Identification Of Glycans From LTL-recognized Glycoproteins In Saliva Of Hbv-induced Hepatocellular Carcinoma Patients

Background: Liver cancer is a kind of malignant tumor with a high morbidity.Up to now,there still exists some deficiency of the primary HCC diagnosis methods,such as low specificity,inconvenience of sampling and incapacity of early diagnosis.Some researches indicate that the glycosylation of proteins from tissue and body fluids altered during the process of tumorigenesis and development.Salivary diagnostics exhibit advantages of easy access,noninvasive collection and dynamic monitoring,but little of this method has been applied to HCC diagnosis.In this study,we analyzed the alterations of salivary protein glycosylation and identified the specific glycan structures among healthy volunteers(HV),viral hepatitis-B patients(HB),hepatic cirrhosis patients(HC)and hepatocellular carcinoma patients(HCC),in order to find the HCC-specific glycans as potential biomarkers.Method: Firstly,215 salivary samples(HV: n=51,HB: n=51,HC: n=68,HCC: n=45)were tested by a small-scale lectin microarrays which is exclusively used in chronic liver diseases in order to choose a lectin which could specifically recognize the HCC-specific glycans.Secondly,120 salivary samples(HV: n=30,HB: n=30,HC: n=30,HCC: n=30)were enrolled to verify the specificity of the candidate lectin by saliva microarrays and lectin blotting.Thirdly,by isolating these specific glycoproteins,we investigated the structures of their glycans,and expect to find the HCC-specific glycans.Results: Firstly,by the analysis of lectin microarrays,the glycan structures of Fuc?1-2Gal?1-4Glc NAc and Fuc?1-3(Gal?1-4)Glc NAc which were recognized by Lotus Tetragonolobus Lectin(LTL)exhibited significant increased expression in saliva of HCC compared with other three groups.Secondly,the test of saliva microarrays and lectin blotting also verified the abundance of Fuc?1-2Gal?1-4Glc NAc and Fuc?1-3(Gal?1-4)Glc NAc showed a high expression level in in saliva of HCC compared with other three groups(p<0.0001).Thirdly,the LTL-specific glycoproteins of saliva from HV,HB,HC and HCC were isolated by LTL-magnetic particle compounds(LPMCs).Furthermore,the N-glycans and O-glycans of these specific glycoproteins were released by enzyme digestion of PNGase F and oxidation of Na Cl O,respectively.The N-/O-glycans were identified by MALDI-TOF/TOF-MS,and 21/20,25/18,29/19 and 28/24 N/O-glycan peaks were identified and annotated with proposed structures in saliva of HV,HB,HC,and HCC.Because of the specificity of LTL,there were 16/8,18/8,19/10 and 18/16 fucosylated N/O-glycan peaks which contained the structures of Fuc?1-2Gal?1-4Glc NAc and Fuc?1-3(Gal?1-4)Glc NAc in each corresponding group.Of these,there were 8 fucosylated O-glycan peaks(m/z 652.216,828.269,833.314,995.367,1144.295,1157.420,1163.417 and 1193.402)that were found only in HCC group.In conclusion,it indicated that there were specific fucosylated O-glycans that only expressed in saliva of HCC patients.These distinctive discoveries may have potential to facilitate the study of biomarkers for HCC based on precise alterations of specific fucosylated O-glycans in saliva.