Proteomic Analysis of Isolated Plasma Membrane Fractions from the

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Proteomic Analysis of Isolated Plasma Membrane Fractions from the Mammary Gland in Lactating Cows Qiongxian Yan, Shaoxun Tang, Zhiliang Tan,* Xuefeng Han, Chuanshe Zhou, Jinhe Kang, and Min Wang Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Research Center of Livestock & Poultry Sciences, South-Central Experimental Station of Animal Nutrition and Feed Science in Ministry of Agriculture, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan 410125, People’s Republic of China S Supporting Information *

ABSTRACT: The mammary gland of dairy cows is a formidable lipid-synthesizing machine for lactation. This unique function depends on the activities of plasma membrane (PM) proteins in mammary cells. Little information is known about the expression profiles of PM proteins and their functions during the lactating process. This study investigated the proteome map of PM fractions of mammary gland in lactating cows using 1D-Gel-LC-MS/MS and identified 872 nonredundant proteins with 141 unknown proteins, wherein 215 were PM-associated proteins. Most of the PM-associated proteins were binding, transport, and catalytic proteins such as annexin proteins, heat shock proteins, integrins, RAS oncogene family members, and S100 calcium binding proteins. The PM-associated pathways such as caveolae-mediated endocytosis, leukocyte extravasation, aldosterone signaling in epithelial cells, and remodeling of epithelial adherens junctions were also significantly over-represented. Proteomic analysis revealed the characteristics and predicted functions of PM proteins isolated from the lactating bovine mammary gland. These results further provide experimental evidence for the presence of many proteins predicted in the annotated bovine genome. The data generated here also provide a reference for the PM-related functional research in the mammary gland of lactating cows. KEYWORDS: bovine, mammary gland, Nano-LC-ESI-MS/MS, plasma membrane, proteomics

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INTRODUCTION The mammary gland has a unique metabolism function that allows it to synthesize fatty acids, proteins, and other energy substrates and secrete them into milk.1 In bovines, the lactating mammary gland is a formidable triacylglycerol-synthesizing machine.2 It also represents an ideal model for studying synthesis or secretion regulation of milk components. It is made up of a branching network of ducts that end in alveoli. The alveolar epithelial cells are where blood nutrients are transformed into milk components, which is followed by secretion into the alveolar lumen. The luminal epithelial cells and contractile myoepithelial cells serve as channels for milk transport during lactation. Specifically, these processes take place at the plasma membrane (PM) of mammary cells. The specific lactation function of the bovine mammary gland is related to the PM proteins of mammary cells. It has been demonstrated that some of the PM proteins are involved in the binding, transport, and intercellular metabolism of milk precursors originating from the circulating plasma pool and constitute >75% of current drug targets.3 Recent research on the bovine mammary gland2 has identified that CD36 (fatty acid translocase) and FABPpm (PM-associated fatty acid binding protein) facilitate the movement of fatty acids across the PM and ATP-binding cassette and that subfamily G (ABCG5 and ABCG8) may play a potential role in lipid trafficking and excretion during lactation, as well as control of sterol concentrations in milk. Application of immunofluoresence confocal laser microscopy has shown that some monocarboxylate transporters (MCT1, MCT2, MCT4, and MCT8) are also localized on the PM of mammary alveolar © 2015 American Chemical Society

epithelia cells in cows, and this existence implies their possible involvement in the transport of essential elements required for milk synthesis and secretion.4 Despite the importance of PM proteins, they are largely uncharacterized in the mammary gland of lactating cows. Thus, sufficient knowledge about the profile of PM proteins is essential to understand their functional roles during the lactation processes. Profiling the PM proteome is challenging due to the strong hydrophobicity and low abundance of PM proteins. Fortunately, tandem mass spectrometry is currently the most powerful tool for PM proteome analysis on the condition that the PM fraction has been efficiently purified. Differential centrifugation combined with density gradient ultracentrifugation and two-phase aqueous partition5 are traditional methods to enrich the PM. Immunoisolation, biotin−avidin, and agglutinin-affinity purification have also been used to enrich the PM from rat liver,6 cancer cells,7 and prostate cells,8 respectively. Recently, cationic colloidal silica nanoparticles were labeled to enrich the PM fraction of kidney vascular endothelial cells.9 The PM isolation from mammary gland is very complicated because of the distensibility of the tissue arising from the large quantities of connective tissues. Highly purified PM from the mammary gland has been obtained by a density perturbation procedure in lactating rats.10 Keenan et al.11 reported the first successful isolation of the PM fraction Received: Revised: Accepted: Published: 7388

May 5, 2015 July 28, 2015 August 3, 2015 August 3, 2015 DOI: 10.1021/acs.jafc.5b02231 J. Agric. Food Chem. 2015, 63, 7388−7398

Article

Journal of Agricultural and Food Chemistry

each step. The PM fractions were stored in a final volume of 2 mL at −80 °C. Electron Microscopy. Fresh PM (0.1 mL) and 3% glutaraldehyde in PBS were mixed, incubated overnight at 4 °C, and centrifuged at 14500g for 15 min. The resulting pellets were fixed in 2% OsO4, dehydrated through a graded series of ethanol (50, 70, 90, and 100% in turn) and acetone (100%), embedded in Epon overnight at 37 °C, and then solidified at 60 °C for 48 h. After cooling, the samples were sliced into thin sections with thickness of 70 nm and observed under a transmission electron microscope (Hitachi JSM-1230; JEOL, Tokyo, Japan) at 100 kV. Western Blotting. Proteins of the homogenates and PM fractions were solubilized in a lysis buffer (LB1: 5 M urea, 2 M thiourea, 10% glycerol, 50 mM Tris-HCl, 2.0% OG, 2.5% SB3-10, 1 mM PMSF, pH 8.0), incubated at 37 °C for 30 min and room temperature for 1 h, respectively. Then the suspension was centrifuged at 20000g for 1 h, and the concentration of protein in the supernatant was determined by RC DC protein assay kit (catalog 500-0120, Bio-Rad, Hercules, CA, USA). The extracted proteins were separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (Bio-Rad, USA). The membranes were blocked overnight at 4 °C and then incubated with anti-Na+/K+-ATPase α1 subunit (Novus Biologicals, LLC, Littleton, CO, USA), anti-flotillin-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and anti-prohibitin and anti-actin (Abcam, Hong Kong, China) for 2.5−3 h at room temperature. After being washed three times with PBST (0.1% Tween 20 in PBS), the membranes were incubated with peroxidase-conjugated goat antimouse, anti-rabbit, and donkey anti-goat IgG (H + L) for 1.5 h, washed three times with PBST, and detected by WesternBright ECL Western Blotting HRP Substrate (APGBio, Shanghai, China). Densitometric analysis of immunoblots was performed using an AlphaImager 2200 digital imaging system (Digital Imaging System, Kirchheim, Germany). Experiments were performed in triplicate and replicate three times. Enzyme Activity Determination. The determination of organelle marker enzyme activity of the purified PM fractions was performed according to the standard protocols of the following kits: the 5′nucleotidase assay kit (A041, Nanjing, China), the bovine Na+/K+ATPase ELISA kit (catalog no. CSB-eq 027407BO, Wuhan, China), the bovine monoamine oxidase, succinodehydrogenase, aryl sulfatase, β1,4-GTase ELISA kits (JRDUN Biotechnology Co. Ltd., Shanghai, China), and the bovine NADPH cytochrome c reductase assay kit (BGI, Guangdong, China). Preparation of PM-Associated Proteins. To compare the solubilization efficiency of PM proteins, two lysis buffers (one is LB1 as previously described; the other is LB2, containing 4% SDS, 10 mM Tris-HCl, 20% glycerol, and 100 mM dithiothreitol, pH 6.8) were used to dissolve the pooled PM fractions isolated from 12 homogenates. The isolation procedure of proteins and determination of protein concentration were the same as that of LB1. The isolated proteins were used for further in-solution digestion and SDS-PAGE separation. The PM proteins were separated by 6% SDS-PAGE and then stained by Coomassie brilliant blue R250 solution (Zomanbio, Beijing, China) overnight. Each protein gel lane was divided into four to five equal parts and removed by a sterilized knife. In-Gel Digestion of Samples. Protein slices in fresh Coomassie brilliant blue R250-stained gel were excised and destained twice with 200 μL of 50 mM NH4HCO3 and 50% acetonitrile (ACN). Then 200 μL of 10 mM dithiothreitol (DTT) in 50 mM NH4HCO3 was added for protein reduction at 56 °C. After the removal of supernatant, 40 μL of 55 mM iodoacetamide in 100 mM NH4HCO3 was immediately added for protein alkylation in the dark. Finally, protein slices were dried twice with 200 μL of ACN. Afterward, the dried pieces of gels were incubated with ice-cold digestion solution (12.5 ng/μL trypsin and 20 mM NH4HCO3) for 20 min and then transferred into a 37 °C incubator for digestion overnight. Finally, peptides in the supernatant were collected after extraction twice with 200 μL of extract solution (5% formic acid in 50% ACN).

from bovine mammary gland. Later, ficoll and sucrose density gradient centrifugations were proven to obtain a high purification of the PM fraction from lactating bovine mammary gland.12 Differential velocity centrifugation has been used to isolate the membrane fraction of mammary tissues from Holstein cows.13 Recently, proteomics technologies have been successfully applied to reveal proteome profiles of lactating bovine mammary tissue,13−15 milk fat globule membrane,16 and cultured mammary epithelial cells from humans17 and Bos indicus,18 but it has not been used to elucidate the proteome profile of PM from lactating bovine mammary gland. Therefore, this experiment was designed to isolate and identify the PM of bovine mammary gland by discontinuous sucrose density gradient centrifugation and by liquid chromatography−tandem mass spectrometry (LC-MS/MS), respectively. Investigating the composition of PM proteins in the mammary gland of lactating cows, and subsequent identification of regulatory proteins involved in synthesis and secretion pathways of milk components on the subcellular level, would extend our understanding of the milk synthesis and secretion mechanisms of the bovine mammary gland.

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MATERIALS AND METHODS

Preparation of Plasma Membranes of the Bovine Mammary Gland. Plasma membranes were isolated from bovine mammary gland homogenates by discontinuous sucrose density gradient centrifugation. Twelve mammary gland tissue blocks were obtained from the right or left fore-quarter of the mammary gland of four female HolsteinFriesian dairy cows (500−530 kg, 6 years old) in their fourth parity and midlactation period. This experiment was conducted according to the animal care guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha city, Hunan province, China (no. KYNEAAM-2013-0009). Three technical replicates per cow were prepared for the isolation of plasma membranes. Approximately 6−8 g of fresh, mixed mammary gland tissue was weighed for each technical replicate and washed in ice-cold 0.9% NaCl, until the blood, milk, and connective tissues were removed. The tissue samples were then minced in 5 volumes of icecold homogenization buffer (0.25 M sucrose, 10 mM HEPES, pH 7.5) and homogenized by a Fisher Scientific PowerGen 125 homogenizer (Thermo Fisher Scientific Inc., USA). The homogenate was filtered through one layer of nylon gauze, and the filtrates were centrifuged at 1000gmax for 20 min in a Himac CR21 centrifuge (Hitachi High Technology, Japan). The resulting pellet was resuspended in 3 mL of buffer A (0.3 M sucrose, 50 mM Tris-HCl, 3 mM MgCl2, pH 7.5) and 9 volumes of buffer B (1.98 M sucrose, 50 mM Tris-HCl, 3 mM MgCl2, pH 7.5) to achieve a concentration of 1.8 M sucrose. Thirty milliliters of suspension liquids was transferred into one each of SW28 rotor tubes and overlaid with buffer C (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.5) to fill the tubes. They were then centrifuged at 70900gav for 90 min in a Beckman Optima L-XP100 ultracentrifuge (Beckman Coulter Inc., USA). The band at the interface was collected, resuspended in buffer C, and centrifuged at 20000gmax for 10 min. The resulting pellet was resuspended in a 1.45 M sucrose solution mixed with buffers C and D (2.4 M sucrose, 10 mM HEPES, pH 7.5), overlaid with enough buffer C to fill the SW28 rotor tubes, and then centrifuged at 68400gav for 80 min. The pellicle at the interface was resuspended in buffer C and centrifuged at 17600gmax for 10 min. Isometric buffer C and buffer D were added into the pellet to bring the final concentration of sucrose to 1.35 M, the solution was transferred into an NVT100 rotor tube, overlaid with enough buffer C, and centrifuged at 231000gav for 60 min. The purified plasma membranes were collected from the interface, washed in buffer C and then 10 mM HEPES (pH 7.5), and then centrifuged at 40000gmax for 10 min. All operations were performed at 4 °C, and a piece of protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) was added into every centrifugation tube in 7389

DOI: 10.1021/acs.jafc.5b02231 J. Agric. Food Chem. 2015, 63, 7388−7398

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Journal of Agricultural and Food Chemistry Table 1. Enzyme Activities in Homogenates and Isolated Plasma Membranes (PMs) from Bovine Mammary Gland enzyme

homogenates

PMs

protein (8) mg/mL 5′-nucleotidase, U/mg protein (6) Na+/K+-ATPase, ng/mg protein/h (7) monoamine oxidase, U/mg protein (8) NADPH cytochrome c reductase, nmol/min (7) succinodehydrogenase, IU/mg protein (10) aryl sulfatase β1,4-GTase, U/mg protein

2.10 ± 0.550 0.143 ± 0.053 0.182 ± 0.053 0.071 ± 0.019 4.14 ± 1.19 4.11 ± 0.842 NDb ND

1.11 ± 0.370 1.58 ± 0.694 1.64 ± 0.464 0.282 ± 0.141 47.8 ± 17.1 6.31 ± 1.07 ND ND

relative specific activitya 11.3 9.09 2.37 11.3 1.54

± ± ± ± ±

2.49 0.920 0.015 1.89 0.103

a

Relative specific activities were calculated as the ratio of the specific activity of the plasma membranes to that of the homogenates. The specific activity of the homogenates was normalized to 1. bND, not detectable. In-Solution Digestion of Samples. The membrane proteins isolated by LB1 and LB2 were in-solution digested using the method previously described19 with minor modification. In each experiment, the concentrated proteins were heated at 100 °C for 10 min. After the sample was cooled to room temperature on ice, DTT was introduced into the solution to reach a final concentration of 10 mM, and the sample was incubated at 57 °C for 30 min. To prevent disulfide bond formation, cysteine residues were alkylated by iodoacetamide, which was added to the sample solution at 20 mM final concentration. The 30 min incubation in the dark, and at room temperature, was performed for cysteine derivatization. The reaction was quenched by the addition of DTT at a 10 mM final concentration in solution for 10 min. After iodoacetamide deactivation, the sample solution was diluted 10-fold with 50 mM NH4HCO3 buffer. Trypsin was added to the sample at a ratio of (1:50) and incubated overnight at 37 °C. All digested peptide mixtures were passed over a C18 column to remove extra DTT and salt. Tryptic peptides were eluted from the column with 80% ACN in 0.1% trifluoroacetic acid, and the peptide solution was divided equally. Thereafter, each aliquot was dried in a vacuum centrifuge for later use. Strong Cation Exchange (SCX) Chromatography. The peptide mixtures from in-gel and in-solution digestion were fractionated by SCX chromatography on a 20AD high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) using a polysulfethyl column (2.1 × 100 mm, 5 um, 200 Å; Nest Group, Southborough, MA, USA). The mixed sample was diluted with a loading buffer (10 mM KH2PO4 in 25% ACN, pH 2.6) and loaded onto the column. Buffer A was identical in composition to the loading buffer, and buffer B was formatted using buffer A containing 350 mM KCl. Separation was performed using a linear binary gradient of 0−80% buffer B in buffer A at a flow rate of 200 μL/min for 60 min. The absorbance at 214 and 280 nm was monitored, and a total of 40 SCX fractions were collected along the gradient. Automated Nano-LC-ESI-MS/MS Analysis of Peptides. All of the eluted fractions and extracted solutions mentioned above were dried thoroughly using a vacuum centrifuge and then resuspended with 40 μL of 5% ACN in 0.1% formic acid, separated by Nano-LC, and analyzed by online electrospray tandem mass spectrometry as follows. The experiments were performed on an LC-20AD system (Shimadzu) connected to an LTQ Orbitrap mass spectrometer (Thermo Electron, Bremen, Germany) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Auburn, CA, USA). The separation of the peptides took place in a 15 cm reverse phase column (100 μm i.d., Michrom Bioresources). The peptide mixtures were injected onto the trap column with a flow of 60 μL/min, subsequently eluted with a gradient of 5−45% solvent B (95% ACN in 0.1% formic acid) over 90 min, and then injected into the mass spectrometer at a constant column-tip flow rate of 500 nL/min. Eluted peptides were analyzed by MS and data-dependent MS/MS acquisition, selecting the eight most abundant precursor ions for MS/MS with a dynamic exclusion duration of 1 min. Database Searching. The .mzXML files in the LTQ Orbitrap mass spectrometer were converted to.mgf files by MassMatrix Mass Spec Data File Conversion Tools and submitted for database sequence

searches using ProteinPilot software (version 4.0, Applied Biosystems). The following parameters were set in the searching: trypsin as enzyme, fixed modification of carbamidomethyl-labeled cysteine, no special factors, biological modification, thorough identification search, SwissProt database. Other parameters, such as tryptic cleavage specificity, precursor ion mass accuracy, and fragment ion mass accuracy, were built-in functions of ProteinPilot software, and the Paragon method was adopted. The detected protein threshold was set at 1.3, responding to the minimum identification probability of ≥0.95, and the competitor error margin was 2.00. Data Analysis and Bioinformatics. Swiss-Prot accession numbers of identified proteins were submitted to the retrieve ID/ mapping tool of UniProt (http://www.uniprot.org) to generate FASTA formatted protein sequences. The prediction of transmembrane helices in identified proteins was carried out using the transmembrane hidden Markov model (TMHMM) algorithm, available at http://www.cbs.dtu.dk/servies/TMHMM. The average hydrophobicity for identified proteins was calculated using ProtParam software (http://us.expasy.org) by submitting each protein accession. The proteins exhibiting positive grand average of hydropathicity (GRAVY) values were recognized as hydrophobic, and those with negative values were deemed hydrophilic.20 The subcellular location and function of all the identified proteins were elucidated by gene ontology (GO) component and function terms respectively, which were online annotated by DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov) against the background of Bos taurus. Functional enrichment analyses of cellular components, molecular functions, and biological processes were also performed via DAVID database. In the enrichment analysis, modified Fisher’s exact tests and false-discovery rate were used for statistical analysis. The significantly (p < 0.05 and false-discovery rate 0, % no. of proteins with plasma membrane-associated locations, %

parameter

LB1a

LB2

total

0.156

0.035

445 132 (29.7)

575 182 (31.7)

872 195 (22.4)

99 (22.2)

124 (21.6)

143(16.4)

135 (30.3)

162 (28.2)

215 (24.7)

a

LB, lysis buffer. bTM, transmembrane domain. cGRAVY, grand average of hydropathicity.

hundred and sixty-two of the 872 proteins were recognized by DAVID, and the remaining 10 proteins were not recognized and defined as unknown proteins. The transmembrane domains of the identified proteins were predicted with TMHMM. One-hundred and ninety-five (22.4%) were integral membrane proteins with at least one predicted transmembrane domain, and the percentage of these within LB2 (31.7%) was slightly greater than that of LB1 (29.7%). The GRAVY index represented the hydrophobicity property of proteins. Onehundred and forty-three (16.4%) proteins had positive values, and the percentage of LB1 (22.2%) was greater than that of LB2 (21.6%). Two-hundred and fifteen proteins (24.7%) of 872 identified proteins belonged to the PM proteins according to their GO annotations and the published literature; 30.3 and 28.2% of all identified proteins were from LB1 and LB2, respectively, including a list of known PM marker proteins such as sodium/potassium-transporting ATPase subunit α-1, flotillin-1, vesicle-associated membrane proteins, and 5′-nucleotidase. Protein Classification and Enrichment Analysis. Among the identified proteins, 215 (30.6%) proteins were classified as PM proteins, with a smaller number classified as proteins in mitochondria (25.2%), endoplasmic reticulum (7.7%), Golgi apparatus (2.4%), nuclei (6.4%), and ribosome (7.6%). There were 20.1% unknown proteins without cellular component terms (Figure 4a). The PM-associated proteins were further classified according to their molecular functions. As Figure 4b displays, 100 proteins (47.4%) and 41 proteins (19.4%) were binding proteins and transporter molecules, respectively, 32 proteins (15.2%)

Figure 2. Western blotting analysis of the plasma membranes (PM) isolated by subcellular fractionation. Eighty micrograms of the mammary gland homogenate and plasma membrane proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. The blots were probed with antibodies against organelle-specific proteins: anti-Na+/K+-ATPase and anti-flotillin-1 for plasma membranes (PM); anti-prohibitin for mitochondria; anti-actin for cytoplasm. H represents homogenates.

enrichment of plasma membranes, we detected the PM-specific proteins Na+/K+-ATPase and flotillin-1 and found that these proteins were enriched about 4- and 5-fold, respectively, in PM in comparison with mammary gland homogenate. Meanwhile, both mitochondria and cytoplasm-specific proteins prohibitin and actin were about 14-fold less in PM than in mammary gland homogenate. Separation and Identification of PM Proteins. The overall separation profiles of PM proteins isolated by two different lysis buffers are shown in Figure 3. The staining patterns of the two samples were similar for most bands, but differences in the intensities of a few bands were apparent. Gel slices cut from SDS-PAGE gel and proteins isolated by lysis buffers were digested in gel and in solution, respectively, separated by SCX chromatography, and further analyzed by automated Nano-LC-ESI-MS/MS. As shown in Table 2, PM protein yield of LB1 was 4.5-fold greater than that of LB2. The total number of nonredundant proteins identified from LB1 and LB2 was 872 (Supporting Information Supplementary Table 1). Of these 445 were found in LB1 and 575 were found in LB2; therefore, there were 381 proteins in common. Eight7391

DOI: 10.1021/acs.jafc.5b02231 J. Agric. Food Chem. 2015, 63, 7388−7398

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Journal of Agricultural and Food Chemistry

gland. With respect to cellular components, the top 10 cellular component terms were significantly over-represented, including PM, vesicle, melanosome, pigment granule, cytoplasmic membrane-bounded vesicle, cytoplasmic vesicle, membranebounded vesicle, PM part, internal side of PM, and cell surface (Figure 5a). Similar to the cellular components, the top 10 overrepresented molecular function terms were typically associated with the PM, including enzyme binding, GTP binding, guanyl ribonucleotide binding, guanyl nucleotide binding, calciumdependent phospholipid binding, unfolded protein binding, cytoskeletal protein binding, purine nucleotide binding, purine ribonucleotide binding, and ribonucleotide binding (Figure 5b). In biological processes, the top 10 enriched terms were vesicle-mediated transport, protein localization, protein transport, cell adhesion, cellular protein localization, intracellular transport, small GTPase mediated signal transduction, membrane organization, establishment of protein localization, and biological adhesion (Figure 5c). The identified PM proteins were involved in 74 pathways according to the pathway analysis. The top 15 pathways that were significantly enriched included caveolae-mediated endocytosis signaling, leukocyte extravasation signaling, clathrinmediated endocytosis signaling, RhoGDI signaling, ephrin receptor signaling, signaling by Rho family GTPases, ephrin B signaling, virus entry via endocytic pathways, aldosterone signaling in epithelial cells, lipid antigen presentation by CD1, eNOS signaling, remodeling of epithelial adherens junctions, sertoli cell sertoli cell junction signaling, PPARα/ RXRα activation, and tight junction signaling (Figure 5d).

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DISCUSSION Discontinuous sucrose gradient centrifugation, with its advantage of a reproducible and wide range of applications, was employed in this study to isolate the plasma membranes from lactating bovine mammary gland. However, this method often encounters difficulties in separating particular domains of PM from other cellular organelles, and from each other,21 which also appeared in the current study. The sheet-like plasma membranes were low in relative activities of marker enzymes and protein yield (0.185 mg per g wet tissue). This was greater than the yield of 0.032 mg obtained from plasma membranes isolated from lactating cow mammary gland,12 but less than that of isolated PM from lactating rat mammary gland by two-phase polymer partitioning (0.3 ± 0.1 mg).5 The marker enzymes used in the present study, 5′-nucleotidase and Na+/K+-ATPase, are considered to be reliable as PM markers of lactating mammary cells.12 Their activities in the PM were only 9 or 11 times greater than in the homogenates; perhaps the contamination of cytoplasmic proteins originating from the outer mitochondrial membranes caused this result. Western blotting results also confirmed that the plasma membranes were relatively enriched as indicated from an increased amount of PM marker proteins Na+/K+-ATPase and flotillin-1 and a lower amount of mitochondria and cytoplasm-specific proteins prohibitin and actin. All of these results indicate that plasma membranes isolated from bovine mammary gland and purified by discontinuous sucrose gradient centrifugation can be used for identification of proteins specific to the PM. In the current investigation, two different detergents were employed to solubilize more PM proteins. LB1 contains zwitterionic detergent SB3-10 and nonionic detergent n-octylβ-D-glucopyranoside (OG), and LB2 contains ionic detergent

Figure 4. Classification of proteins identified in isolated bovine mammary gland plasma membrane. The subcellular localization of the identified proteins (a), functional characterization of plasma membrane proteins (b), and biological processes of plasma membrane proteins (c) were classified by GO terms annotated by DAVID Bioinformatics Resources 6.7 against the background of Bos taurus.

corresponded to catalytic activity, and 23 proteins (10.9%) were associated with structural proteins. Other proteins were involved in ion channel activity (2.4%), motor activity (2.3%), and transcription regulator activity (2.4%). The PM proteins were also classified into several categories according to their DAVID annotation (Figure 4c): 36 (16.7%) proteins were involved in cellular processes, 28 (13%) in cell adhesion, 48 (22.3%) in transport, 17 (7.9%) in developmental process, 52 (24.2%) in metabolic process, 7 (3.3%) in immune system process, and 27 (12.6%) in apoptosis, cell cycle, generation of precursor metabolites, and response to stimulus. Seventy-five of 215 PM proteins had transmembrane domains and were involved in various processes associated with milk synthesis and secretion (Table 3), such as nutrient (glucose, amino acids, and fatty acids) transport, endocytosis, lactation, and synthesis of steroid and phospholipid. To access the enrichment degree of plasma membranes and to explore over-represented biological functions associated with the PM proteins, DAVID was used to characterize potential biological functions in the PM proteome of bovine mammary 7392

DOI: 10.1021/acs.jafc.5b02231 J. Agric. Food Chem. 2015, 63, 7388−7398

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Journal of Agricultural and Food Chemistry Table 3. Characterization of Identified PM Proteins with Transmembrane Domain protein name 3-ketodihydrosphingosine reductase 5′-nucleotidase activated leukocyte cell adhesion molecule adipocyte plasma membrane-associated protein amine oxidase [flavin-containing] A aminopeptidase N aquaporin 1 (Colton blood group) ATP-binding cassette subfamily G member 2 β-1,4-galactosyltransferase 1 BOLA class I histocompatibility antigen, α chain BL3-7 butyrophilin subfamily 1 member A1 cadherin-1 carbonic anhydrase 4 catechol O-methyltransferase cation-independent mannose-6-phosphate receptor caveolin-1 caveolin-2 CD36 molecule (thrombospondin receptor) CD46 molecule, complement regulatory protein CD81 molecule CD9 molecule claudin-3 claudin-4 claudin-7 cytochrome b-245 heavy chain cytochrome b5 desmocollin-2 (fragment) disintegrin and metalloproteinase domain-containing protein 10 DnaJ homologue subfamily C member 5 dolichyl-diphosphooligosaccharide−protein glycosyltransferase subunit STT3A dysferlin dystroglycan epithelial cell adhesion molecule inositol 1,4,5-trisphosphate receptor type 1 integrin α-V integrin β-1 integrin β-2 integrin β-6 intercellular adhesion molecule 1 leukocyte surface antigen CD47 lysophospholipid acyltransferase 5 lysosomal-associated membrane protein 1 membrane primary amine oxidase membrane-associated progesterone receptor component 1 mucin 15, cell surface associated mucin-1 peptidyl-prolyl cis−trans isomerase B platelet/endothelial cell adhesion molecule polymeric immunoglobulin receptor protein disulfide-isomerase A4 protein S100-A10 proteolipid protein 2 receptor accessory protein 6 sigma nonopioid intracellular receptor 1 sodium/potassium-transporting ATPase subunit α-1 sodium/potassium-transporting ATPase subunit β-3 solute carrier family 1, member 1 solute carrier family 1, member 5 solute carrier family 16, member 1 solute carrier family 2, member 3

accession

TMa

Q2KIJ5 Q05927 Q9BH13 Q3T0E5 P21398 P79098 P47865 Q4GZT4 P08037 P13753 P18892 Q6R8F2 Q95323 A7MBI7 P08169 P79132 Q66WT7 P26201 Q6VE48 Q3ZCD0 P30932 Q765N9 Q6BBL6 Q3B7N4 O46522 P00171 P33545 Q10741 Q29455 Q2KJI2

1 1 1 1 1 1 6 6 1 2 1 1 1 1 1 1 2 2 1 4 4 4 4 4 6 1 1 1 1 13

oxidation reduction negative regulation of inflammatory response cell adhesion arylesterase activity regulation of neurotransmitter levels ion binding water transport xenobiotic transport; protein dimerization activity epithelial cell development immune response secretion of milk-fat droplets regulation of cell death one-carbon metabolic proces regulation of neurotransmitter levels glycoprotein binding MAPKKK cascade protein dimerization activity regulator of fatty acid transport adaptive immune response positive regulation of cell proliferation; signal transduction negative regulation of cell proliferation response to oxygen levels cell adhesion structural molecule activity generation of precursor metabolites and energy generation of precursor metabolites and energy calcium ion binding regulation of cell growth regulation of cell death protein amino acid glycosylation

A6QQP7 O18738 Q3T0L5 Q9TU34 P80746 P53712 P32592 Q8SQB8 Q95132 Q9N0K1 Q3SZL3 Q05204 Q9TTK6 Q17QC0 Q8MI01 Q8WML4 P80311 P51866 P81265 Q29RV1 P60902 Q6Y1E2 Q32LG5 Q58DH7 Q08DA1 Q3T0C6 Q95135 Q95JC7 Q3MHW6 P58352

1 1 1 6 1 1 1 1 1 5 9 1 1 1 1 2 1 1 1 1 1 4 2 1 10 1 8 9 11 12

plasma membrane repair,calcium ion binding epithelium development positive regulation of cell proliferation cellular calcium ion homeostasis endocytosis positive regulation of cell proliferation regulation of protein amino acid phosphorylation inflammatory response; multicellular organismal development cell adhesion positive regulation of cell proliferation phospholipid biosynthetic process granzyme-mediated apoptotic signaling pathway copper/calcium binding steroid binding − − protein folding regulation of cell migration − cell redox homeostasis calcium ion binding cytokine binding regulation of intracellular transport steroid biosynthetic process ATP biosynthetic process ATP biosynthetic process neuronal/epithelial high affinity glutamate transporter neutral amino acid transporter monocarboxylic acid transporter glucose transporter

7393

predicted functionb

DOI: 10.1021/acs.jafc.5b02231 J. Agric. Food Chem. 2015, 63, 7388−7398

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Journal of Agricultural and Food Chemistry Table 3. continued protein name solute carrier family 44, member 3 stromal interaction molecule 1 suppressor of tumorigenicity 14 protein homologue synaptojanin 2 binding protein Toll-like receptor 2 transmembrane 9 superfamily member 1 transmembrane emp24 domain-containing protein 1 transmembrane emp24-like trafficking protein 10 (yeast) transmembrane protein 168 very-long-chain enoyl-CoA reductase vesicle-associated membrane protein 8 vesicle-associated membrane protein 3 (cellubrevin) vesicle-associated membrane protein-associated protein A vesicle-associated membrane protein-associated protein B V-type proton ATPase 116 kDa subunit a isoform 1

accession

TMa

A5PK40 Q58CP9 Q0IIH7 Q3T0C9 Q95LA9 A4IFE9 Q2TBK5 Q5E971 A0JNG0 Q3ZCD7 Q3T0Y8 Q2KJD2 Q0VCY1 A2VDZ9 Q29466

8 1 1 1 1 9 1 2 11 5 1 1 1 1 6

predicted functionb − positive regulation of calcium ion transport serine-type endopeptidase activity endocytosis regulation of cytokine production autophagy protein transport regulated secretory pathway − steroid biosynthetic process regulation of endocytosis Ca2+-dependent exocytosis signal transducer activity sphingolipid de novo biosynthesis inorganic cation transmembrane transporter activity

a

TM, transmembrane domain. bThe predict function of PM proteins was noted by the molecular function or biological process of the Gene Ontology annotation. −, the protein had no Gene Ontology annotation.

synthase and fatty acid-binding proteins) were also identified in the PM fractions. These proteins provide a substantial fraction of the reducing equivalents of NADPH and long-chain fatty acids taken up from blood, needed for lipid synthesis in the mammary gland.27−31 We also detected 5 of 10 enzymes involved in glycolysis, including hexokinase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ADP-dependent glucokinase, and enolase. This suggests that our lactating bovine mammary glands provide the necessary precursors for the energygenerating pathway required for milk synthesis and secretion. Additionally, we also identified six proteins participating in milk secretion and lactation regulation, including β-casein, κ-casein, lactotransferrin, xanthine dehydrogenase, butyrophilin A1, and fatty acid synthase. We compared the proteome profile of our PM fractions with the proteome profiles of mammary tissues and cells. Of 215 proteins and 188 PM proteins reported in lactating mammary tissue of Friesian cows,14,15 83 proteins (39%) and 48 proteins (26%) matched our data. Seventy-four proteins (25%) of 297 proteins identified in bovine milk fat globule membrane31 and 138 proteins (28%) of 497 proteins reported in bovine mammary epithelial cells18 matched our data, respectively. Relatively less coverage of our data with data from other groups can be attributed to the different lactation period of mammary glands and the fact that the mammary tissue may represent a mixed-cell population that constitutes adipose and stromal tissue, epithelial tissue, macrophages, and lymphocytes.32 It would appear that some newly identified PM-associated proteins of lactating bovine mammary gland have not been reported earlier. The PM proteins were further classified according to their functional characterization (Figure 4b,c). The main groups identified were binding and transport proteins, including annexin proteins (ANXA2, ANXA6, ANXA7, ANXA9, ANXA11), heat shock proteins, integrin, RAS oncogene family members, S100 calcium binding proteins, and solute carrier (SLC) families and members (SCL1A1, SLC1A5, SLC2A3, SLC16A1, SLC25A1, SLC44A3). Different types of annexins identified in the bovine mammary gland in midlactation have roles in various cellular functions including cell proliferation,33 secretion of milk components,34 apoptosis,35 and other

SDS and reductive agent DTT. Although the protein yield of LB1 was slightly greater than that of LB2, the number of total identified proteins, PM proteins, transmembrane proteins, and hydrophobic proteins was less than that of LB2 (Table 2). It could be suggested that the solubilization efficiency of LB2 was higher than that of LB1; thus, LB2 was more suitable for the extraction of PM proteins of the mammary gland. 1D-SDS-PAGE was applied in this study because of its high loading capacity and high solubilizing power of hydrophobic membrane proteins.22 Finally, 215 of the identified 872 proteins were PM proteins. If 20.1% unknown proteins were deducted, the proportion of PM proteins would be greater. Janjanam et al.18 had identified 111 and 37 PM proteins of mammary epithelial cells by 1D-Gel-LC-MS/MS and 2DE-MS/ MS, respectively. It was reported that around 9.1% of 700 protein spots detected by 2DE-MS/MS were localized to PM in the rat mammary gland.23 Peng et al.15 and Yang et al.13 had identified 160 and 183 membrane proteins by 1D-Gel-RPLCMS/MS and 1D-Gel-LC-MS, respectively. Therefore, 1D-SDSPAGE combined with LC-MS/MS had its advantage of highthroughput identification for PM-associated proteins. In the proteomic maps of PM fractions (Supplementary Table 1), many proteins were associated with metabolic pathways and involved in lactation-associated function such as staphylococcal nuclease domain-containing protein 1, annexin A5, and Rab GDP dissociation inhibitor 2. Signorelli et al.24 reported that in lactating sheep there was an increased expression of these three proteins at the lactation peak, which was in agreement with their basic cellular functions in relationship to higher milk yield. Staphylococcal nuclease domain-containing protein 1 was increased in humans in response to lactogenic hormones.25 Apolipoprotein A-I observed in this study’s PM fractions has been involved in delivering cholesterol to milk fat globules,26 and the highest expression of this protein occurred at midlactation.24 The eukaryotic translation elongation factor 2 was also present in the PM fractions. It has been reported that the main limiting factor in the protein synthesis at the end of lactation was the availability of this protein.24,26 The enzymes of the pentose phosphate pathway (transketolase), the tricarboxylic acid cycle (cytosolic NADP-isocitrate, succinate dehydrogenase, and malate dehydrogenase), and de novo lipogenesis (fatty acid 7394

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Figure 5. continued

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Figure 5. Enriched cellular components, molecular functions, biological processes, and pathways in the plasma membrane proteome. The overrepresentation of each category is determined by p value (≤0.05). Each general category is organized by GO algorithms, and only top 10 categories are shown: enriched cellular components in plasma membrane proteome (a), enriched molecular functions in plasma membrane proteome (b), enriched biological processes in plasma membrane proteome (c), and enriched pathways in plasma membrane proteome (d). PM, plasma membrane; CMB, cytoplasmic membrane-bound; ARVC, arrhythmogenic right ventricular cardiomyopathy.

demonstrated that CD36 facilitated the movement of fatty acids across the PM in bovine mammary gland. Also, the expression of CD36 mRNA in the bovine mammary gland has been shown to increase during lactation38 and stimulated by octanoate in the bovine mammary epithelial cells,37 demonstrating that CD36 expression could be related to physiological variations of lipid transport and metabolism within the cell. The relationship between the expressions of other CD antigen proteins and biological function in mammary gland has not been reported; therefore, there is an area for further investigation. In the investigation of the significantly enriched pathways (Figure 5d), it was found that integrin β1 was involved in eight signal pathways. Integrins mediate adhesion-induced phosphorylation events that trigger activation of numerous signaling intermediates such as FAK, Src, ILK, p130CAS, PI3K, JNK, and MARK and regulated Rho-family GTPases, which are central for the control of actin cytoskeleton organization, contractility,

essential signaling events during lactation. SLC1A1 (EAAC1) and SLC1A5 (ASCT2) mediated transport of L-Glu, L-Asp, and + D-Asp and Na -dependent exchange of small neutral amino acids such as Ala, Ser, Cys, and Thr, respectively,36 implying that SLC1A1 and SLC1A5 play important roles in providing materials for milk protein synthesis. Physiological functions regarding other SLC families and members expressed in mammary gland are not characterized yet and need to be investigated in the future. DAVID was used to identify enriched cellular component terms such as PM, vesicle, PM part, internal side of PM, and cell surface (Figure 5a). Functions such as binding (Figure 5b) and transport and adhesion (Figure 5c) were typically associated with the PM proteins. We found that six CD antigen proteins (CD9, CD36, CD47, CD166, PECAM1, ICAM1) were included in the cell adhesion terms. These cell surface molecules often act as receptors or ligands, which usually initiate a signal cascade. Yonezawa et al.37 have 7396

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Journal of Agricultural and Food Chemistry and cell shape changes.27 It was also reported that β1 integrins were critical for the alveolar morphogenesis of a glandular epithelium and for the maintenance of its differentiated function.27 β1 integrins and activation of FAK were essential for ductal morphogenesis, and Rac and ILK appeared to be major mediators of integrin signaling in the regulation of mammary luminal cell lactogenic differentiation.27 Myoepithelial cell contractile activity in lactation required α3β1 integrinmediated activation of Rac, FAK, and PAK1.39 In the bovine mammary gland, milk accumulation decreased the mRNA expression of integrins (β1, α5, α6) involved in cell− extracellular matrix communication and was associated with the induction of apoptosis.27 The integrin-induced intracellular signal transduction in bovine mammary gland remains to be further characterized. In summary, the PM fractions isolated from lactating bovine mammary gland by discontinuous sucrose gradient centrifugation were relatively enriched. Eight hundred and seventy-two nonredundant proteins were identified by 2D-Nano-LC-ESI LTQ-Orbitrap MS/MS, wherein 215 proteins belonged to the PM. Bioinformatic analysis revealed that the main groups of the PM proteins had functions related to binding, transport, and catalysis and were predicted to be involved in caveolaemediated endocytosis, leukocyte extravasation, aldosterone signaling in epithelial cells, and remodeling of epithelial adherens junctions. The results of this study provide the proteome profile of PM from bovine mammary tissue in vivo. This profile will be helpful to further elucidate the composition and functions of PM proteins and provide more knowledge about the molecular events taking place in the lactating bovine mammary gland.

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chemiluminescence; DTT, dithioerythritol; SCX, strong cation exchange

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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.5b02231. Proteins identified by Nano-LC-ESI-MS/MS (Table S1) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*(Z.T.) Phone: + 86 731 84619702. E-mail: [email protected]. Funding

We thank the National Natural Science Foundation of China (Grants 31001023, 31402112, 31101741, 31320103917), the National Science and Technology Support Program (Grant 2012BAD12B02), the project “Strategic Priority Research Program − Climate Change: Carbon Budget and Relevant Issues” of the Chinese Academy of Sciences (Grant XDA05020700), the K. C. Wong Education Foundation of CAS, and the CAS Visiting Professorship for Senior International Scientists (Grant 2010T2S13) for joint financial support. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED PM, plasma membrane; LB, lysis buffer; LC-MS/MS, liquid chromatography−tandem mass spectrometry; 1D, one-dimensional; SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; 2DE, two-dimensional gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; ECL, enhanced 7397

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