Molecular Modification Of Peptide N-glycosidase F And Glycan-binding Protein And Their Application In Protein N-glycosylation

As one of the most important post-translational modifications of proteins,N-glycosylation is widely involved in various important physiological and pathological processes,including cell proliferation and growth,signal transduction,immune response and inflammation.The qualitative and quantitative analysis of N-glycans is an important component of protein N-glycosylation research.However,studies have shown that N-glycans are hard to be direct analyzed as their characteristics of micro-heterogeneity,structural diversity and dynamic changes.Therefore,effective pretreatment methods for the release,enrichment or labeling of N-glycan are needed.It has been reported that many functional proteins can be effectively used in the release,separation and enrichment of glycans.In this paper,these functional proteins were employed and modified for effective research of N-glycosylation.The work is as follows:Peptide N-glycosidase F(PNGase F)is one important enzyme in glycomics research.It can specifically release N-glycan from glycoproteins or glycopeptides under mild conditions,playing an important role in researches of N-glycan's structure and function.Currently,the conventional method to produce His-PNGase F based on the purification of soluble protein,but His-PNGase F is prone to form inactive inclusion body proteins during the expression process,making its production cost relatively high.In order to effectively use the His-PNGase F in the form of inclusion bodies,we optimized the induction conditions to obtain high expression of the recombinant His-PNGase F in the form inclusion body,which accounts for more than 40% of the total bacterial protein,and then used denaturation and renaturation methods to obtain a large amount of target protein.In addition,results displayed that the glutamine tag(Gln-substrate peptide tag,Q tag)co-fused at the end of PNGase F can effectively improve its solubility.Both schemes to express and purify PNGase F can obtain the target protein with a purity of >95%.Enzyme immobilization method is one of the important means to improve the efficiency of PNGase F.In this paper,the immobilization of PNGase F with two different molecular tags obtained from the above expression was studied.The non-oriented and oriented immobilization of His-PNGase F was carried out using the methods of chemical condensation and affinity interaction.It was found that the non-covalent oriented immobilization method can load His-PNGase F by about 120 times compared with the non-oriented method.Non-covalent oriented immobilization of His-PNGase F can retain52.8% enzyme activity after reused for five times.The reduced enzyme activity is partly due to the loss of immobilized PNGase F.The Q tag can mediate the covalent and oriented immobilization of PNGase F under the catalysis of transaminase.The covalently oriented immobilization of PNGase F can still own 78.6% enzymatic activity after reused for five times,showing the advantages of covalently oriented immobilization of PNGase F.In order to further improve the digestion efficiency of immobilized PNGase F,we first proposed a method combining immobilized PNGase F with microwave-assisted digestion,which can effectively release of N-glycans within 5 minutes.The site-directed mutagenesis and crystal structure analysis of PNGase F verified that its active center is composed of several key amino acid residues,D60,E206 and E118.Mutations in its key amino acid can cause loss of deglycosylated activity or change the affinity with the substrate.Based on the previous research,we selected three PNGase F mutants without deglycosylated activity for molecular modification and protein purification,and then used for the specific recognition of glycans.The results show that the R911 protein has relatively strong affinity to recognize substrates,and can enrich part of glycoproteins or glycopeptides,but has no recognition effect on non-glycoproteins or non-glycopeptides.However,the ability of R911 protein to bind to substrates is too weak to be applied to large-scale N-glycan analysis.Hence,we analyzed a mutant of F-box protein that recognizes sugar chains(Fg),and verified that the intact M3N2 structure is necessary for Fg recognition.Fg fused with Glutathione S-transferases(GFg)can be directly immobilized on agarose microspheres modified with glutathione under mild conditions to form immobilized GFg-agarose microspheres.17 and 11 glycopeptides can be identified by GFg-agarose microspheres from the tryptic peptide of immunoglobulin G(Ig G)and horseradish peroxidase(HRP),respectively.GFg-agarose microspheres also show good selectivity and anti-interference in a complex sample.In addition,GFg-agarose microspheres can separate and enrich ribonuclease B(RNase B)containing high mannose glycans and transferrin containing sialic glycans in protein mixtures.Moreover,GFg-agarose microspheres own many advantages,including low cost,simple operation and good biocompatibility.Based on the ability of Fg to recognize N-glycans,we designed a fluorescent probe(GFP-Fg)fused with green fluorescent protein(GFP)and Fg to label the N-glycans on cell membranes.The results of confocal fluorescence imaging and flow cytometry showed that GFP-Fg can specifically label the N-glycans of cells,and the labeling efficiency reaches 99.9%.In summary,based on the structure and function of PNGase F and Fg protein,two types of proteins can be effectively used in the separation,enrichment and labeling of N-glycans.Results show that the oriented immobilization of PNGase F exhibits good enzyme activity and reusability,which provides a new means for the high-throughput release of N-glycans.At the same time,the modified PNGase F and Fg proteins can be specifically applied to the enrichment of N-glycans,and further applied to the labeling of N-glycans on the cell surface.The related research of two types of functional proteins provides a new method for the research of N-glycosylation,which has broad application prospects.