| Sialic acids are essential sugar derivatives positioned at the termini of oligosaccharides,glycolipids or glycoproteins.They are responsible for many crucial biochemical roles,including the regulation of cell survival,reproduction,cell membrane flow,mediation of cellular endocytosis processes and also serve as key recognition elements.They are closely involved in nervous system development,influenza virus invasion and tumor progression,etc.The biosynthesis of sialic acids begins with ManNAc which can be epimerized by GlcNAc 2-epimerase(Gn2E)from GlcNAc.Gn2E catalyzes a reversible reaction,maintaining cellular ManNAc level through epimerization between GlcNAc and ManNAc,in order to regulate the synthesis of sialic acids.Thus,elucidating the mechanism of epimerization catalyzed by Gn2E will improve our understanding of the sialic acid synthetic pathway and to manipulate the biosynthesis of sialic acids.Sialosides can compete with their natural counterparts in receptor binding sites and may thus affect cellular recognition of pathogens,drugs or other cells.Synthetic sialic acid analogues could therefore enable the study of the relationships between sialic acids and their receptors,and improve our understanding of the processes of infection and inflammation.?-GlcNAc modification at the end of glycoproteins,related with arthritis pauperum,cancer,type ? diabetes,senile dementia and other human diseases,can be a biomarker for early diagnosis of some certain diseases.Thus,an effective method for monitoring ?-GlcNAc quantity on certain glycoproteins is of great biological importance for early diagnosis of diseases.Several methods for the detection of ?-GlcNAc have been reported previously,however,they all present deficiencies in terms of low sensitivity,low specificity and false positive results.This study focused on the mechanism and application of Gn2E cloned from Pedobacter heparinus(PhGn2E).Here,we presented the first experimental evidence of the epimerization mechanism and clarified its epimerization process.Based on this understanding of the epimerization mechanism,two applications for PhGn2E were developed:a four-enzyme biosynthesis of sialosides,and a fast quantification of ?-GlcNAc with low detection limit.The concrete research contents are as follow:1.Cloning,recombinant expression,purification and enzymatic characterization of PhGn2EThe Gn2E gene was cloned from P.heparinus.Homology analysis of the amino acid sequences showed that several amino acid sequences are highly conserved across different species,which may play an critical role in the enzyme active site.The enzyme was recombinantly expressed and purified.Analysis of the purified protein by SDS-PAGE showed a single band corresponding to the mass of the expected 47.1 kD.The identity and functionality of the purified enzyme were confirmed by MALDI-TOF-MS and the enzyme activity tests,respectively.Biochemical characterization indicated that the optimal temperature of PhGn2E was 37 ? and the optimal pH lay in the 7.0-10.0 range.PhGn2E showed activity without the presence of metal ions,while tests with a number of detergents and denaturing agents failed to inhibit PhGn2E completely.The presence of 2 mM ATP increased enzyme activity three-folds,arriving at the highest enzymatic activity.Although PhGn2E is not a thermo-stable enzyme,its activity remained at 50%of maximum after 24 h of incubation at 37 ?,indicating the industrial potential of this enzyme.Substrate specificity tests showed that a diverse range of N-acyl GlcN analogues could be epimerized to corresponding N-acyl ManN analogues by PhGn2E.Enzymatic parameter tested at pH 8.0 revealed that,the catalytic efficiency of PhGn2E using GlcN Ac as substrate was higher than that of ManNAc.2.Epimerization mechanism of PhGn2ED2O was used to trace the anomeric and epimeric center of PhGn2E-catalyzed GlcNAc.H-D exchange on the anomeric hydroxyl group and at the C-2 position,together with pyranose ring opening were observed during epimerization,proving a deprotonation/reprotonation mechanism.Site-directed mutagenesis of four highly conserved amino acid residues located in the active center demonstrated that Arg63 and Glu314 were directly involved in the epimerization mechanism.Moreover,the pKas of Arg and Glu are 12.5 and 3.1,respectively.Since the catalytic efficiency of PhGn2E using GlcNAc as substrate was higher than that of ManNAc when tested at pH 8.0,we can conclude that Glu314 deprotonates C-2 of GlcNAc,while Arg63 incorporates the proton on the same carbon but on the opposite face,forming ManNAc.On the other hand,Arg63 deprotonates C-2 of ManNAc followed by reprotonation by Glu314 obtaining GlcNAc.3.Application of PhGn2ESynthesis of sialosides:PhGn2E was applied in one-pot four-enzyme reaction to synthesize sialiosides.First,ManNAc analogues were synthesized from the corresponding GlcNAc analogues through epimerization by PhGn2E.The products of this reaction were condensed with pyruvate in a sialic acid aldolase-catalyzed reaction to form Neu5Ac analogues,which were then conjugated to CMP by CMP-sialic acid synthase.Finally,the activated Neu5Ac analogues were linked to X-Gal,forming Neu5Ac a2,6-X-Gal analogues which were easily detected on HPLC.According to the above mentioned principle,we successfully synthesized different sialosides with one-pot four-enzyme reaction.The synthesized silosides provides substrate candidates for the investigation of cell-cell,pathogen-cell and drug-cell interactions.Quantification of terminal ?-GlcNAc in sugar chains:To detect P-GlcNAc at the end of sugar chains,we first synthesized simple anomeric substituted GlcNAc derivatives containing both a-linked and ?-linked GlcNAc.Commercial ?-glucosaminidase was applied to specifically hydrolyze ?-linked GlcNAc forming free GlcNAc which was epimerized by PhGn2E to form ManNAc.ManNAc was then oxidized by ManNAc dehydrogenase to form the corresponding lactone,accompanied by the reducing NAD+ into NADH which was recorded by plate reader.This method was also applied to quantify ?-GlcNAc in pNP-core 2.Comparing the result of both the newly developed method and HPLC,we could demonstrate that the developed method was accurate and stable. |
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