Study On The Minimal Sugar Units Of N-Glycosidase PNGase H~+ And Its Application In The Research Of Legumes Glycans

N-glycosidase(PNGase)is the main tool enzyme which release N-linked oligosaccharides from glycopeptide or glycoprotein for further analysis.This enzyme has been widely used in the research of sugar chain structure information,sugar chain structure and function,the effect of sugar chain on the function of glycoprotein,and protein structure analysis.PNGase H+is a newly discovered N-glycosidase that acts on both glycopeptides and glycoproteins and also acts on N-linked glycans containing a-1,3 fucose core structures.In this paper,in order to further study this enzyme,substrates of different sugar unit sizes were synthetised by enzymatic methods.The PNGase H+ minimum sugar unit and its application in the analysis of legumes plant glycoprotein N-linked oligosccharide chain were studied.The detail contents were as follows:1.Substrate synthesis of different sugar unit size based on GNGNSuitable substrates are the basis for enzyme activity assays and enzymatic characterization studies.The research was based on GNGN(dabsyl-Gly-Glu-Asn-(GlcNAc4Man3)-Arg)and several enzymes β-N-Acetyl-Hexosaminidase、α-Mannosidase、β-Mannosidase to get four substrate dabsyl-Gly-Glu-Asn-(GlcNAc2Man3)-Arg、dabsyl-Gly-Glu-Asn-(GlcNAc2Man)-Arg、dabsyl-Gly-Glu-Asn-(GlcNAc2)-Arg、dabsyl-Gly-Glu-Asn-(GlcNAc)-Arg by the method of gradual enzymatic synthesis.Besides dabsyl-Gly-Glu-Asn-(GlcNAc2Gal)-Arg was obtained by One-pot multi-enzyme method withβ4GalT1 and CGIUGE.Five substrates were obtained in total.2.Study on minimal sugar unit of PNGase H+based on synthetic substratesThe substrate specificity of PNGase H+was studied based on five synthetic substrates.The reaction of PNGase H+ with the synthetic substrate can be detected by HPLC because of its ultraviolet-labeled group at the amino acid position.The sugur of Dabsyl-Gly-Glu-Asn-(GlcNAc2Man3)-Arg、dabsyl-Gly-Glu-Asn-(GlcNAc2Man)-Arg、dabsyl-Gly-Glu-Asn-(GlcNAc2)-Arg>Dabsyl-Gly-Glu-Asn-(GlcNAc2Gal)-Arg four substrate can be released by PNGase H+.However,(dabsyl-Gly-Glu-Asn-(GlcNAc)-Arg cannot be released.The conclusion was that PNGaseH+can hydrolyze glycopeptides with sugar unit more than one.3.Application of PNGase H+in legumes gleans analysisThe application of PNGase H+in the research of plant glycoproteins was studied by HPLC and MALDI-TOF-MS/MS based on red beans,soybeans,red beans and black beans.Peaks were collected and detected by MALDI-TOF-MS to obtain possible corresponding structures compared with Glycoworkbench software.On this basis,MALDI-TOF secondary mass spectrometry analysis of the N-glycans structures can be used to verify the predicted structure.And quantitative analysis was performed for relative content.Man3GlcNAc2Xyl1,Man4G1cNAc2,Man3GlcNAc2Xy11Fucl,Man5G1cNAc2,Man6G1cNAc2,Man7G1cNAc2,Man8G1cNAc2 were obtained in red bean and mung bean.The content was 3.3%,4.5%,3.6%,2.8%,19.2%、46.8%,19.8%respectively for red bean.The content was 3.1%、4.6%、3.5%、2.7%、18.4%、47.3%、20.3%respectively for mung bean.Man6G1cNAc2,Man7G1cNAc2,Man8G1cNAc2 were obtained in soybean and black bean.The content was 18.7%、26.5%、54.8%respectively for soybean.The content was 18.2%、26.3%、55.5%respectively for black bean.It was compared for the content of N-glycans in four beans.The majority of N-glycans is high mannose structure in four beans.The percentage composition of Man7G1cNAc2 in red bean and mung bean was the highest,and the percentage composition of Man8G1cNAc2 in soybean and black beans was the highest.