Glycan Profiling Of Serum Haptoglobin In Patients Bearing Hepatocellular Carcinoma And Its Potential Clinical Application

As one of the most common post-translation modifications(PTMs),glycosylation is involved in fundamental molecular and cell biology processes occurring in cancer,such as cell signalling and communication,tumour cell dissociation and invasion,cell–matrix interactions,tumour angiogenesis,immune modulation and metastasis formation.The relationship between glycoprotein and diseases especially tumor has drawn more and more attention.Haptoglobin(Hp)is an acidic glycoprotein tetramer mainly secreted and degraded in the liver,whose glycan structures are altered during the cancer process.In this study,serum Hp was purified from healthy individuals and patients with hepatocellular carcinoma(HCC).Antibody overlay lectin microarray and lectin blot were used to determine glycan profiling of serum Hp,establishing the specific glycopattern of serum Hp from HCC.Forthemore,a rapid ELISA assay based on lectin-coated magnetic beads was developed to assess glycosyationalteration of Hp as glycobiomarker and its potential application in HCC diagnosis.Part One Glycan profiling of serum Hp in HCC patientsGlycopatterns of serum Hp in healthy individuals and HCC patients were compared and analyzed by antibody overlay lectin microarray.The results demonstrated an increased affinity of 9 lectins to the glycan of serum Hp from HCC: LCA,UEA-I,AAL,SNA-I,Con A,CALSEPA,RCA I,PHA-L and Jacalin,and a decreased affinity of 2 lectins: LEL and PHA-E.It was suggested serum Hp containing ?-fucose and core fucose,?2,6 linked sialic acid residues,?-Man,?1,4-Gal and ?1,6-Glc NAc branched N-glycan structures,as well T/Tn antigen and sialyl-T antigen were increased in HCC,and that glycan containing ?1,4-Glc NAc branching and bisecting Glc NAc structures were decreased.PHA-E?AAL?LCA?SNA-I?Jacalin and PHA-L were used for further validation by lectin blot,implying aberrant glycosylation of serum Hp is closely related to the occurrence and development of HCC.Part Two Rapid AAL@Magbeads-based Hp ELISA system and its potential clinical applicationHepatocellular carcinoma(HCC)is one of the most common cancers worldwide with the characteristics of high mortality and poor prognosis.Increased fucosylated Hp(Fuc-Hp)was found in HCC patients by us previously.In this study,a high-throughput method was developed for sensitive quantification of Fuc-Hp in HCC patients.Firstly,a comparison of normal lectin ELISA and reverse lectin ELISA indicated that the reverse lectin ELISA had more advantages.Secondly,Aleuria Aurantia Lectin coated magnetic beads(AAL@Magbeads)based on reverse lectin ELISA was developed to analyze Fuc-Hp from serum samples.In addition,protein ELISA was used to measure Hp protein expression.Hp protein level and fucosylated Hp were detected in 260 healthy controls and HCC patients.Additionally,Fuc-Hp ratios(Fuc-Hp/Hp=Fuc-Hp level/ Hp protein level;ELISA Index=OD value of Fuc-Hp/ OD value of protein Hp)were established to reflect Hp fucosylation level on its protein level.The results showed that serum Fuc-Hp,Fuc-Hp/Hp and ELISA Index were higher significantly in HCC and AFP-HCC patients than those in healthy controls,and improved sensitivity,specificity and accuracy could be obtained when combined with AFP.In summary,Fuc-Hp/Hp and ELISA Index could be potential glycobiomarkers for the surveillance and diagnosis of HCC even with a low level of AFP.Moreover,combination of AFP and Fuc-Hp ratios could improve the sensitivity and specificity of HCC diagnosis.Conclusions1.Nine enhanced affinity and two reduced affinity of lectins were screened out by antibody overlay lectin microarray and lectin blot,and the specific glycan pattern of serum Hp from HCC was established through oligosaccharides structure analysis.2.A novel ELISA strategy based on AAL@Magbeads was established to quantify fucosylation of serum Hp,providing candidate biomarkers for AFP-and AFP+ HCC.3.Serum Fuc-Hp was significantly increased in HCC patients,it implied Fuc-Hp could act as the candidate glycobiomarker and had the potential value.Combination of AFP and Fuc-Hp ratios could improve the ability of prediction and diagnosis of HCC.Novelty of Project1.The specific glycan pattern of serum Hp from HCC could serve as potential glycobiomarkers and might provide novel diagnostic indicators.2.A novel strategy based on lectin@Magbeads ELISA was provided for quantitative analysis glycosylation of target protein.