title: Identification of a primary biliary cirrhosis associated protein as lysosome-associated membrane protein-2.
abstract: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and abnormality of hepatobiliary transport might contribute to its pathogenesis. In this study, we aimed to isolate and identify new molecules associated with PBC. With hepatocyte canalicular membrane vesicles (CMVs) of PBC patients as immunogens, we screened the monoclonal antibody 1F9 (mAb1F9), whose antigen dominantly recognized the subapical domains in hepatocytes in normal livers. Immunohistochemistry revealed that the expression of mAb1F9 antigen (mAb1F9-Ag) significantly increased in PBC livers compared with control groups including normal livers, cirrhosis or cholestasis other than PBC. Interestingly, the augmented expression of mAb1F9-Ag was correlated with the severity of PBC, and ursodeoxycholic acid treatment may significantly improve the recovery of mAb1F9-Ag. In addition, redistribution of mAb1F9-Ag was found in 46% of PBC. mAb1F9-Ag was isolated and analyzed with mass spectrometry, which indicated lysosome-associated membrane protein 2 (LAMP-2) as the candidate. Further studies showed that mAb1F9 recognized LAMP-2 immunoprecipitates and vice verse, mAb1F9 reacted with recombinant LAMP-2. mAb1F9 and LAMP-2 antibody exhibited similar staining pattern and displayed similar subcellular localization. Together, the identity of mAb1F9-Ag is LAMP-2, suggesting that LAMP-2 may assist in the differentiation of PBC and predict a poor outcome in patients with PBC.
  
  
  CONCLUSIONS
  This manuscript describes the expression of a specific antibody, named mAb1F9. The antigen recognized by mAb1F9 may assist in the differentiation of primary biliary cirrhosis (PBC) and predict a poor outcome in patients with PBC. Through antigen identification, we confirm the identity of mAb1F9-Ag as lysosome-associated membrane protein 2 (LAMP-2). The clinical relevance of the manuscript is well regarded since markers are rare and usually not successful for PBC diagnosis and treatment.
authors.affiliation: Division of Hepatology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi''an 710032, Shaanxi Province, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.forename: Jingbo
authors.initials: J
authors.lastname: Wang
authors.forename: Yongquan
authors.initials: Y
authors.lastname: Shi
authors.forename: Xinmin
authors.initials: X
authors.lastname: Zhou
authors.forename: Xuechang
authors.initials: X
authors.lastname: Wang
authors.forename: Zengshan
authors.initials: Z
authors.lastname: Li
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Huang
authors.forename: Jianhong
authors.initials: J
authors.lastname: Wang
authors.forename: Zheyi
authors.initials: Z
authors.lastname: Han
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Ruian
authors.initials: R
authors.lastname: Wang
authors.forename: Daiming
authors.initials: D
authors.lastname: Fan
authors.forename: Ying
authors.initials: Y
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002454:Cell Differentiation; D004285:Dogs; D005786:Gene Expression Regulation; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008105:Liver Cirrhosis, Biliary / Q000378:metabolism*; D008107:Liver Diseases / Q000378:metabolism; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D011379:Prognosis; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014018:Tissue Distribution; D014580:Ursodeoxycholic Acid / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014580:Ursodeoxycholic Acid
keywords: 4′,6-diamidino-2-phenylindole; AE2; AMA; BSEP; CMVs; Canalicular membrane vesicles (CMVs); DAPI; HBV; LAMP-1; LAMP-2; Lysosome-associated membrane protein 2 (LAMP-2); MALDI-TOF/TOF; MDR1; MDR3; MRP2; MS; Monoclonal antibody 1F9 (mAb1F9); PBC; PBS; PFIC2; PMF; Primary biliary cirrhosis (PBC); RT-PCR; SDS-PAGE; SS; Sjögren's syndrome; UDCA; anion exchanger 2; anti-mitochondria antibody; bile salt export pump; canalicular membrane vesicles; hepatitis B virus; lysosome-associated membrane protein 1; lysosome-associated membrane protein 2; mAb1F9; mAb1F9 antigen; mAb1F9-Ag; mass spectrometry; matrix assisted laser desorption/ionization-tandem time-of-flight analysis; monoclonal antibody 1F9; multidrug resistant protein 2; multiple drug resistant protein 1; multiple drug resistant protein 3; peptide mass fingerprinting; phosphate buffer saline; primary biliary cirrhosis; progressive familial intrahepatic cholestasis type 2; real-time polymerase chain reaction; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ursodeoxycholic acid