Study On The N-glycosylation And Its Effect On Molecular Structure Stability Of Chicken Egg Yolk Immunoglobulin

Glycosylation is one of the most important post-translation modification of proteins.Immunoglobulins are members of glycoproteins family, and micro-heterogeneity exists in their N-glycans. The most common structures of thease glycans are complex biantinary oligosaccharides. IgG-Fc fragment would keep their active conformation due to the exsitance of oligosaccharides. Additionally, different glycoforms could modulated the ADCC or CDC function and prolong their serum half-life time. It was reported that glycosylation could increase the thermal stability of IgGs, especially contribution to CH2 domain. Proteins are prone to proteolysis, while the glycosylation has an steric hindrance effect and might cover the proteolytic cleavage sites. Therefore, glycosylation could increase the resistance of IgG to proteolysis. The glycosylation modification also occurs in IgY. Comparing to mammalian IgG, different glycosylated sites and glycoform may have an different effect on the structure and function of IgY molecule. This paper systematically studied the effect of N-glycosylation on the structual stability of IgY molecular. The results are as followings:1. IgY was isolated and identified, and the method to prepare deglycosylated IgY under the non-denaturing condition was established. The crude IgY was preciptated by3.5% PEG6000 and 8.5% PEG6000, continuously. A further purification was performed on gel filtration chromatography, and only one separation peak appeared in filtration chromatography. The reducing SDS-PAGE showed two bands in the gel, 70 kDa and 25 kDa respectively. After MALDI-tof Msand MASCOT search analysis, the corresponding characteristic ion peaks of IgY hydrolysis fragments were obtained, and the score was above 35, and the coverage rate of the peptides reached more than 36%. HPLC performance showed that the purity of IgY, after purified by 12% PEG6000 and gel filtration chromatography, was 92.01% and met the purity of protein structure research.Lately, the effects of PNGase F amount and incubation time on the degree of deglycosylation of IgY were investigated. Results showed that with the increasement of PNGase F amount and incubation time, the PAS stainning band color of sample becamegradually light. A optimization conditions for deglycosylation was 800 NEB units: 1 mg IgY with 48 h incubation time at 37 ℃. Then anion exchange chromatography technique was used to isolate and pool deglycosylated IgY. Two separation peaks appeared in the chromatography, and the products of peak one and peak two corresponded to deglycosylated and native IgY respectively.2. The effect of glycosylation on the structure stability of IgY molecule was studied.FTIR spectra showed that a drastic decreasement in 1200 cm-1~900 cm-1, suggesting that sugar chains were removed. The secondary structures of deglycosylated and native IgY were investigated by CD spectra. Some significant changes were observed, the content ofα-helix decreased from 30.9% to 2.2%, β-sheet decreased from 41.8% to 23.3%, β-turn increased from 22.3% to 25%, and random curl increased from 5% to 28.5%. Intrinsic fluoresence spectra indicated that microenvirment polarity of tryptophan residues changed after deglycosylation, the hydrophobic property of IgY was enhenced and the conformation of IgY molecule transformed from a stretching station to a compacted station. It can be inferred that N-gylcans could keep the conformation stability of IgY.The GndHCl induced denaturation experiment presented that the resistance ability of IgY to GndHCl could improve 0.6 M GndHCl, which is 0.1 M GndHCl higher than that of IgG1. These results may be due to more glycosylation sites and different distribution domains of glycans. It indicated that glycosylation has a greater contribution to IgY molecule stability.3. The effect of glycosylation on the thermal-induced aggregation of IgY was studied.The result of SDS-PAGE and SEC-HPLC experiment indicated that polymer aggregates appeared after 6 h incubation at 55 ℃ for IgY, and its amount increased rapidly with the increasement of temperature and time. As for deglycosylated IgY, high molecule weight aggregates appeared after 6.5 h at 50 ℃, and the content of polymer aggregates rased rapidly with delayed incubation time and temperature prolonging. The above experiment demonstrated that glycosylaion could enhence the thermal stability of IgY, approximately5 ℃, and help to resist thermal-induced aggregation. This result might be meaningful to pasteurization during production of IgY.4. The digestion conditions for producing IgY Fab fragment was optimized. Pepsin digested IgY at a ratio of 1:10(w/w) at 37 ℃ for 3 h. Papain digested IgY at a ratio of1:10(w/w) at 37 ℃ for 3 h. The effect of glycosylation on the resistance of IgY to proteolytic cleavage was investegated. When the ratio of enzyme and substrate was 1:40(w/w), the intact molecule bands of deglycosylated IgY disappeared, which were digested by pepsin, papain and chymotrypsin after 6 h. But after digestion by pepsin, papain and chymotrypsin for 6 h, some clear intact molecule bands could still be found for native IgY.5. The glycosylation sites, glycoform and the contribution of glycans to IgY molecule mass were analyzed. Two glycopeptides were detected: SGSTYSLSSRVNVSGTDWR and PMVLQEHFNGTYSASSAVPVSTQDWLSGER, containing the glycosylation sites Asn308 and Asn407, respectively. Both of the two glycopeptides belonged to IgY heavy chains. Two different glycoforms were found: high-mannose glycans and complex glycans and high-mannose glycans was dominant. Additionally, there were termianl galactose and NeuAc modification in IgY glycans. The molecule weight of N-glycans was 5.5922 kDa, possessing 3.29 % of intanct molecule weight.