Site-specific N-glycosylation Analysis Of Human Sperm And Seminal Plasma Glycoproteins

Semen is composed of sperm and seminal plasma.Sperm is the main carrier of genetic material.Seminal plasma provides the nutrition and environment required by sperm.The normal physiological and biochemical functions of sperm and seminal plasma are inevitable prerequisites for successful fertilization.Glycosylation is one of the most common posttranslational modifications of proteins and is involved in the whole process of spermatogenesis,maturation and fertilization.Several studies have shown that glycoproteins in sperm and seminal plasma play important roles in the fertilization process.Therefore,this study was focused on analyzing glycoprotein,site-specific N-glycosylation,and glycan composition and structures of human sperm and seminal plasma by using the newly established complete glycopeptide enrichment method and Struc GP software developed in our laboratory recently.By analyzing glycoproteins and site-specific glycan structures in healthy fertile sperm and seminal plasma,a total of 369 glycosylated proteins in sperm,2777 intact glycopeptides,and 401 glycan structures were identified in male sperm samples.Among these glycan structures,the high-mannose type accounts for 3%,the hybrid type accounts for 41.8%,and the complex type glycan reaches 53.2%.Through qualitative analysis of glycoproteins and site-specific glycan structures in seminal plasma,a total of 369 glycosylated proteins,3267 intact glycopeptides,and 780 glycan structures were found in seminal plasma samples.In the structure of seminal plasma glycan chains,high mannose type accounts for 2.31%,hybrid type accounts for 40.59%,and complex type accounts for 56.17%.Sperm and seminal plasma contain many special glycan structures.Comparing the differences of glycoproteins and glycan structures between sperm and seminal plasma,we found that the proportion of high mannose and bisected glycan in sperm is higher than that in seminal plasma.The glycan structure of sialic acid in sperm is also more abundant than in seminal plasma,while the structure of fucose glycan chain in seminal plasma is more abundant than in sperm.In order to further investigate the function of glycoproteins in sperm and seminal plasma,we performed GO function analysis on glycoproteins in sperm and seminal plasma through DAVID,the results showed that glycoproteins in sperm and seminal plasma mainly were associated with enzymatic activation and protein connection function.Glycoproteins in sperm are mainly involved in sperm capacitation,sperm egg recognition and fertilization processes;and glycoprotein functions in seminal plasma mainly involve in metabolic processes,cell adhesion and proteolysis for assisting sperm fertilization.In this study,the site-specific glycoprotein and glycan structure of healthy fertile sperm and seminal plasma were analyzed to establish the site-specific glycoproteins and glycan map of human sperm and seminal plasma.Analyzing the function of specific glycoproteins using bioinformatics methods,revealed the roles of important glycoprotein and glycan during fertilization process,and explains the biological function of sperm and seminal plasma at the level of glycoproteomics and glycomics.This study provided a very comprehensive data basis for future research relating to sperm and seminal plasma glycoproteomics.