic and functional perspective.During the past two decades,the researches on the structure of PNGases mainly focus on PNGase F and cytoplasmic PNGases.The three dimensional structures of other types of PNGase are still remain unknown.Therefore,determing the structure of PNGase A like N-glycanase is critical for a comprehensive understanding of the relationship between the structure and function.In this thesis,we hereby present the discovery and characterization of a novel bacterial N-glycanase from Dyella japonica.Multiple sequence alignments and site-directed mutagenesis techniques were used to find the activity site.The substrate binding site was found by analyzing the structure of the protein.Besides,PNGase Dj was used to release N-glycans from human and radish glycoproteins.The concrete research contents are as follows:1.Gene synthesis,recombinant expression,purification and enzymatic characterization of PNGase DjThe PNGase Dj gene was synthesized from Dyella japonica and transformed into different competent cells.The expression temperatures and time were optimized.Using BL21 cell and express at 15? for overnight was selected to do the large scale expression.Purified enzyme was analyzed by SDS-PAGE and confirmed by MALDI-TOF-MS.Biochemical characterization indicated that the optimal pH of PNGase Dj was 4.0 and the optimal temperature was 37?.PNGase Dj showed activity without the presence of metal ions.PNGase Dj is not a thermo-stable enzyme,but 0.5%BSA can increase the stability of the enzyme.Substrate specificity tests showed that PNGase Dj could liberate all types of N-glycans including the one with core a 1,3 fucose structures from both natural and denatured glycoproteins.2.Study on the activity sites of PNGase DjA previous mutagenesis study of the active sites suggested that aspartic acid and glutamic acid were essential amino acids for PNGase F activity.Therefore,four homologous PNGases with the same function were selected for multiple sequence alignments.In total,13 conserved sites containing aspartic acid or glutamic acid were found.Site-directed mutagenesis was performed and the relative activities of the mutants were tested.Asp101 and Glu235 showed only 2.2%and 1.3%relative activities when compared with the wild type.This result indicated that Asp101 and Glu235 maybe involved in the catalytic activity of PNGase.The binding abilities between the enzyme and substrate were also test for these two muntants.The result revealed that site-directed mutagenesis had no effect on their binding ability.The preliminary screening of the separation conditions showed that 0.1 M acetic acid can be used to separate the enzyme with substrate.3.Crystallization screening and sturacture analysis of PNGase DjFuther optimization was performed to purify PNGase Dj.The initial hits were??appeared in phosphate/citrate buffer(pH 4.2)with 40%ethanol(v/v)and 5%PEGIOOO(w/v).The concentration and the ratio of two precipitants were changed to optimize the conditions.Hanging drop was used to get the crystal.The optimal condition is phosphate/citrate buffer(pH 4.2)with 40%ethanol(v/v)and 5%PEG1500(w/v).Under this condition,seeding was used to get the crystal of PNGase Dj and the complex for both wild type and two mutants.The resolution is 1.08 A and the space group is P1 for both apo and complex.A selenomethionine derivatised crystal was used to solve the phase.The structure of the native protein was solved using the Se-Met structure as a model.Similarly the structures of the co-crystals of(wild type and mutant)Dj PNGase and one of the carbohydrates MMXF or MUXF were solved using apo-structure of Dj PNGase as a model.The complex structure indicated that Asp101 and Glu235 were essential amino acids for PNGase Dj activity.4.Application of PNGase DjThe protein extracted from human milk and radish was used as the substrate to test the activity of PNGase Dj.The released N-glycan structures were analyzed and confirmed by MALDI-TOF-MS.By comparing the differences of iV-glycans profile released from the same substrate by PNGase Dj,PNGase F and PNGase H+,it was found that in the presence of all types of iV-glycans,PNGase Dj would preferentially release N-glycans without high mannose structure.In addition,PNGase Dj showed the highest efficiency toward the structures which contained core ?1,3 fucose.By analyzing the structure of N-glycans released from radish,14 structures were found,including high mannose,hybrid and complex type.5 were high mannose type,8 of 14 contained xylose,3 of 14 had both xylose and core ?1,3 fucose and 6 of 14 had 1 or 2 terminal GlcNAc.