Study On Glycosylation Proteomics Of Human Seminal Plasma

N-linked glycosylation, one of the most common types of PTMs, play important roles in protein folding, cell–matrix interactions, and cell–cell interactions. N-linked glycoproteins are mainly secreted or located on the extracellular side of plasma membrane. Seminal plasma is a mixture of secretions from several male accessory glands. The seminal plasma contains many secreted proteins which are important for sperm function and male fertility. We assume that N-linked glycoproteins are enriched in seminal plasma. The quantity of proteins in seminal plasma is more than serum and urine, which could be access by comparative or qualitative proteomics. It is suggested that N-glycosylated proteins in human seminal plasma can also be a source of biomarkers for male diseases.N-linked glycosylation is important for protein structure and functions, which involved in protein folding, cell–matrix interactions, and cell–cell interactions. These functions are similar as seminal plasma functions in female reproductive tract. Currently, several proteins in human seminal plasma are known to be glycosylated. For example, seven Con A binding proteins were N-glycosylated. N-glycosylated proteins in human seminal plasma can also be a source of biomarkers for male diseases. For example, prostate-specific antigen(PSA), secreted by prostate, is a well-known biomarker for prostate cancer. However, the expression level of PSA cannot distinguish benign prostate hyperplasia from prostate cancer. But N-glycosylated PSA can be an efficient biomarker for prostatic cancer. However, our knowledge of the global composition of N-linked glycoproteins in human seminal plasma remains limited.In this study, we employed N-linked glycosylated peptide enrichment, combined with LC–MS/MS analysis, and establish the first large scale N-linked glycoproteome of human seminal plasma. Combined with the results of five biological replicates, a total of 720 N-glycosylated sites on 372 glycoproteins were identified. Analysis of variations among five individuals revealed similar compositions of N-glycosylated proteins in seminal plasma.Thus to investigate the functional significance of glycosylated proteins in seminal plasma, we performed functional enrichment analysis based on GO and KEGG using Topp Gene. The 372 glycoproteins were converted to 365 Entrez gene identities for convenient functional annotation. For enriched cellular components, the most significantly enriched term is extracellular region part(138 genes). There were 76 genes prone to locate in lysosome, which contains acid hydrolases and is involved in secretion; 61 genes locate in cellular membrane. A total of 160(43%) glycoproteins were found to be secreted proteins. For biological processes, the most significantly enriched term is biological adhesion(78 genes). It is suggested that the function of N-glycosylated proteins in seminal plasma is involve in semen liquefaction, sperm motility and capacitation, fertilization. We provided resources for future screening of candidate biomarkers, including 11 epididymal specific markers, 10 testicular specific markers, 4 seminal vesicle specific markers and 3 prostate specific markers, especially in the level of PTMs, for the diagnosis of male infertility.In second part of our study, we focus on correlation between quantity of glycosylated proteins and sperm motility. Sperm number(64.6±33.8 VS 88.4±44.1)×106/ml and morphology(normal rate: 9.4±5.3 VS 12.9±5.6)% are significantly reduced by comparing control group with asthenospermia. The concentration of fructose in seminal plasma is positive correlated with sperm progressive motility(r=0.21) and motile rate(r=0.25). We quantify 4 glycosylated proteins, LTF, FN1, SEMG2 and WFDC2, using western blot. We find LTF is associated with sperm progressive motility, the quantity of LTF in asthenospermia group is significantly reduced by comparing with control group(P<0.01). LTF is secret by seminal vesicle and prostate, which indicated that secretion of seminal vesicle and prostate is important for sperm progressive motility. The future work will focus on the mechanism of LTF in regulation of sperm motility.