Heterogeneities Of Site-specific N-glycosylation In HCC Tumors With Low And High AFP Concentrations

Hepatocellular carcinoma(HCC)is still one of the malignant tumors with high morbidity and mortality in China and worldwide.The patient with early diagnosis of HCC can achieve 70% five-year survival rate through surgical treatment,but due to the occult onset of hepatocellular carcinoma,most patients cannot get timely diagnosis and treatment.Although alpha fetoprotein(AFP)as well as core fucosylated AFP(AFP-L3)have been widely used as important biomarkers for the early diagnosis and evaluation of HCC,the AFP level has a huge variation among HCC patient populations and HCC patients with different AFP levels show differences in molecular,tumorigenesis,and clinical angiography.Many studies have shown that abnormal glycosylation is a basic feature of the occurrence and development of HCC.Therefore,understanding the intrinsic heterogeneities of HCC associated with AFP levels is essential for the molecular mechanism studies of HCC with different AFP levels as well as for the potential early diagnosis and personalized treatment of HCC with AFP negative.In this study,an integrated glycoproteomic and proteomic analysis of low and high AFP level of HCC tumors was performed to investigate the intrinsic heterogeneities of site-specific glycosylation associated with different AFP levels of HCC.By large-scale profiling and quantifying more than 4,700 intact glycopeptides from 20 HCC and 20 paired paracancer samples,we identified many commonly altered site-specific glycans from HCC tumors regardless of AFP levels,including decreased modifications by oligo-mannose and sialylated bi-antennary glycans,and increased modifications by bisecting glycans.By relative quantifying the intact glycopeptides between low and high AFP tumor groups,the great heterogeneities of site-specific N-glycans between two groups of HCC tumors were also uncovered.We found that several sialylated but not core fucosylated tri-antennary glycans were uniquely high-regulated in low AFP level of HCC tumors,while many core fucosylated bi-antennary or hybrid glycans as well as bisecting glycans were uniquely increased in high AFP tumors.For analysis of serum,1626 intact glycopeptides were identified from the low and high AFP HCC sera.By integrating quantitative data of proteomics and glycoproteins in HCC serum,it was found that the site-specific glycosylation expression difference between tumors and adjacent cancers was greater than protein expression,which was consistent with the results in HCC tissues.In addition,through structure analysis of N-glycans on identified glycopeptides,we found that sialylated but not core fucosylated tri-antennary glycans were uniquely high-regulated in low AFP level of HCC sera,and the bisecting glycans were specifically increased in high level of AFP HCC sera,which is consistent with the glycosylation alterations in tissue.These results revealed that HCC with low and high AFP showed heterogeneity in both tissue and serum,and these differences were very similar in HCC tissue and serum.The data provide a valuable resource for future HCC studies regarding the mechanism,heterogeneities and new biomarker discovery.