Purification Of Complex N-Glycan And Synthesis Of Glycopeptides

Glycoproteins play an important role in both physiological and biochemical aspects; however, it is difficult to obtain homogeneous glycoproteins with current technology, which hampers the research on structure and function of glycoproteins. In vitro engineering conjugates the modified sugar chains that have the similar structure as the glycans from human resource to glycosylation sites on peptides via the synthetic activity of enzyme. Since large-scale synthesis of glycan is hard to accomplish both chemically and enzymatically, purification from natural resource becomes the efficient method of N-glycan preparation. In the dissertation, the methods for purification of complex-type N-glycan from egg yolk and two tentative enzymatic methods of glycopeptide synthesis are presented.Egg yolk, containing sialyl complex-type N-glycan, is abundant resource of N-glycan for in vitro glycopeptides synthesis. The structure similarity between sugar chains from egg yolk and those from human makes it significant to isolate N-glycan from egg yolk. By modifying Akira Seko's method, new methods have greatly shortened the purification time and improved the purification efficiency. Starting from 100 egg yolks, purification of complex-tye N-glycan cost about five and a half months and yielded 240 mg SGP and 12 mg FSG according to Akira Seko's method, four and a half months and 300mg SGP and 15 mg FSG according to modified method 1. two and a half months and 340 mg SGP according to modified method 2. By modified method 2, the purification efficiency was improved by 41%. while the purification time was reduced to only 45% of the original method, and the cost of reagents was saved as well. Compared with SGP that bears six amino acids, SG the complex-type oligosaccharide linked to a sole Asn, is easy to protect and modify chemically. The protected and/or modified SG can be a substrate for solid-phase synthesis of glycopeptides with desired sequences. When 1 mg SGP was hydrolyzed by pronase and purified by Graphic Carbon, 0.73 mg SG was obtained, while purification efficiency was about 80%. The current market price for SG is about RMB 20.000/mg.Under certain conditions the cysteine protease can constitute amide bond to synthesize polypeptides by exhibiting its reverse reaction activity. Based on similarity of catalytic center and mechanism, the experiment was designed to explore whether Png1p from Saccharomyces cerevisiae can constitute amide bond and synthesize glycopeptides with a reverse reaction. After 2 h of reaction, synthesized octapeptide with erythropoietin glycosylation sites began to lose protecting methyl groups onγ-carboxyl of Asp. When the reaction time reached 4 h, half of the octapeptide lost methyl groups. If methyl ester of Asp turns to carboxy, the octapeptide will lose the activity of being a substrate for glycopeptide synthesis. Therefore, the reaction time of glycopeptide synthesis should be controlled within 4 h. Acetone concentration greater than 8% significantly reduced the enzyme activity of Png1p, while more than 10% of acetone abolished enzyme activity. Hence, the concentration of acetone for enhancing the reaction rate was selected at 8%. In 8% acetone, Png1p was found active within 2 h. Based on the above optimum conditions, 8% acetone and 2 h were selected as optimized condition for enzymatic synthesis of glycopeptides. However, no synthesized glycopeptides was detected by LC-MS.Experiments show that recombinant AtPng1p from Arabidopsis, when expressed in Escherichia, coli, functions as a transglutaminase but not a PNGase. However, Andreas Diepold et al proved that recombinant AtPng1p from Arabidopsis, when expressed in Saccharomyces cerevisiae. is a PNGase rather than a transglutaminase. Combining these two phenomena, we can draw a conclusion: recombinant AtPng1p expressed in different microorganisms show different activities—transglutaminase activity in the prokaryotic E. coli and PNGase activity in the eukaryotic S. cerevisiae. It is speculated that the enzyme expressed in the eukaryotes is glycosvlated and results in its PNGase activity, but is non-glycosylated in the prokaryotes and shows transglutaminase activity. In the experiment of synthesizing glycopeptides via AtPng1, a new peak was detected by HPLC, but the molecular weight corresponding to expected synthesized glycopeptides was not detected by LC-MS. The results may be influenced by both the substrate preference of enzyme and the product detection methods.