Identification of a primary biliary cirrhosis associated

J O U RN A L OF P ROT EO M IC S 9 1 ( 2 01 3 ) 5 6 9 –5 79

Available online at www.sciencedirect.com

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Identification of a primary biliary cirrhosis associated protein as lysosome-associated membrane protein-2 Lu Wanga,1 , Jingbo Wanga,⁎,1 , Yongquan Shia , Xinmin Zhoua , Xuechang Wanga , Zengshan Lib , Xiaofeng Huangc , Jianhong Wanga , Zheyi Hana , Tingting Lid , Min Wanga , Ruian Wangb , Daiming Fana , Ying Hana,⁎ a

Division of Hepatology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'’an 710032, Shaanxi Province, China Department of Pathology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China c Center of Electron Microscope, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China d Department of Gastroenterology, Yan'an University Affiliated Hospital, Yan'an 716000, Shaanxi Province, China b

AR TIC LE I N FO

ABS TR ACT

Article history:

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and

Received 9 June 2013

abnormality of hepatobiliary transport might contribute to its pathogenesis. In this study, we

Accepted 26 August 2013

aimed to isolate and identify new molecules associated with PBC. With hepatocyte canalicular

Available online 2 September 2013

membrane vesicles (CMVs) of PBC patients as immunogens, we screened the monoclonal antibody 1F9 (mAb1F9), whose antigen dominantly recognized the subapical domains in

Keywords:

hepatocytes in normal livers. Immunohistochemistry revealed that the expression of mAb1F9

Primary biliary cirrhosis (PBC)

antigen (mAb1F9-Ag) significantly increased in PBC livers compared with control groups

Canalicular membrane vesicles

including normal livers, cirrhosis or cholestasis other than PBC. Interestingly, the augmented

(CMVs)

expression of mAb1F9-Ag was correlated with the severity of PBC, and ursodeoxycholic acid

Monoclonal antibody 1F9

treatment may significantly improve the recovery of mAb1F9-Ag. In addition, redistribution of

(mAb1F9)

mAb1F9-Ag was found in 46% of PBC. mAb1F9-Ag was isolated and analyzed with mass

Lysosome-associated membrane

spectrometry, which indicated lysosome-associated membrane protein 2 (LAMP-2) as the

protein 2 (LAMP-2)

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.

Abbreviations: PBC, primary biliary cirrhosis; CMVs, canalicular membrane vesicles; mAb1F9, monoclonal antibody 1F9; mAb1F9-Ag, mAb1F9 antigen; UDCA, ursodeoxycholic acid; MALDI-TOF/TOF, matrix assisted laser desorption/ionization-tandem time-of-flight analysis; MS, mass spectrometry; PMF, peptide mass fingerprinting; LAMP-2, lysosome-associated membrane protein 2; BSEP, bile salt export pump; PFIC2, progressive familial intrahepatic cholestasis type 2; MRP2, multidrug resistant protein 2; MDR1, multiple drug resistant protein 1; MDR3, multiple drug resistant protein 3; AE2, anion exchanger 2; SS, Sjögren's syndrome; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate buffer saline; AMA, anti-mitochondria antibody; HBV, hepatitis B virus; RT-PCR, real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LAMP-1, lysosome-associated membrane protein 1. ⁎ Corresponding authors. Tel.: +86 29 84771506; fax: + 86 29 82539041. E-mail addresses: [email protected] (J. Wang), [email protected] (Y. Han). 1 These two authors contributed equally to this work. 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.08.019

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J O U RN A L OF P ROTE O M IC S 9 1 ( 2 01 3 ) 5 6 9 –57 9

Biological significance 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. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology [1,2]. Although it is classified as an autoimmune disease, immunosuppressive regimes tested thus far appear to lack beneficial effects in modifying the natural progression of PBC [3–5]. In contrast, administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid known to induce bicarbonate-rich choleresis, may improve the clinical course of PBC [6]. Hepatocyte exhibits a striking polarity with distinct canalicular and sinusoidal plasma membrane domains that have unique morphological, functional and molecular characteristics [7]. Hepatocyte canalicular domain is characterized by numerous microvilli and specialized for transport activities which are carried on in bile secretion. Its structure or function impairment could directly lead to bile metabolism disorders. Mutation of bile salt export pump (BSEP) could lead to the inherited cholestatic disorder progressive familial intrahepatic cholestasis type 2 (PFIC2) [8,9]. Absence of multidrug resistant protein 2 (MRP2), responsible for transport of divalent bile acids, could cause Dubin-Johnson syndrome, an inherited liver disorder characterized by conjugated hyperbilirubinemia [10]. Meanwhile, reduced transporter expression may contribute to impaired excretory liver function in patients with cholestatic liver diseases [11,12]. Given that PBC is a prototypic cholestatic liver disease, several researchers have investigated the contribution of hepatobiliary transport to its pathogenesis. In PBC patients, BSEP, multiple drug resistant proteins 1and 3 (MDR1, MDR3) are increased in protein and mRNA levels [13,14], whereas MRP2 are unchanged in early stage and redistributed into intracellular structures in end stage [15]. Notably, a significant reduction of sodium-independent Cl−/HCO−3 anion exchanger 2 (AE2), involved in biliary bicarbonate secretion and intracellular pH homeostasis, was found in liver and blood mononuclear cells from PBC patients [16,17], and Ae2a,b−/− mice may indeed develop some characteristics that resemble PBC [18]. Interestingly, AE2 expression in salivary glands was also reduced in PBC patients with Sjögren's syndrome (SS) [19], which suggests that reduced AE2 expression may be implicated in polyglandular exocrine failure with accompanying PBC. Although several researchers have theoretically investigated the function and regulation of hepatobiliary transporters in patients with PBC, a complete description of the cholestasis of PBC is still unavailable. In this study, we prepared hybridomas using canalicular membrane vesicles (CMVs) isolated from PBC liver homogenates, and screened PBC molecular markers. Monoclonal antibody 1F9 (mAb1F9) dominantly recognized the subapical domain in hepatocytes in normal livers. However, mAb1F9 antigen

(mAb1F9-Ag) in PBC patients explored the unique characteristics: protein and mRNA expression increased, and diffuse cytoplasmic staining was occasionally observed. Notably, mAb1F9-Ag expression was correlated with the histological stage of PBC and ursodeoxycholic acid (UDCA) treatment may significantly improve the recovery of mAb1F9-Ag. Finally, mAb1F9-Ag was confirmed to be human lysosomal-associated membrane protein 2 (LAMP-2).

2.

Materials and methods

2.1.

Human subjects

Human wedge-biopsied or surgically resected liver tissues were collected from the liver disease file of Xijing Hospital (Xi'an, Shaanxi, China). All patients who agreed to be involved in the study signed an informed consent. PBC was diagnosed based on its clinical, serological and histological characteristics and staged histologically according to Ludwig's classification. All cases of other liver diseases were also clinically and pathologically proven. The protocols used in the study were approved by the hospital's Ethics Committee.

2.2. Isolation of liver CMVs from PBC patients and production of hybridomas CMVs were prepared from PBC surgically resected liver homogenates using differential and density gradient centrifugation method [20,21]. The morphological and biochemical characteristics of isolated CMVs were confirmed by transmission electron microscopy and enzyme assay respectively as described in our published paper [21]. Hybridomas were produced as follows. BALB/c mice were immunized with 100 μg isolated CMV proteins of PBC patients in complete Freund's adjuvant (Sigma, MO), and boosted twice with 50 μg proteins in incomplete Freund's adjuvant (Sigma, MO) at 3-week intervals. After spleen cells of immunized mice were fused with SP2/0 myeloma cells, hybridoma cell supernatants were screened with immunohistochemistry assay. Positive hybridomas were cloned by limiting dilution and collected for ascites production. The ascites was purified according to the protocol of the Antibody Purification Kit (Pierce, IL).

2.3.

Immunohistochemistry

Patients were grouped as follows: (1) 115 subjects with PBC (stage I in 22 subjects and stage II, III or IV in 26, 20 or 47, respectively), which can be divided into different subgroups (63 untreated and 52 UDCA-treated cases, or 82 anti-mitochondria


antibody (AMA)-positive and 33 AMA-negative cases); (2) a first control group referred to as the normal liver group, formed by 39 subjects with histologically normal livers; (3) a second control group referred to as the cirrhosis group, which included 61 subjects with cirrhosis of two different etiologies: alcoholic cirrhosis and hepatitis B virus (HBV) induced cirrhosis; and (4) a third control group referred to as the cholestasis group, which included 32 subjects with intrahepatic cholestasis (including Gilbert's disease Dubin-Johnson syndrome, drug induced cholestasis and primary sclerosing cholangitis) and 8 with extrahepatic biliary obstruction. Formalin-fixed, paraffin-embedded sections were deparaffinized with xylene, rehydrated in graded ethanol (100% to 85%) and then treated for 15 min in a microwave oven with 0.001M ethylene diamine tetraacetic acid /Tris-base (pH 8.0) for antigen retrieval. After endogenous peroxidase and goat serum block, tissues were incubated in mAb1F9 1:1000 or mouse anti-LAMP-2 monoclonal antibody 1:500 (Abcam, MA) and horseradish peroxidase labeled goat antimouse IgG (Dako, Denmark), then visualized with diaminobenzidine (Dako, Denmark). The immunoreactivity of mAb1F9-Ag was evaluated on a scale ranging from 0 to 12 according to the following histological scoring method. The score was obtained after the intensity score (0 to 3: 0, no signal; 1, slight; 2, moderate; 3, intense) multiplied by the distribution score (0 to 4: 0, staining of 0–1%; –1%; 1, staining of 2–25%; 2, 3, or 4, 50, 75, or more than 75% staining, respectively). The resulting score was graded as negative (−, score: 0), weak (+, 1–4), moderate (++, 5–8), or strong (+++, 9–12) for further non-parametric tests. Each slide was scored by two pathologists in a double-blind fashion.

2.4.

Immunofluorescence

Cells cultured on glass coverslips or ice-frozen live tissues were fixed with phosphate buffer saline (PBS) containing 2.5% paraformaldehyde, then incubated with first antibodies: mAb1F9 and rabbit anti-CD13 antibody (Santa Cruz, CA) or rabbit anti-LAMP-2 polyclonal antibody (Abcam, MA) and second antibodies: FITC-conjugated anti-mouse IgG and CY3-conjugated anti-rabbit IgG (Santa Cruz, CA). Cells or tissues were stained with 4′,6-Diamidino-2-phenylindole (DAPI) before visualization with a laser confocal microscope (Fluoview-FV10i; Olympus, Tokyo).

2.5.

Immunoelectron microscopy

The fresh liver tissues were fixed with ice-cold mixture of 4% paraformaldehyde, 0.05% glutaraldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (pH 7.4) for 3 h at 4 °C. Serial liver sections of 50 μm thickness were prepared and placed PBS containing 25% sucrose and 10% glycerol for 1 h. After a freeze-thaw treatment, sections were immersed in PBS containing 5% BSA and 5% NGS to block nonspecific immunoreactivity, incubated with mAb1F9 1:1000 and goat anti-mouse IgG conjugated to 1.4 nm gold particles at 1:100 dilution (Nanoprobes, NY). Sections were postfixed in 2% glutaraldehyde and silver enhancement was carried out in the dark with HQ Silver Kit (Nanoprobes, NY). After fixing with 0.5% osmium tetroxide in 0.1 M phosphate buffer for 1 h, immunolabeled sections were dehydrated in graded ethanol

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series, then in propyleneoxide, and finally embedded in Epon 812. After mounting the ultra thin sections on mesh grids (six to eight sections/grid), they were counterstained and observed under a JEM-1230 electron microscope (JEOL Ltd., Tokyo).

2.6.

Immunoprecipitation

Protein enrichment was conducted using a classic immunoprecipitation assay kit (Pierce, IL). According to the manufacturer's instruction, cell layer was washed with ice-cold PBS and lysed in NP40 lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol, pH 7.4) before being scraped off. Cell lysates supernatant was collected by centrifugating at 13,000 ×g for 10 min, incubated with mAb1F9 or mouse anti-LAMP-2 monoclonal antibody (10 μg/ml) or control (isotype matched mouse IgG) for 1 h and then added to Protein A/G Plus Agarose (20 μl/ml) overnight. After washing and recovering the immune complex, samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), according to conventional methods. The gel was stained either with silver using a Fast Silver Stain Kit (Beyotime, Haimen) or by Western blotting.

2.7. In-gel digestion and matrix assisted laser desorption/ ionization-tandem time-of-flight (MALDI-TOF/TOF) analysis The protein band recognized and matched to that detected on Western blot was excised from the silver-stained gel, digested with trypsin and then analyzed by combined peptide mass fingerprinting (PMF) and mass spectrometry (MS)/MS as described previously [22,23]. Briefly, the protein band was destained, washed, shrunk by dehydration and then digested with trypsin. Extracted peptides were mixed with MALDI matrix (5 mg/ml CHCA diluted in 0.1% TFA/50% ACN) and spotted on to the 384-well stainless steel MALDI target plates. The molecular masses of the tryptic peptides were determined in the positive-ion mode on an ABI 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MA). The MS spectra were recorded in the positive reflector mode in a mass range from 800 to 4000 using a 200-Hz ND:YAG laser operating at 355 nm. For one main MS spectrum 25 subspectra with 125 shots per subspectrum were accumulated using a random search pattern. For MS calibration, autolysis peaks of trypsin ([M + H] + 842.5100 and 2211.1046) were used as internal calibrates, and up to 10 of the most intense ion signals were selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals. In MS/MS mode, 50 subspectra with 50 shots per subspectrum were accumulated for one main MS spectrum using a random search pattern. Using the individual PMF spectra, peptides exceeding a signal-to-noise ratio of 20 that passed through a mass exclusion filter were submitted to fragmentation analysis. The 10 most and 10 least intense ions per MALDI spot, with signal-to-noise ratios >50, were selected for subsequent MS/MS analysis in the 1-kV mode. Air served as the collision gas. The data were calibrated using the ABI 4700 Calibration Mixture (Applied Biosystems, CA). The National Center for Biotechnology Information (NCBI, Homo sapiens, 20110710) was searched for matches to the MS/MS spectra using GPS-Explorer Software 3.6, with MASCOT as the database search engine, with the following parameter settings:

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the mass accuracy was 100 ppm, one missed trypsin cleavage was allowed, carbamidomethylation was set as a fixed modification, the oxidation of methionine was allowed as a variable modification, and the MS/MS fragment tolerance was set to 0.4 Da. A GPS Explorer protein confidence index ≥95% was used for further manual validation.

2.8.

granular, clubbed, and cytoplasmic along the hepatocyte membranes (Figs. 1A and 3A). In addition, staining of the brush border of the interlobular bile ducts was also observed

Cell transfection

Madin-Darby canine kidney (MDCK) cells were maintained on cell plates at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium (Gibco, NY) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cell transfection was performed with Lipofectamine2000 (Invitrogen, NY), as described in the manufacturer's protocol. Briefly, cells were plated and grown to 70% to 90% confluence without antibiotics and then transfected with 1 mg Myc-DDK-tagged human LAMP-2 plasmids (OriGene Technologies, Beijing). Cells were harvested and tested for Western blot assay after 48 h of transfection.

2.9.

Real-time polymerase chain reaction (RT-PCR)

Total RNA extraction, complementary DNA synthesis and PCR were performed according to the manufacturer's protocol. The primer sequences were designed using Primer Express Software (PE Applied Biosystems, CA) as follows: LAMP-2A, 5′-CTT ATA TGT GCA ACA AAG AGC AGA C-3′-GGA AGT TGT CGT CAT CTG CAC TG-3′-CTT ATA TGT GCA ACA AAG AGC AGA C-3′-ATT AGA ATG GTG TCA TCA TCC AGC-3′-GGA GTC AAC GGA TTT GGT-3′-GTG ATG GGA TTT CCA TTG AT-3follows: 50 °C for 2 min, 95 °C for 30 s, and then 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The relative expression between target genes and GAPDH was calculated using the 2 − ΔΔCT method. The specificity of the amplification products was confirmed by ethidium bromide-stained 1.5% agarose gels.

2.10.

Statistical analysis

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., IL). Kruskal–Wallis H test for multi-groups and Mann–Whitney U test for two groups was used to compare the difference of groups for immunoreactivity score data with various clinical pathological parameters. Statistical analysis of mRNA values were performed using Student's t test and one-way analysis of variance (ANOVA) with multiple comparisons (Scheffé's test). Correlation significance was assessed using Spearman's rank test. All p values were two tailed.

3.

Results

3.1. Distribution of mAb1F9-Ag in liver and other normal human tissues With the isolated CMVs from PBC patients as immunogens to immunize mice and prepare hybridomas, we obtained the monoclonal antibody, termed as mAb1F9, which may assist in the diagnosis of PBC from other liver diseases. Sections from normal livers revealed a distinctive staining of the hepatocytes. The cellular staining pattern was chiefly

Fig. 1 – Distribution of mAb1F9-Ag in human liver tissue. A, Liver tissue, showing clear staining at the apical part of the hepatocytes and the brush border of interlobular bile duct epithelium at high magnification. B, Co-localization of mAb1F9-Ag (green) and CD13 (red), a marker of hepatocyte canalicular membrane. C, mAb1F9-Ag subcellular localization in hepatocyte by immunoelectron microscopy. mAb1F9-Ag was positive on single or small clusters of irregularly shaped vesicular structures (triangle) with 50–200 nm diameter near cholangiole (C). Original magnification of A, ×200, of B, ×600.


at high magnification (Fig. 1A). Next, we observed the co-localization of mAb1F9-Ag and CD13, a characteristic of strict distribution on the hepatocyte canalicular membrane. The results showed that the green-labeled mAb1F9-Ag structures surrounded the red CD13 staining with no coexistence, which indirectly illustrated the subapical localization of mAb1F9-Ag in the hepatocytes (Fig. 1B). Immunoelectron microscopy further demonstrated that mAb1F9-Ag was positive on the membrane of single or small clusters of irregularly shaped vesicular structures with 50–150 nm cup-shaped vesicles immediately underneath the hepatocyte canalicular membrane (Fig. 1C). No gold particle was detected on the nucleus, endoplasmic reticulum, mitochondria or plasma membrane. Immunoglobulin subclass determination results showed that the isotype of mAb1F9 was confirmed to be IgG1/κ type.

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As many cells like hepatocytes also exhibit distinct polarity, we further explored the subcellular localization of mAb1F9-Ag in other organelles. It was shown that mAb1F9-Ag was restricted to the brush border of small intestine and proximal tubule epithelial cells (Fig. 2A–B). It was also positive on the apical region of salivary, gastric and pancreas gland epitheliums (Fig. 2C–E), as well as epithelial cells of large bile, endometrial and mammary duct (Fig. 2F–H). However, it explored no polarity distribution on gland epitheliums in prostate, epinephros, thyroid, parathyroid, pituitary and testicle (Fig. 2I–N).

3.2. Augmented expression and redistribution of mAb1F9-Ag in PBC mAb1F9-Ag expression was demonstrable in 114 of 115 patients with PBC, 60 of 61 patients with cirrhosis, 44 of 45

Fig. 2 – Distribution of mAb1F9-Ag in human tissue array by immunohistochemistry analysis. A, small intestine; B, kidney; C, sialaden; D, stomach; E, pancreas; F, bile duct; G, uterus; H, mammary gland; I, epinephros; J, prostate; K, thyroidea; L, parathyroideum; M, pituitary; N, testicle. mAb1F9-Ag was restricted to the brush border of small intestine and proximal tubule epithelial cells (A–B), the apical region of salivary, gastric and pancreas gland epitheliums (C–E), as well as epithelial cells of bile, endometrial and mammary duct (F–H). Original magnification of A and B, × 600, of C–N, × 400.

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patients with cholestasis, 37 of 39 normal controls. When considering immunoreactivity scores as described above in the Materials and methods section, mAb1F9-Ag abundance in PBC patients was significantly higher than that in both normal livers and livers with diseases other than PBC (cholestasis or cirrhosis) (p < 0.001, Table 1; Fig. 3A–H). Such increased immunoreactivity was evident in both hepatocytes and bile ducts (both terminal and interlobular). What's more, in PBC patients, the mAb1F9-Ag abundance in late-stage group (Scheuer stages III and IV) was increased compared with that in early-stage group (Scheuer stages I and II), indicating a correlation between mAb1F9-Ag expression and PBC histological stage. Further data analysis demonstrated that significant difference was also observed between UDCA-treated and untreated cases (p = 0.004, Table 1). However, no significant difference in staining was observed between AMA-positive and AMA-negative cases (p = 0.274, Table 1). In 46% (53/115) of PBC patients, especially PBC stages II–IV, the staining was broadened, irregular or even diffused in the cytoplasm of hepatocytes (Fig. 3I). The rate of mAb1F9-Ag redistribution in late stages (49/67, 73.13%) was much higher than that in early stages (4/48, 8.33%). Diffuse cytoplasmic staining was validated by indirect immunofluorescence, with the green-labeled mAb1F9-Ag had a strikingly increased presence in the cytoplasm of the hepatocytes (Fig. 3J). Immunoelectron microscopy further confirmed that in addition to lysosomal proliferation, mAb1F9-Ag was positive not only at the vesicular structures in a close proximity to the apical domain but also in the lysosomes near the nucleus. Note that the number of immunogold particles in the remaining cytoplasm and/or in the lysosome lumen was also increased (Fig. 3K).

Table 1 – Expression of mAb1F9-Ag in patients with PBC and control groups. Histological type

Patient groups Normal liver a Cirrhosis a Cholestasis a PBC Histological stage PBC stage I PBC stage II PBC stage III PBC stage IV Treatment Untreated PBC UDCA treated PBC AMA AMA (+) AMA (−)

n

Immunoreactivity score of mAb1F9-Ag −

+

++

+++

39 61 45 115

2 1 1 1

18 22 17 26

17 28 20 43

2 10 7 45

22 26 20 47

1 0 0 0

16 8 2 0

5 16 10 12

0 2 8 35

63 52

1 0

9 17

21 22

32 13

82 33

1 0

19 7

33 10

29 16

p value

p p p p

< < = =

0.001 0.001 0.002 0.003

p < 0.001

3.3. Identification of mAb1F9 immunoreactive protein as LAMP-2 We identified the protein reacting with mAb1F9 through immunoprecipitation from mAb1F9-Ag-positive cell lines (a hepatoma cell line, HepG2 and a human proximal tubule kidney cell line, HK2) (Supplementary Fig. 1A, B), followed by MALDI-TOF/TOF MS. SDS-PAGE showed that a protein band with approximate molecular mass 110 kDa in HepG2 cells was consistently precipitated and recognized exclusively by mAb1F9 (Fig. 4A). The immunoreactive protein band was excised from the silver-stained gel and digested with trypsin. The resulting peptide fragments were analyzed by MALDI-TOF/TOF MS and a peptide fingerprint was obtained (Fig. 4B). Fig. 4C displays an MS/MS spectrum acquired for the peptide of EQTVSVSGAFQINTFDLR with Ion Score of 85. Using the MASCOT engine against NCBI database, the protein band was found to contain LAMP-2 amino acid sequences (NCBI accession number, gi|307110; Protein Score 139). The matched peptides are highlighted and underlined with a sequence coverage of 16% (Fig. 4D). The above results were also obtained in HK2 cells (Supplementary Fig. 2).

3.4.

Confirmation of mAb1F9-Ag as LAMP-2

To confirm mAb1F9-Ag as LAMP-2, the antigen cross-reactivity was tested by blotting the immunoprecipitated proteins with each of the two antibodies (Fig. 5A). Both mAb1F9 and anti-LAMP-2 antibody blotted the proteins immunoprecipitated by either mAb1F9 or anti-LAMP-2 antibody, whereas anti-LAMP-1 antibody did not. To determine whether mAb1F9 recognized the heterologously expressed LAMP-2 protein in vitro, we transiently transfected MDCK cells, which negatively express mAb1F9-Ag (Supplementary Fig. 1C) with a vector that expresses human LAMP-2. As shown in Fig. 5B, both mAb1F9 and anti-LAMP-2 antibody detected the heterologously expressed LAMP-2 protein. In contrast, anti-LAMP-1 antibody did not. Next, we analyzed the distribution of LAMP-2 and mAb1F9 in HepG2 and HK2 cells, and determined if they colocalized (Fig. 5C). Double-labeled cells were clearly observed, which showed that the green-stained mAb1F9-Ag almost completely overlapped with the red-stained LAMP-2. Regions of colocalization appeared yellow in the micrographs. Paired immunohistochemistry was performed to determine whether LAMP-2 had the same expression pattern when probed with mAb1F9 in successive sections of normal human tissues (Supplementary Fig. 3). The immunostaining characteristics of mAb1F9-Ag in these tissues were almost identical to those of LAMP-2.

p = 0.004

3.5. p = 0.274

Kruskal–Wallis test was for comparison among multi-groups, p < 0.001. Mann–Whitney test was for further comparison between two groups. Differences were considered statistically significant at p < 0.05. a Compared with PBC group.

mRNA expression of LAMP-2 in PBC

LAMP-2 undergoes alternative splicing which leads to at least three isoforms (LAMP-2A, LAMP-2B, and LAMP-2C) [24]. Finally, we analyzed mRNA expression of these three isoforms by RT-PCR from liver samples of 57 patients with PBC and 30 patients with other liver diseases. In our study, only LAMP-2A and LAMP-2B mRNA could be detected in the liver, whereas LAMP-2C did not. Both LAMP-2A (43.8 ± 15.4 VS 20.5 ± 8.9) and LAMP-2B (4.6 ± 1.8 VS 2.8 ± 1.2) genes were upregulated in PBC


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Fig. 3 – Expression of mAb1F9-Ag in different liver diseases. A–D, mAb1F9-Ag expression in normal liver, fatty liver, cirrhosis and cholestatic liver disease by immunohistochemistry analysis. (A) normal liver, (B) fatty liver, (C) Hepatitis B virus-induced cirrhosis, and (D) Gilbert's syndrome. E–H, Augmented expression of mAb1F9-Ag in patients with PBC. (E) PBC/stage I, (F) PBC/ stage II, (G) PBC/stage III, (H) PBC/stage IV. I, Redistribution of mAb1F9-Ag in PBC/stages II–IV. J, Co-localization of CD13 (red) and mAb1F9-Ag (green) in PBC/stages III–IV. K, Subcellular localization of mAb1F9-Ag in PBC/stages III–IV. (C) cholangiole, (G) Golgi's apparatus, and (N) nucleus. mAb1F9-Ag was positive not only at the vesicular structures (triangle) in a close proximity to canalicular membrane but also in the lysosomes near nucleus and none-organelle structure (arrow). Original magnification of A–I, × 400, of J, × 600.

livers compared to controls (Fig. 6A, C). In the group of patients with PBC, LAMP-2A and LAMP-2B mRNA levels were higher in patients with stages III and IV than in those with stages I or II (p < 0 .01). A significantly positive correlation was also found in PBC patients between histological stage and levels of LAMP-2A mRNA (r = 0.73, p < 0 .001) or levels of LAMP-2B mRNA (r = 0.79, p < 0 .001), suggesting that the regulation of LAMP-2 is impaired in PBC (Fig. 6B, D).

4.

Discussion

In this study, we prepared hybridomas using the isolated CMVs from PBC liver homogenates, and obtained the mAb1F9. It was firstly revealed that the molecule mAb1F9-Ag (LAMP-2) explored the characteristic features in PBC: (i) LAMP-2 was increased in protein and mRNA levels in PBC livers; (ii) augmented expression of LAMP-2 was correlated with the severity of PBC; (iii) UDCA treatment may significantly improve

the recovery of LAMP-2; and (iv) redistribution of LAMP-2 was found in 46% of PBC. LAMP-2, along with LAMP-1, is a heavily glycosylated type 1 membrane protein [25]. These two different classes of LAMPs are encoded by two different but evolutionarily related genes. In humans, the X chromosome carries the gene encoding LAMP-2, which undergoes alternative splicing that results in at least three different splice variants (LAMP-2A, LAMP-2B, and LAMP-2C), all of which have identical lumenal regions [24,26]. Newly synthesized LAMP-2 is transported to the trans-Golgi from the rough endoplasmic reticulum [27]. After leaving the trans-Golgi, it shuttles between lysosomes, endosomes, and the plasma membrane [28]. The LAMP-2 protein is expressed in a variety of tissues [29]. Although LAMP-2 has established roles in vasculitis [30], adhesion [31], and cellular homeostasis, including autophagocytosis [32] and antigen presentation [33], the function of LAMP-2 is still uncertain. The most significant result of our study is the finding of increased LAMP-2 immunoreactivity in PBC livers. This augmentation in LAMP-2 expression in PBC livers may be a result of

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Fig. 4 – Identification of mAb1F9 immunoreactive protein as LAMP-2. A, The protein band with molecular mass ~110 kDa (arrow) was recognized by mAb1F9 in hepG2 cells by immunoprecipitation. (N) protein A/G plus agarose, (C) control agarose resin. B, Mass spectra of mAb1F9-banded antigen digested with trypsin by MALDI-TOF/TOF MS. Only peptide fragments with m/z between 800 and 4000 and signal/noise ratios higher than 50 were chosen. C, An MS/MS spectrum acquired for the sequence of EQTVSVSGAFQINTFDLR (Ion Score 85, C.I.% 100). D, Comparison of mAb1F9-Ag peptide mass fingerprinting with NCBI public database. The matched peptides are highlighted and underlined with a sequence coverage of 16%.

inflammatory changes, increased production of proinflammatory cytokines, or cholestatic damage to cells expressing the molecule. However, no changes in immunoreactivity were observed in association with conditions with obvious inflammatory reactions, such as viral cirrhosis, or cholestatic conditions other than PBC. Moreover, if elevated LAMP-2 expression in PBC was a phenomenon secondary to inflammation, it would be difficult to explain the increased expression of LAMP-2 in areas of the liver parenchyma distant from the inflammatory infiltrate. AMA is considered to be a specific autoimmune marker of PBC, produced many years before clinical disease onset [1,34]. Nevertheless, AMA does not predict the severity of PBC or any clinical subset of the disease [35]. In our study, there was no statistically significant difference between the AMA-positive and AMA-negative cases, but the difference in the histological stage of diagnosis was statistically significant. The latter difference suggests that LAMP-2 may be pathogenic and/or that the increase in LAMP-2 immunoreactivity may be one of the independent prognostic markers able to predict, at the time of diagnosis, a poor outcome in patients with PBC. PBC is considered the result of the immune-mediated destruction of intrahepatic bile ducts, but little is known regarding its etiology. It is intriguing that PBC lesions are restricted to bile ducts because the increased LAMP-2 staining in PBC was observed not only in the bile ducts but also in the hepatocytes. Sidney et al. [36] found that the diffuse cytoplasmic staining of copper in hepatocytes was present in 8 of 17 patients with PBC and the presence of copper generally correlated with

the stage of PBC, suggesting staining for copper may assist in diagnosis of PBC, but the destruction of bile ducts did not seem to affect the distribution of copper. In addition, PBC is frequently associated with SS, a disease characterized as autoimmune epithelitis and primarily affecting exocrine glands and other organs [37], with a prevalence ranging from 69% to 81% [38,39]. In this study, we found that LAMP-2 was positive in various gland epithelial cells including salivary, which might account for the polyglandular exocrine failure in PBC patients along with SS. Although the initial injury was believed to affect interlobular bile ducts in PBC, this disease should be considered as the whole liver impairment, even widespread throughout the body. Specific transporters expressed in the liver and intestine play a critical role in driving the enterohepatic circulation of bile acids [40]. We observed the polarized expression of LAMP-2 in the liver, small intestine, and kidney, indicating a possible role of LAMP-2 in bile acid homeostasis. Fibroblasts deficient in LAMP-2 exhibited an unesterified cholesterol accumulation in the late endosomes and lysosomes [41], indicating that LAMP-2, specifically its lumenal domain, plays a critical role in endosomal/lysosomal cholesterol export [41]. Furthermore, the subapical compartments, in which LAMP-2 is located in our study, participate in the targeting of the most important apical bile transporters, BSEP and MRP2, to the apical membrane [42]. UDCA, the only FDA approved drug to treat PBC, appears to promote the rate of transport of intracellular bile acids across the liver cell and into the canaliculus in PBC [43]. In our study, LAMP-2 increase could be improved after UDCA


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Fig. 5 – Confirmation of mAb1F9-Ag as LAMP-2. A, Immunoprecipitation of cell-derived antigens by mAb1F9 or LAMP-2 antibody. mAb1F9 and LAMP-2 were reacted with each other, but LAMP-1 did not. B, mAb1F9 recognized the heterologously expressed LAMP-2 protein in vitro. WCL, whole cell lysates. C, Co-localization of mAb1F9-Ag (green) and LAMP-2 (red) in cells. Original magnification of C × 1000.

treatment, suggesting that LAMP-2 should be involved in PBC defective hepatobiliary transport, but extensive mechanistic research in this field is still needed.

5.

abnormalities in the expression of LAMP-2 may assist in the differentiation of PBC from other liver diseases. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2013.08.019.

Conclusions Conflict of interest statement

In conclusion, we found a marked increase of LAMP-2 immunoreactivity in PBC, and the increase of LAMP-2 generally correlated with the stage of the disease. It is suggested that

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately

578


Fig. 6 – RT-PCR analysis of LAMP-2A and LAMP-2B mRNA expression in human liver tissue. LAMP-2A and LAMP-2B gene expression in samples from 57 patients with PBC and 30 patients with other liver diseases were determined by qPCR as described under Materials and methods. A and C, Both LAMP-2A and LAMP-2B mRNA levels were significantly upregulated in PBC livers compared to controls; B and D, A significantly positive correlation was also found in PBC patients between histological stage and levels of LAMP-2A mRNA (r = 0.73, p < 0 .001) or levels of LAMP-2B mRNA (r = 0.79, p < 0 .001). Data are means ± SD.

influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Identification of a primary biliary cirrhosis associated protein as lysosome-associated membrane protein-2”.

Acknowledgments We are grateful to Yongzhan Nie and Yingying Liu for their excellent guidance during the experiments. This study was supported by the National Natural Science Foundation of China (No. 81200290, 81070326, 30971339 and 81071873), the Foundation of Shaanxi Province (No. 2011KTCL03-09 and 2011ZDKG-71), and the Doctoral Foundation of The Fourth Military Medical University (2012D12).

REFERENCES [1] Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005;353:1261–73. [2] Talwalkar JA, Lindor KD. Primary biliary cirrhosis. Lancet 2003;362:53–61. [3] Gong Y, Gluud C. Colchicine for primary biliary cirrhosis. Cochrane Database Syst Rev 2004:CD004481.

[4] Hendrickse MT, Rigney E, Giaffer MH, Soomro I, Triger DR, Underwood JC, et al. Low-dose methotrexate is ineffective in primary biliary cirrhosis: long-term results of a placebo-controlled trial. Gastroenterology 1999;117:400–7. [5] Dickson ER, Fleming TR, Wiesner RH, Baldus WP, Fleming CR, Ludwig J, et al. Trial of penicillamine in advanced primary biliary cirrhosis. N Engl J Med 1985;312:1011–5. [6] Beuers U, Spengler U, Zwiebel FM, Pauletzki J, Fischer S, Paumgartner G. Effect of ursodeoxycholic acid on the kinetics of the major hydrophobic bile acids in health and in chronic cholestatic liver disease. Hepatology 1992;15:603–8. [7] Zegers MM, Hoekstra D. Sphingolipid transport to the apical plasma membrane domain in human hepatoma cells is controlled by PKC and PKA activity: a correlation with cell polarity in HepG2 cells. J Cell Biol 1997;138:307–21. [8] Jansen PL, Muller M. Early events in sepsis-associated cholestasis. Gastroenterology 1999;116:486–8. [9] Strautnieks SS, Byrne JA, Pawlikowska L, Cebecauerova D, Rayner A, Dutton L, et al. Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology 2008;134:1203–14. [10] Kartenbeck J, Leuschner U, Mayer R, Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology 1996;23:1061–6. [11] Shoda J, Kano M, Oda K, Kamiya J, Nimura Y, Suzuki H, et al. The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function. Am J Gastroenterol 2001;96:3368–78.


[12] Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, et al. Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology 2001;33:633–46. [13] Zollner G, Fickert P, Silbert D, Fuchsbichler A, Marschall HU, Zatloukal K, et al. Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis. J Hepatol 2003;38:717–27. [14] Takeyama Y, Uehara Y, Inomata S, Morihara D, Nishizawa S, Ueda S, et al. Alternative transporter pathways in patients with untreated early-stage and late-stage primary biliary cirrhosis. Liver Int 2009;29:406–14. [15] Kojima H, Nies AT, Konig J, Hagmann W, Spring H, Uemura M, et al. Changes in the expression and localization of hepatocellular transporters and radixin in primary biliary cirrhosis. J Hepatol 2003;39:693–702. [16] Prieto J, Qian C, Garcia N, Diez J, Medina JF. Abnormal expression of anion exchanger genes in primary biliary cirrhosis. Gastroenterology 1993;105:572–8. [17] Medina JF, Martínez-Ansó E, Vázquez JJ, Prieto J. Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis. Hepatology 1997;25:12–7. [18] Salas JT, Banales JM, Sarvide S, Recalde S, Ferrer A, Uriarte I, et al. Ae2a, b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis. Gastroenterology 2008;134:1482–93. [19] Vazquez JJ, Vazquez M, Idoate MA, Montuenga L, Martinez-Anso E, Castillo JE, et al. Anion exchanger immunoreactivity in human salivary glands in health and Sjogren's syndrome. Am J Pathol 1995;146:1422–32. [20] Mazzone A, Tietz P, Jefferson J, Pagano R, LaRusso NF. Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes. Hepatology 2006;43:287–96. [21] Wang L, Wang J, Zhou X, Li J, Shi Y, Han Z, et al. CM2 antigen, a potential novel molecule participating in glucuronide transport on rat hepatocyte canalicular membrane. Eur J Histochem 2012;56:e26. [22] Yue QX, Cao ZW, Guan SH, Liu XH, Tao L, Wu WY, et al. Proteomics characterization of the cytotoxicity mechanism of ganoderic acid D and computer-automated estimation of the possible drug target network. Mol Cell Proteomics 2008;7:949–61. [23] Chen Z, Zeng M, Song B, Hou C, Hu D, Li X, et al. Dufulin activates HrBP1 to produce antiviral responses in tobacco. PLoS One 2012;7:e37944. [24] Eskelinen E-L, Cuervo AM, Taylor MRG, Nishino I, Blum JS, Dice JF, et al. Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2. Traffic 2005;6:1058–61. [25] Carlsson SR, Roth J, Piller F, Fukuda M. Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan. J Biol Chem 1988;263:18911–9. [26] Hatem CL, Gough NR, Fambrough DM. Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains. J Cell Sci 1995;108(Pt 5):2093–100. [27] Akasaki K, Michihara A, Fujiwara Y, Mibuka K, Tsuji H. Biosynthetic transport of a major lysosome-associated

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

[43]

579

membrane glycoprotein 2, Lamp-2: a significant fraction of newly synthesized Lamp-2 is delivered to lysosomes by way of early endosomes. J Biochem 1996;120:1088–94. Schroder B, Wrocklage C, Pan C, Jager R, Kosters B, Schafer H, et al. Integral and associated lysosomal membrane proteins. Traffic 2007;8:1676–86. Furuta K, Yang X-L, Chen J-S, Hamilton SR, August JT. Differential expression of the lysosome-associated membrane proteins in normal human tissues. Arch Biochem Biophys 1999;365:75–82. Kain R, Exner M, Brandes R, Ziebermayr R, Cunningham D, Alderson CA, et al. Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis. Nat Med 2008;14:1088–96. Sawada R, Lowe JB, Fukuda M. E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels. J Biol Chem 1993;268:12675–81. Tanaka Y, Guhde G, Suter A, Eskelinen E-L, Hartmann D, Lullmann-Rauch R, et al. Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature 2000;406:902–6. Zhou D, Li P, Lin Y, Lott JM, Hislop AD, Canaday DH, et al. Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens. Immunity 2005;22:571–81. Gershwin ME, Ansari AA, Mackay IR, Nakanuma Y, Nishio A, Rowley MJ, et al. Primary biliary cirrhosis: an orchestrated immune response against epithelial cells. Immunol Rev 2000;174:210–25. Miyakawa H, Tanaka A, Kikuchi K, Matsushita M, Kitazawa E, Kawaguchi N, et al. Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens. Hepatology 2001;34:243–8. Goldfischer S, Popper H, Sternlieb I. The significance of variations in the distribution of copper in liver disease. Am J Pathol 1980;99:715–30. Tsianos EV, Hoofnagle JH, Fox PC, Alspaugh M, Jones EA, Schafer DF, et al. Sjogren's syndrome in patients with primary biliary cirrhosis. Hepatology 1990;11:730–4. Hatzis GS, Fragoulis GE, Karatzaferis A, Delladetsima I, Barbatis C, Moutsopoulos HM. Prevalence and longterm course of primary biliary cirrhosis in primary Sjögren's syndrome. J Rheumatol 2008;35:2012–6. Selmi C, Meroni PL, Gershwin ME. Primary biliary cirrhosis and Sjogren's syndrome: autoimmune epithelitis. J Autoimmun 2012;39:34–42. Dawson PA, Lan T, Rao A. Bile acid transporters. J Lipid Res 2009;50:2340–57. Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M, Blanz J, et al. Role for LAMP-2 in endosomal cholesterol transport. J Cell Mol Med 2011;15:280–95. Wakabayashi Y, Dutt P, Lippincott-Schwartz J, Arias IM. Rab11a and myosin Vb are required for bile canalicular formation in WIF-B9 cells. Proc Natl Acad Sci U S A 2005;102:15087–92. Jazrawi RP, de Caestecker JS, Goggin PM, Britten AJ, Joseph AE, Maxwell JD, et al. Kinetics of hepatic bile acid handling in cholestatic liver disease: effect of ursodeoxycholic acid. Gastroenterology 1994;106:134–42.