O-glycosylation Analysis Of Serum Haptoglobin In Patients With Liver Diseases And Its Clinical Significance

Glycosylation is a functional,complex form of protein post-translational modifications(PTMs),which plays an important role in cell adhesion,molecular transport and clearance,receptor activation,signal transduction,tumorigenesis and metastasis,and so on.The heterogeneity of glycosylation responds significantly to tumors and changes rapidly in diseases,which indicates glycans/glycoprotein biomarkers with great potential.Haptoglobin(Hp)is an important differential glycoprotein found in serum of liver diseases by traditional two-dimensional electrophoresis technique in our previous studies,and its N-glycosylation modification has been studied in detail.It was found that glycosylation site of Asn241 occupancy and the overall fucosylation increased significantly in serum of patients with hepatocellular carcinoma(HCC),which suggested that the potential application value of Hp as a candidate glyco-biomarker for the diagnosis of HCC.However,the study of O-glycosylation of Hp is limited.In this study,serum Hp from normal controls,liver cirrhosis(LC)and HCC patients were purified by antibody affinity chromatography.High-resolution tandem MS(Orbitrap Fusion)was used to identify the O-intact glycopeptides of serum Hp,including O-glycosylation sites and the O-glycosylation of serum Hp in liver diseases was confirmed by combined lectin blot.The structural characteristics and dynamic changes of glycoprotein glycans are closely related to its function and course of diseases,it is important to identify and confirm the O-glycosylation modification of Hp in liver diseases to reveal its biological function of glycosylation modification and to explore the potential clinical application value.Part one O-intact glycopeptides identification of serum Hp in patients with liver diseasesThe purpose of this part is to identify O-intact glycopeptides of serum Hp in patients with liver disease.Here,we used antibody affinity chromatography to purify serum Hp from normal controls,LC and HCC patients and its subunit separated by SDS-PAGE.N-glycans of Hp were removed by PNGase F digestion.High-resolution tandem MS(Orbitrap Fusion)combined with Byonic software were used to analyze O-intact glycopeptides and site-specific structural informations of O-glycans.The O-intact glycopeptides HYEGST316/317 VPEKK of Ser/Thr O-glycosylation site was identified in normal controls,LC and HCC patients of serum Hp by LC-MS/MS.The O-glycoforms was H1N1S1.Another glycoforms H1N1S2 at this site was also identified in LC patients.The results showed that there was O-glycosylation modification in serum Hp and there were differences in the identified O-intact glycopeptides from different liver diseases.It is very important to confirm that the O-glycosylation modification of Hp in liver diseases to reveal its biological function of glycosylation modification and to explore the potential clinical application value of glycosylation modification.Part two Lectin blot analysis of serum Hp in liver diseasesThis part is aimed at verifying O-glycosylation modification of serum Hp.Serum Hp was purified from patients with LC and HCC.Jacalin,GSL-I,VVA,ACA and WFA of affinity O-glycans structure were selected for lectin blot analysis.The results demonstrated the specific affinity ability of serum Hp-β to lectin Jacalin,GSL-I,VVA,ACA and WFA in HCC patients were significantly higher than that in patients with LC(P<0.05)and an increased affinity to Jacalin,VVA and ACA of serum Hp-α2 in HCC patients(P<0.05).Based on the structure of lectins to the O-glycans,it was suggested that serum Hp containing GalNAcα-Ser/Thr(Tn antigen),Galβ1-3GalNAca-Ser/Thr(T antigen)and sialyl-T antigen(sT antigen),GlcNAcβ1-3GalNAca-Ser/Thr(core3),GalNAc and α-Gal,GalNAcαl-3/6Gal and GalNAcβ4GlcNAc were increased in HCC,implying the abnormal O-glycosylation of serum Hp is closely related to the occurrence and development of HCC.Conclusions1.The O-intact glycopeptide of HYEGST316/317 VPEKK was identified in serum Hp purified from normal controls and patients with LC and HCC,and O-glycosylation site on S316/T317,the O-glycoforms was H1N1S1.Besides,another glycoforms with H1N1S2 at this site was also identified in serum Hp from LC patients.2.An significantly increased specific combination with lectin to the O-glycans structure of serum Hp-P in HCC patients compared with LC patients:Jacalin,GSL-I,VVA,ACA and WFA,as well as specific affinity of lectins of Jacalin,VVA and ACA to serum Hp-α2 was significantly increased in HCC patients.Novelty of ProjectThe O-glycosylation modification was identified and verified in serum Hp and the abnormal O-glycosylation modification was found in serum Hp of different liver diseases.Potential Clinical SignificanceIt is very important to confirm of O-glycosylation modification of serum Hp in liver diseases to reveal its biological function involved in glycosylation modification and to explore the potential clinical application value as glyco-biomarkers for liver diseases.