Isolation And Identification Of Primary Biliary Cirrhosis-associated1F9Antigen

【Background】Primary biliary cirrhosis (PBC) is characterized by the chronic intrahepaticcholestasis, which is related to the abnormality of hepatocyte canalicular surfaces.In our previous study, hepatocyte canalicular membrane fraction isolated from thelivers of PBC patients was used as immunogens to produce hybridoma and1F9monoclonal antibody was obtained. Further studies demonstrated that, to comparewith normal human liver,1F9antigen expressed much differently in PBC patients.In normal human liver,1F9antigen exclusively shown a polarized distribution atthe side of the hepatocyte canalicular membrane. While in the liver of PBC,1F9antigen expressed highly and the polarized distribution disappeared, indicatingthat it may contribute to pathological diagnosis of PBC. However, the precise1F9antigen remains enigmatic.【Objective】The present study aimed:(1) to further determine the clinical pathological significance of1F9antigen in PBC.(2) to isolate and dentify the1F9antigen.【Materials and methods】Liver tissue specimens (all were biopsied or surgically resected) werecollected from the liver disease file of Xijing Hospital. The liver specimensenrolled in this study were PBC，viral hepatitis b, drug induced liver disease,alcoholic liver disease, nonalcoholic fatty liver disease and “histologicallynormal” livers. Distribution and expression of1F9antigen were detected byimmunohistochemistry.1F9antigen was enriched using immunoprecipitation inHuman hepatocellular liver carcinoma cell line (HepG2) and Human proximaltubules cell line (HK2) respectively, both followed by confirmation with silverstaining and Western blot. The target protein bands were detected byMALDI-TOF-TOF and LC-MS/MS followed by bioinformatics analysis. Westernblot, immunohistochemistry, immunofluorescence, prokaryotic expression systemand pre-embedding immunogold-silver cytochemistry provided evidences forfurther analysis.【Results】This study provided further evidences that1F9antigen may play someimportant roles in PBC. Distribution characteristics of1F9antigen in six kinds ofliver tissues were detected by immunohistochemistry. In normal human livertissues and several other non-cholestatic liver tissues,1F9antigen exclusivelylocalized at the side of hepatocyte canalicular membrane. However, in the liversof PBC,1F9antigen expressed much highly and the polarized distributiondisappeared. Immunostaining analysis demonstrated that the over-expression of1F9antigen was correlated with the severity of PBC.The isotype of1F9monoclonal antibody was comfirmed to be IgG1/κ type,and1F9antigen was identified to be LAMP2:①Approximately a110kDa protein immunoreactive with1F9antibody in HepG2cells and HK2cells wasidentified as LAMP2, a major protein component of the lysosomal membrane, byMALDI-TOF-TOF and LC-MS/MS.②The antigenic cross-reactivity was testedby blotting the immunoprecipitated protein with each of1F9, LAMP1(also aprincipal lysosome-associated membrane protein) and LAMP2antibodies. Both1F9and LAMP2antibodies recognized the~110kDa protein immunoprecipitatedby either1F9or LAMP2antibody, but LAMP1antibody did not.③Immunohistochemical analysis was conducted to compare the staining pattern of1F9antigen and LAMP2in normal human tissue microarrays and PBC tissues.The immunostaining characteristics of LAMP2in these tissues were identical to1F9antigen.④To confirm the co-localization of1F9antigen and LAMP2,subcellular localization was further determined by immunofluorescence. Theresults showed that1F9antigen stained green was completely overlapping withLAMP2stained red in HepG2cells and HK2cells, respectively.⑤Transienttransfection of LAMP2plasmids or LAMP1plasmids into the LAMP2and1F9antigen negatively expressed cell line, Madin-Darby canine kidney cell line(MDCK). Both1F9and LAMP2antibodies could recognize theheterologously-expressed LAMP2, but not LAMP1.⑥Pre-embeddingimmunogold-silver cytochemistry was used to provide further evidences.Immunogold particles indicative of LAMP2immunoreactivity were much similarwith that of1F9antigen.【Conclusions】The present study assessed the distribution of1F9antigen in several livertissues, and analyzed the clinical pathological significance of1F9antigen in PBC.With a series of assays,1F9antigen was confirmed to be LAMP2. As describedpreviously, LAMP2was involved in regulating the hepatocyte transport systems, which suggested that the abnormality of LAMP2might be responsible for thecholestasis in PBC patients.