Proteomic Analysis of Lysosomal Membrane Proteins in Bovine

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Proteomic Analysis of Lysosomal Membrane Proteins in Bovine Mammary Epithelial Cells Illuminates Potential Novel Lysosome Functions in Lactation Chaochao Luo,†,‡,§,# Shengguo Zhao,†,‡,§,# Wenting Dai,†,‡,§ Nan Zheng,*,†,‡,§ and Jiaqi Wang*,†,‡,§ State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, ‡Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture, Institute of Animal Science, and §Key Laboratory of Quality & Safety Control for Milk and Dairy Products of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China

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S Supporting Information *

ABSTRACT: Lactation of bovine mammary epithelial cells (BMEC) is a complex biological process that involves in various organelles. Studies have shown that lysosome and lysosomal membrane proteins (LMP) plays an important role in lactation of BMEC. But the LMP of BMEC remains poorly understood. To obtain a global view of the LMP of BMEC and the affect of lysosome on lactation, the LMP of BMEC was identified using sequential windowed acquisition of all theoretical mass spectra (LC-SWATH/MS). 1214 LMP were identified and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. Gene ontology annotation of these identified proteins showed that both previously reported casein synthesis-related LMP, such as LAMTOR1, 2, 3, and rRagC, and newly identified casein and milk fat synthesis-related LMP, such as EIF4E and ACAA1, were found. KEGG pathway analysis of these identified proteins showed that some pathways involved in lactation, such as PI3K-Akt, mTOR, insulin, PPAR, and JAK-STAT pathway, were found. The lysosomal location of five proteins (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by laser confocal microscopy, and all five were associated with the lysosomal membrane. These findings help to elucidate lysosome functions in the regulation of lactation. The results implicate lysosomes as important organelles in regulation of lactation of BMEC that have been previously undervalued. KEYWORDS: bovine mammary epithelial cell, lysosomal membrane proteinomics, LC-SWATH/MS, lysosome novel function

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INTRODUCTION Lysosomes are capsule bubble cellular organelles surrounded by a single membrane.1 The lysosome lumen contains at least 60 different hydrolase enzymes including proteases, nucleases, phosphatases, and lipases.2 Their main functions are the degradation and recycling of endogenous components such as aging organelles and misfolded proteins, and exogenous constituents such as food vacuoles and pathogenic bacteria following phagocytosis, through endocytosis, autophagy, and other cellular trafficking pathways.3,4 However, in addition to these classic functions, lysosomes are now known to participate in the regulation of signaling pathways and cell physiology, and these new functions are fulfilled by lysosomal membrane proteins (LMPs).5−7 The identification of LMP is therefore key for study of the new functions of lysosomes, but LMPs remain poorly understood because of limitations in detection technologies. Liquid chromatography tandem mass spectrometry (LCMS/MS)-based proteomics is the method of choice for largescale identification of proteins in a sample. Discovery proteomics is usually performed using data-dependent acquisition (DDA).8 As DDA is a semistochastic process, the set of peptides identified across samples is not reproducible.9 A new method, sequential windowed acquisition of all theoretical mass spectra (SWATH), was recently described, which is performed in data-independent acquisition (DIA) mode. In this approach, sequencing cycles through fixed precursor © XXXX American Chemical Society

isolation windows (for example, 25 m/z) using a quadrupole-time-of-flight mass spectrometer, achieving essentially complete peptide fragment ion coverage for precursors in the tryptic peptide mass range.10,11 The essential feature of SWATH-MS is the use of prior knowledge regarding fragmentation and chromatographic behavior of target peptides. This information is used for scoring signal groups extracted from SWATH-MS data sets to identify and quantify peptides automatically and at large scale.12,13 In the present study, LMPs in bovine mammary epithelial cells (BMECs) were identified using LC-SWATH/MS. We designed this study to provide a basic understanding of LMPs, and to look for new proteins on the lysosomal membrane to provide insight into novel functions of lysosomes, including the regulation of lactation in BMECs.

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MATERIALS AND METHODS

All experiments involving dairy cattle were conducted according to the principles of the Chinese Academy of Agricultural Sciences Animal Care and Use Committee (Beijing), who approved all experimental protocols. Cell Culture and Experimental Scheme. Primary BMECs were obtained and cultured as previously reported.14 In brief, mammary Received: August 23, 2018 Revised: November 11, 2018 Accepted: November 13, 2018

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DOI: 10.1021/acs.jafc.8b04508 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

transferred to a new centrifuge tube. A sample (100−200 μL) of the supernatant was kept for subsequent assays. The sample was centrifuged at 20 000g for 20 min. The supernatant liquid was removed and the pellet collected in a minimal volume of extraction buffer (∼0.4 mL per 108 cells). The pellet well was suspended in a single microcentrifuge tube by using a pellet pestle. This material was a crude lysosomal fraction (CLF) containing a mixture of mitochondria, lysosomes, peroxisomes, and endoplasmic reticulum. To further enrich the lysosomes in the CLF, diluting CLF with a solution containing 19% Optiprep density gradient medium solution with a protein concentration of 0.5−1.0 mg of protein/mL for cell culture extracts. This solution was defined as the diluted OptiPrep fraction (DOF). The DOF was centrifuged by density gradient centrifugation (150 000g for 4 h) on a multistep OptiPrep gradient. The tube will show multiple bands floating in the gradient and the heavy and light lysosomes solution were obtained. Further purification of the obtained fractions by addition of calcium chloride to a final concentration of 8 mM and low-speed (5000g for 15 min) centrifugation. This step will precipitate the rough endoplasmic reticulum and any mitochondria that are in the fraction, and the purified LMP, were obtained. The protein concentration of purified LMP was analyzed using the Bradford Regent kit (P0006, Beyotime, Beijing), and protein purity was probed by Western blotting (WB) using LAMP-2 (lysosome marker), VDAC1 (mitochondria marker), GM130 (Golgi marker), calnexin (endoplasmic reticulum marker), and anti-β-tubulin (cytoplasm protein marker). The enrichment fold of LAMP-2 was calculated. The total volume of cell lysate was 5 mL, and 100 μL of cell lysate was split and used in WB analysis with 10-fold dilution. After the LMPs were isolated, the final volume of LMP solution was 0.25 mL, and 25 μL of LMP solution was split and used in WB analysis with 100-fold dilution. In this study, three independent LMP samples were extracted and mixed prior to detection by mass spectrometry. Western Blotting. The WB was performed as previously reported.16 For each WB analysis, 20 μL of each sample [the total protein of whole cell lysate (WCL) and LMP solution was 112.12 and 1.232 μg, respectively] was used, and the gray value of LAMP-2 protein band was analyzed with ImageJ2X software. The primary antibodies used in this study were as follows: anti-LAMP-2 antibody (1:200, sc-8100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VDAC1 antibody (1:200, sc-32063, Santa Cruz Biotechnology), anti-GM130 antibody (1:200, sc-30100, Santa Cruz Biotechnology), anticalnexin antibody (1:200, sc-11397, Santa Cruz Biotechnology), anti-β-tubulin antibody (1:1000, 2146, Cell Signaling Technology, Danvers, MA, USA), and anti-CSN2 antibody (1:500, 251309, Abbiotec, San Diego, USA). Identification of LMP. Proteins were identified using the SWATH model.13 The main process of identification was as follows: The protein sample (100 μg) was adjusted to 100 μL, diluted in 500 μL of 50 mM NH4HCO3, and then digested by 2 μg of tryspin at 37 °C for 12 h. The digested sample was acidified with an equal volume of 0.1% formic acid and then added to Strata-X C18 column; the digested sample was collected, added to Strata-X C18 column again, and repeated three times (the Strata-X C18 column was first activated with 1 mL of methanol and then balanced with 1 mL of 0.1% formic acid). The Strata-X C18 column was washed two times with 1 mL solution (containing 0.1% formic acid and 5% acetonitrile). The column was washed with 1 mL of solution (containing 0.1% formic acid and 80% acetonitrile) and then 1 mL of filter liquid was collected and drained with a vacuum concentrator. Subsequently, dry sample was dissolved with 20 μL of 0.5 M triethylamine carbonate buffer (TEAB, 0.1 M trolamine, pH 7−8). The operation of LC-MS/MS DDA Model mass spectrometry was as previously reported.17 The instruments used for MS/MS were as follows: mass spectrometer was AB SCIEX nano LC-MS/MS (Triple TOF 5600 plus, Concord, Canada); the analysis column was AB SCIEX column (75 m diameter, filling 3 m, 120 Å, ChromXP C18 column material, 10 cm); the nanospray needle was NEW objective (20 μm diameter, the diameter of the injection needle was 10 μm); and the trap column was Eksigent

gland tissue was dissected from healthy midlactation Holstein dairy cow, disinfected with 75% alcohol, soaked in D-hank’s buffer supplemented with 1000 U/mL penicillin, 1000 U/mL streptomycin (10378016, Gibco, Camarillo, CA, USA), and 12.5 μg/mL amphotericin B1 (R01510, Gibco), and cut into 1 mm3 pieces with sterile surgical scissors. The pieces were washed with D-Hank’s buffer several times and plated onto rat tail collagen (200110-10, Shengyou, Hangzhou, China) in the bottom of 10 cm cell culture dishes (351092, Corning, Tewksbury, MA, USA). The appropriate Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (11330057, Gibco, Grand Island, NY, USA) was added to each dish and supplemented with 10% fetal bovine serum (FBS) (10099141, Gibco), 100 U of penicillin, 100 U of streptomycin, and 2.5 μg/mL amphotericin B1, and dishes were cultured at 37 °C with 5% CO2. When the confluence of cells reached 80%, samples were removed and fibroblast cells and BMECs were segregated with different concentrations of trypsin. Fibroblast cells were first digested and removed with 0.25% trypsin, and BMECs were digested with 0.15% trypsin-supplemented 0.02% ethylenediaminetetraacetic acid (EDTA) (C0201, Beyotime, Beijing). After ∼3−4 h of digestion, purified BMECs were obtained. The purity and function of casein synthesis of BMECs were determined with cytokeratin 18 (CK18, epithelial cells marker) and β-casein (CSN2, a representative protein of casein) and the operation process of determining the purity and function of casein synthesis of BMECs were performed as previously reported.15 A schematic diagram of the experimental process is shown in Figure 1.

Figure 1. Schematic diagram of the experimental process. Isolation and Purity Detection of LMP. Purified BMEC (8−10 generations) were plated into 10 cm cell culture dishes at a density of 1.0 × 105 cells/mL medium and cultured in DMEM/F12 medium supplemented with 10% FBS, 100 U of penicillin, and 100 U of streptomycin. At 90% confluence (about 24 h after cells were plated), cells were harvested and the LMP was isolated with a lysosome isolation kit (LYSISO1, Sigma, Los Angeles, USA) according to the manufacturer’s instructions. In brief, BMECs were planted into 100 mm cell culture dish (430293, Corning) and cultured with DMEM/ F12 medium supplemented with 10% FBS. When the confluence of cells was about 90%, the media were discarded and cells were washed with ice cold phosphate buffer saline (PBS). The cells were trypsinized, then the cells were centrifuged for 5 min at 600g, and the the supernatant was discarded. The cells were resuspended in icecold PBS, counted, and centrifuged for 5 min at 600g at 4 °C, and the supernatant was discarded. The wash step was repeated once again (without the cell count). The supernatant was discarded. The packed cell volume (PCV) should be 1.5−3.0 mL. Extraction buffer (2.7 PCV) was added and the solution vortexed to achieve an even suspension. The cells were broken in a 7 mL Dounce homogenizer using Pestle B (small clearance). The cells were checked under a microscope using Trypan Blue solution staining to ascertain the degree of breakage. Normally 80−85% of breakage was the best. The sample was centrifuged at 1000g for 10 min. The supernatant was B


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Figure 2. Purity identification of BMEC and LMP. (A) The expression of CK18 in BMEC was tested by immunofluorescence. The CK18 was visualized with Alexa Fluor 488-conjugated secondary antibodies (green) and the nucleus was visualized with DAPI (blue). (B) The expression of CK18 and CSN2 were tested by WB. The gray value of LAMP-2 in WCL was defined as “1” and the result was reported as mean ± SD. (C) The marker proteins in WCL, LMP solution, and cytoplasm were tested by WB. LAMP2, lysosome marker; VDAC1, mitochondria marker; GM130, Golgi marker; β-tubulin, cytoplasm protein marker; WCL, whole cell lysis; Cyt, cytoplasm.

Figure 3. Sequential windowed acquisition of all theoretical mass spectra (SWATH) analysis of LMP. (A) Distribution of unique peptides in identified proteins. (B) Distribution of the length of peptides in the identified proteins. (C) Protein coverage of peptides in the identified proteins. (D) Venn diagram of proteins identified in the present study and designations as LMP reported in previous work. Chromxp Trap Column (3 μm C18-CL, 120 Å, 350 μm × 0.5 mm). The sample dates were identified using the SWATH model and then analyzed using the Protein Pilot. For the database identified by Protein Pilot software v. 4.2 (AB Sciex), the confidence level of map and proteins is more than 95%, and at least one unique peptide included in each protein was considered as trusted protein. The protein pilot search parameters are as follows: type of search, MS/MS ion search; Cys alkylation, iodoacetic acid; instrument, AB-Sciex5600; ID focus, biological modifications; search effort, thorough ID; protein mass, unrestricted; database, UniProt Bovinebos (31 817 protein ̅ sequences).

Bioinformatics Analysis of Identified LMP. The biological process and molecular function of identified proteins were analyzed by gene ontology (GO) annotation with DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/) as previously reported.18,19 Pathway analysis of identified proteins was subsequently performed with KEGG pathway database (http://www.genome.jp/kegg/pathway.html). Verification of Lysosomal Localization of Interested Proteins. The lysosomal localization of proteins of interest was verified by immunofluorescence (IF). Cells were plated onto sterile coverslips in six-well plates at a density of 1.0 × 104 cells/mL. When C


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Figure 4. Gene ontology (GO) annotation of the 1214 identified proteins. Biological processes (A), molecular functions (B), and cellular component (C) identified by GO annotation. the confluence reached 50%, the medium was removed and cells were washed several times with PBS and then fixed with ice-cold methanol for 10 min at 4 °C. After the cells were washed three times with Trisbuffered saline with Tween-20 (TBST), cells were blocked with blocking buffer (P0102, Beyotime, Beijing) for 1 h at 37 °C and incubated with lysosomal marker protein antibody (LAMP-2) and target protein antibody (Protein kinase C alpha type, PRKCA; Eukaryotic translation initiation factor 4E, EIF4E; Acetyl-Coenzyme A acyltransferase 1, ACAA1; GTPase HRas, HRAS; and Thrombospondin-1, THBS1) overnight at 4 °C. Cells were then washed three

more times with TBST and incubated with Alexa Fluor 488- or 647conjugated secondary antibody at 37 °C for 1 h in the dark. After the cells were washed three more times with TBST, the cells were incubated with 10 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, C1002, Beyotime) at 37 °C for 15 min in the dark and then washed three times with TBST. Cells were fixed on slides with antifade mounting medium (P0126, Beyotime) and observed by laser scanning confocal microscopy (LSM780, ZEISS, Germany). Colocalization was analyzed with ImageJ software20 using at least 10 cells for each colocalization experiment. Antibodies used in this experiment were as D


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Figure 5. KEGG pathway analysis of the 1214 identified proteins.

In 2013, a list of LMPs was reported by Chapel et al.21 and is reproduced here in Table S3. Of the 1201 proteins identified (not including the 13 uncharacterized proteins) in the current work, 642 overlapped with Chapel et al.’s report, while 559 proteins did not overlap (Figure 3D). Of these 559 nonoverlapping proteins, 355 were reviewed and 204 have only a gene name and are not included in the UniProt database (Table S4). GO Annotation. The 1214 proteins were analyzed with GO annotation, and these proteins were found to participate in various biological processes involved in generation of precursor metabolites and energy (71 proteins), protein transport (67 proteins), translation (62 proteins), intracellular signaling cascade (55 proteins), transmembrane transport (45 proteins), small GTPase mediated signal transduction (38 proteins), nitrogen compound biosynthetic process (32 proteins), purine nucleotide metabolic process (29 proteins), ATP metabolic process (26 proteins), endocytosis (17 proteins), secretion (16 proteins), fatty acid metabolic process (15 proteins), cell proliferation (14 proteins), glucose catabolic process (13 proteins), and post-transcriptional regulation of gene expression (12 proteins; Figure 4A; Table S5). The molecular functions of the 1214 proteins were analyzed and included nucleotide binding (215 proteins), purine ribonucleotide binding (159 proteins), ATP binding (97 proteins), structural molecule activity (68 proteins), GTP binding (65 proteins), ATPase activity (35 proteins), cofactor binding (33 proteins), enzyme binding (26 proteins), lipid binding (24 proteins), GTPase activity (16 proteins), aminoacyl-tRNA ligase activity (7 proteins), translation elongation factor activity (6 proteins), SH3 domain binding (5 proteins), and phosphatase binding (5 proteins). (Figure 4B; Table S6). The cellular component of the 1214 proteins were analyzed and included extracellular exosome (523 proteins), plasma membrane (187 proteins), mitochondrion (148 proteins), nucleoplasm (123 proteins), cytosol (115 proteins), endoplasmic reticulum (75 proteins), Golgi apparatus (64 proteins), lysosomal membrane (41 proteins), intracellular ribonucleoprotein complex (23 proteins), early endosome (20 proteins), lysosome (20 proteins), endosome (19 proteins), late endosome (13 proteins), early endosome membrane (9 proteins), and lysosomal lumen (3 proteins) (Figure 4C; Table S7).

follows: anti-LAMP-2 antibody (1:100, sc-8100, Santa Cruz Biotechnology); anti-PRKCA antibody (1:100, sc-208, Santa Cruz Biotechnology), anti-THBS1 antibody (1:100, sc-14013, Santa Cruz Biotechnology), anti-HRAS antibody (1:100, 18295-1-AP, Proteintech, Chicago, USA), anti-ACAA1 antibody (1:100, 12319-2-AP, Proteintech), anti-EIF4E antibody (1:100, sc-13963, Santa Cruz Biotechnology), and anti-CK18 antibody (1:100, sc-28264, Santa Cruz Biotechnology). All of these antibodies were diluted with primary antibody dilution buffer (P0103, Beyotime). Alexa Fluor 488 or 647-conjugated secondary antibodies (bs-0294M-AF488 or bs0295M-AF647, Bioss, Beijing) were diluted with secondary antibody dilution buffer (P0108, Beyotime).

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RESULTS Identification of BMEC and LMP Purity. The purified BMEC were determined with CK18 and CSN2, and the results are shown in Figure 2A,B. The CK18 (Figure 2A) and CSN2 (Figure 2B) were detected only in purified BMEC, but not in fibroblast; it suggests that the purified BMECs were obtained and they have the function of casein synthesis. The purity of LMP was tested by WB, and the results are shown in Figure 2C. In whole cell lysate (WCL), lysosome (LAMP-2), mitochondria (VDAC1), Golgi (GM130), and cytoplasm (βtubulin) markers were detected, but in the LMP fraction, only the lysosome marker (LAMP-2) was detected. Meanwhile, VDAC1, GM130, and β-tubulin were detected in the cytoplasm (Cyt). This suggests that the LMP fraction was not contaminated with other subcellular organelle components, and confirmed that it could be used for SWATH analysis. Protein concentration analysis gave the values of total cell lysate and LMP solutions were 56.06 and 6.16 μg/μL, respectively. SWATH Analysis of LMPs. LMPs were analyzed using the SWATH method, which identified 1286 proteins (Table S1, Supporting Information), of which 1214 (94.4%) included at least one unique peptide (Figure 3A; Table S2). Of the 1214 proteins, the average peptide length was 17.66 amino acids residues, and the amino acids residues numbers of most peptides were from 7 to 35 (Figure 3B). The average protein coverage (percentage of the protein sequence is covered by the identified peptides) was 23.3%, and the protein coverage of 567 proteins (46.7%) was equal to or greater than 20.0% (Figure 3C). E


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Figure 6. Colocalization of five proteins of interest with LAMP-2. Cell nuclei are dyed with DAPI (blue), LAMP-2 was visualized with Alexa Fluor 488-conjugated secondary antibodies (green), and proteins of interest (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) were visualized with Alexa Fluor 594-conjugated secondary antibodies (red). Colocalization of proteins with LAMP-2 is indicated by a white color.

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KEGG Pathway Analysis. The 1214 proteins were subjected to KEGG pathway analysis and found to be involved in PI3K-Akt signaling pathway (35 proteins), MAPK signaling pathway (24 proteins), GnRH signaling pathway (14 proteins), estrogen signaling pathway (13 proteins), mTOR signaling pathway (11 proteins), fatty acid metabolism (10 proteins), AMPK signaling pathway (9 proteins), cell cycle (8 proteins), PPAR signaling pathway (8 proteins), insulin signaling pathway (7 proteins), TGF-beta signaling pathway (5 proteins), fatty acid elongation (4 proteins), DNA replication (4 proteins), prolactin signaling pathway (4 proteins), and JakSTAT signaling pathway (4 proteins) (Figure 5; Table S8). Verification of Lysosomal Localization. To verify the results of mass spectrometry, the lysosomal localization of five proteins of interest (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by IF with a laser confocal microscope. The result showed that all five overlapped extensively with the lysosomal marker LAMP-2 (Figure 6), indicating their presence on the lysosomal membrane.

DISCUSSION Proteomics analysis is mainly performed using LC-MS technology, and the type of MS and mode used for data collection are crucial for analysis. For a long time, MS data collection mainly used the DDA model, even though there are disadvantages including poor sensitivity, resolution, and scanning speed, which make it unsuitable for proteomics.22 In recent years, the new SWATH-MS analysis model has been developed and applied in proteomics analysis. In the SWATH model, MS is performed in DIA mass spectrum acquisition mode, and information on all ions is obtained by ultra-highspeed scanning, meaning complete quantitative and qualitative data can be obtained in a single experiment, and the sensitivity, resolution, and scanning speed of the SWATH method are superior to those of DDA.23,24 In the present study, lysosomal fractions of BMEC were extracted with a lysosome isolation kit, and LMP were identified by SWATH. We identified 1214 proteins, of which 13 were uncharacterized with no orthologues of known function. Several proteomic analyses of the lysosome membrane have been reported in the past 10 years. In previous research studies,6,21,25−27 density gradient centrifugation is the most F


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pathways involved in cell proliferation, and casein and milk fat synthesis, such as PI3K-Akt pathway, AMPK pathway, estrogen pathway, insulin pathway, Jak2-STAT5 pathway, and PPAR pathway, were found. The lysosomal membrane location of five proteins of interest, PRKCA, EIF4E, ACAA1, HRAS, and THBS1, was confirmed by IF using laser confocal microscopy. These five proteins are involved in a variety of important biological functions, and in BMEC they participate directly or indirectly in cell proliferation and lactation. PRKCA, EIF4E, THBS1, and HRAS are involved directly or indirectly in the mTOR signaling pathway, one of the main signaling pathways involved in cell proliferation, growth, and differentiation in mammals.35−37 In BMEC, the mTOR signaling pathway regulates cell proliferation and milk protein synthesis.38 ACAA1 is involved in the PPAR signaling pathway, one of the main pathways regulating fat synthesis and differentiation of adipose cells.39 This result suggests that, in BMECs, lysosome is an important organelle for lactation; it is involved not only in cell proliferation and milk protein synthesis but also in milk fat synthesis and differentiation of adipose cells. In summary, in the present work, we identified many new LMPs in BMECs, and further investigation of these proteins will assist in elucidating the novel functions of the lysosome, including cell proliferation, milk protein synthesis, milk fat synthesis, and differentiation of adipose cells. Our findings suggest that lysosomes are important organelles with functions in regulation of lactation of BMECs that have been previously undervalued.

common method that is used to isolate lysosome, and the enrichment fold of some lysosomal marker proteins, such as LAMP-1, LAMP-2, and β-hexosaminidase, is used to evaluate the result of isolation of lysosome, and the enrichment fold is usually between 60 and 80. But in another research study, Han et al.20 reported that they isolated lysosome with a lysosome isolation kit made by Sigma, and then they analyzed the purity of the extract by detecting the marker protein of some organelles (such as calnexin for endoplasmic reticulum, GM130 for Golgi, and LAMP2 for lysosome). In the present study, we isolated the lysosome with a lysosome isolation kit and analyzed the purity of the extract with the marker protein of endoplasmic reticulum, Golgi, lysosome, and cytoplasm; the result was consistent with Han’s research. In addition, the enrichment fold of LAMP-2 in the extract was calculated in the present study, the enrichment fold of LAMP-2 was 67.34, and it was within the scope of previous research studies. In a pioneering study performed by Bagshaw et al.,25 215 LMP from Triton WR1339 density-shifted lysosomes were identified in rat liver. Zhang et al.26 subsequently reported lysosomal trafficking regulators in the lysosomal membrane of mouse liver, and Schroder et al.6 identified 86 lysosomal candidate proteins in human placental lysosomes. Later, Della Valle et al.27 analyzed LMP in native and density-shifted lysosomes by comparative proteomic analysis in rat liver. In a recent study by Chapel et al.,21 proteomic identification LMP in rat liver identified an impressive 2385 proteins, the most extensive list published to date for lysosomes, phagosomes, or lysosome-related organelles. In the present study, we identified 1214 proteins, including 13 uncharacterized proteins with no orthologues of known function. Of the 1201 characterized proteins, 642 overlapped with those in Chapel et al.,21 while 559 were nonoverlapping. The experimental materials in previous studies were rat or mouse liver cells, or human cells, while BMECs were used in the present work, which are a specific cell type that perform lactation; hence, differences in LMP may be related to differences in species and cell type. Lysosomes are cytoplasmic organelles that degrade and recycle macromolecules and cellular components by endocytosis, autophagy, and other cellular trafficking pathways. In the last 10 years, with the development of proteomics, an increasing number of new proteins have been associated with the lysosomal membrane, and new functions of lysosomes have been discovered.28,29 Recent research showed that the cytosolic surface of the lysosome membrane is a major site of action for mTOR signaling complexes.30,31 Lysosomes are also involved in many diseases, such as Sanfilippo disease, Salla disease, and neuronal ceroid lipofuscinosis.32,33 These results suggest that, in addition to their classic degradatory and recycling functions, lysosomes may have critical roles in many other cellular processes and signaling pathways. In the present study, 1214 proteins were identified as LMP and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. The cell proliferation and lactation of BMEC is a complex biological process that involves various cellular signaling pathways. Previous studies have shown that mTOR pathway, insulin pathway, Jak2-STAT5 pathway, and PPAR pathway are the main signaling pathways that regulate the cell proliferation and lactation in BMEC, and in these pathways, the activation of mTOR pathway is regulated by LMP.34 In the present study, the result of KEGG pathway analysis of the 1214 identified proteins showed that besides mTOR pathway, various other

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04508.

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List of all identified proteins (1286 list) (XLS) List of all identified proteins (unique peptide ≥1) (1214 list) (XLS) List of all identified proteins in Chapel’s report (XLS) List of 559 no overlapping proteins (XLS) Biological processes analysis of 1214 identified proteins (XLS) Molecular function of 1214 identified proteins (XLS) Cellular component of 1214 identified proteins (XLS) Pathway analysis of 1214 identified proteins (XLS)

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-10-62816069. Fax: +86-10-62897587. E-mail: [email protected]. Jiaqi Wang: Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, China. *Tel.: +86-10-62816069. Fax: +86-10-62897587. E-mail: [email protected]. Nan Zheng: Institute of Animal 470 Science, Chinese Academy of Agricultural Sciences, No. 2 471 Yuanmingyuan West Road, Haidian District, Beijing, 100193, 472 China. ORCID

Nan Zheng: 0000-0002-5365-9680 Jiaqi Wang: 0000-0001-8841-0124 G


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Chaochao Luo and Shengguo Zhao contributed equally to this paper. Funding

This study was financially supported by the National Dairy Industry and Technology System (CARS-36), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS12). Notes

The authors declare no competing financial interest.

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Proteomic Analysis of Lysosomal Membrane Proteins in Bovine

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