Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry

Abstract

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive^™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

Introduction

The small intestine is important for nutrient digestion and absorption, while also functioning as a major immunological defense barrier that can recognize and respond to external antigens. In addition, intestinal epithelial cells are normally involved in initiating and coordinating the intestinal immune response, mainly by producing signaling molecules [39]. Cell culture models can serve as powerful tools, provided they are functionally and structurally equivalent to the respective tissue, and due to their accessibility, homogeneity, and reproducibility, immortalized intestinal cell lines are used as in vitro models to understand intestinal host–pathogen interactions [45]. Studies correlating human intestinal processes often use the human colon cell line Caco-2 for its high morphological, structural, and biochemical similarities to small intestinal epithelial cells; however, it poses limitations related to its functional resemblance [12,31]. IPEC-J2 cells are non-transformed columnar epithelial cells extracted from the jejunal region of the small intestine of neonatal piglets [3] and have the strong advantage of being similar to primary intestinal epithelial cells [12].

Owing to the close similarities between swine and human intestinal functions, studies using IPEC-J2 cells can provide valuable insights into the pathogenesis of zoonotic enteric infections that also affect humans [36], as well as the function of the intestinal epithelium. Recent morphological and transcriptional reports have supported the utility of IPEC-J2 cells in understanding intestinal processes involved in metabolic and cellular functions, but have focused mostly on host–pathogen interactions [4,11,20,24,35]. Although extensive morphological and transcriptional data on porcine intestinal epithelial cells are available [4,35], the characterization and quantization of the total proteins produced in this intestinal cell line are limited. Several studies in IPEC-J2 cell line used gene transcripts rather than its proteome in investigating at the cellular level, which does not account for post-translational modifications and does not functional in some cases [11,12,20]. Other studies related to the IPEC-J2 proteome provided limited insights into a handful of proteins that might help in understanding the immunomodulatory mechanisms and regulation of vital functions in the gastrointestinal tract [4].

The aim of this study was to describe the global proteome of the IPEC-J2 cell line, to provide a basis for understanding its role in bacterial colonization and host–microbe interactions. A comprehensive analysis using label-free Q Exactive™ Orbitrap mass spectrometry (MS) was performed to obtain a quantitative proteomic profile of the IPEC-J2 cell line. In addition, the porcine genome sequence was used as a reference to compare the detected proteins to the porcine gene counterparts [13].

 

Materials and Methods

Intestinal Epithelial Cell Line and Preparation

The IPEC-J2 cell line comprises non-transformed intestinal cells isolated from the jejunal epithelium of neonatal piglets [4]. The cells were grown in Dulbecco’s modified Eagle’s medium/F-12 Ham containing 0.12% NaHCO3, 15 mM HEPES, pyridoxine, and L-glutamine (Sigma-Aldrich, USA), supplemented with 100 U/ml erythromycin, 0.5 mmol/l sodium pyruvate, and 5% fetal bovine serum (Sigma-Aldrich), and maintained in an atmosphere of 5% CO2 at 37℃ and 95% relative humidity. Cells were passaged every 3–4 days (seeding at a 1:3 ratio), and the medium was changed every other day.

To prepare the proteomic samples, IPEC-J2 cell cultures grown until confluence were harvested by centrifugation at 10,000 ×g for 10 min at 4℃, washed with phosphate-buffered saline (pH 7.0), and re-dissolved in lysis buffer (10 mM Tris-EDTA and 0.5% Triton X-100, pH 7.8) supplemented with Pierce protease inhibitor (Thermo Fisher Scientific, USA). The cells were disrupted by vortexing for 5 min. Four biological replicates were prepared for protein extraction and proteomic analysis. After vortexing, crude lysates were cleared by centrifugation at 10,000 ×g for 20 min at 4℃. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, USA).

UHPLC-Q Exactive™ Orbitrap Mass Spectrometry

Sample preparation was performed based on aprevious method [44]. Peptides were separated by ultra-high performance liquid chromatography (UHPLC) (Dionex UltiMate 3000; Thermo Fisher Scientific), followed by separation of the tryptic digestates using reversed-phase chromatography. Fraction reconstitution was conducted in solvent A (water/acetonitrile, 98:2 (v/v); 0.1% formic acid). The UHPLC was equipped with an Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper C18, 5 μm, 100 Å) to capture the sample, which was washed with 98% solvent A for 6 min at a flow rate of 6 μl/min and then continuously separated using an Acclaim PepMap 100 capillary column (75 μm × 25 cm, nanoViper C18, 3 μm, 100 Å) at a flow rate of 300 nl/min. The liquid chromatography analytical gradient was loaded at 2–35% solvent B over 90 min, then 35–95% over 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min. The ensuing peptides were electrosprayed through a coated silica tip (PicoTip emitter; New Objective, USA) at an ion spray voltage of 2,100 eV.

The UHPLC was connected via a heated electrospray ionization source (HESI-II) to the quadrupole-based Q Exactive™ Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific). A resolution of 70,000 (200 m/z) was used to acquire the MS spectra, with a mass range of 350–1,800 m/z. Ion accumulation was performed at a maximum injection time of 100 msec. Eluted samples were used for MS/MS events, which measured the 10 most abundant peaks (i.e., top 10 method) in a data-dependent mode through the high mass accuracy Orbitrap after ion activation/dissociation, with higher energy C-trap dissociation at 27 collisions and a mass range of 100–2,000 m/z.

Protein Database Search and Protein Identification

All identified proteins from the IPEC-J2 samples were cross-referenced against the latest Sus scrofa genome database in NCBI (Accession No. SAMN02953785). The false discovery rate of all peptide identifications was set at less than 1%. All proteins with at least two unique peptides were considered. All RefSeq protein GI identifiers from the pig genome for the IPEC-J2 proteome dataset were used, and the results were used to extract the best hits for subsequent analysis.

Proteins were quantified using a label-free approach based on spectral counts. The Xcalibur Qual Browser was used to process all spectra of the modified peptides after the full data scan, which was curated using Protein Discoverer software (ver. 1.4; Thermo Scientific). From the protein GI numbers, different file formats were obtained using the Biological DataBase Network website (http://biodbnet.abcc.ncifcrf.gov/db/db2db.php), including ENSEMBL Protein ID, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations. For GO molecular function, biological processes, and subcellular localization, the GO PANTHER database (http://www.pantherdb.org/) was used. The functional pathway analysis was conducted using the KEGG Pathway mapping tool, along with the KEGG S. scrofa taxon identifier 9823, for an organism-specific curation. All identified proteins located in the extracellular region, matrix, and membrane according to GO annotation were analyzed using the SignalP 4.1 Server for the presence of signal peptides, SecretomeP 2.0 Server for the prediction of mammalian secretory proteins with a nonclassical secretion system, and TMHMM Server (ver. 2.0) for transmembrane protein prediction, which were freely accessed on the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk/services/) [2,6,32].

 

Results and Discussion

UHPLC coupled to Orbitrap MS allows high-throughput identification and quantification of proteins and was recently used to determine the global proteomes of Vibrio metschnikovii and Lactobacillus mucosae LM1 [17,30]. The global proteomic profile of IPEC-J2 cells corresponds to previous microarray and mRNA expression data [4,35,39]; however, proteomic data provide greater coverage and insight into the biological activities of cell lines. Initially, Orbitrap MS was used to identify a total of 1,667 proteins from at least one proteomic sample (data not shown). We assumed that the proteins present in all samples were significantly detected in IPEC-J2 cells. From this, 1,517 high-confidence proteins were identified from the intracellular proteome of IPEC-J2 cells using the UHPLC-MS/MS method, accounting for 7.35% of all S. scrofa genes (1,517 proteins from 19,116 genes) (Fig. S1). These 1,517 proteins were further characterized into functional categories by pathway analysis. GO software classified the proteins into three functional groups: molecular function, biological processes, and cellular localization (Fig. 1 and Fig. S2).

Fig. 1.Functional categories of protein-encoding genes in the IPEC-J2 cell line. Proteins were categorized into (A) molecular function (black) and (B) biological processes (grey) according to the GO database. The number of proteins is labeled beside each bar.

A total of 1,046 GO terms (out of 1,517 proteins) fell under the same category. Most of the IPEC-J2 proteins identified display functions related to binding (322 proteins), catalytic activity (417 proteins), and structural molecule activity (113 proteins) (Fig. 1A). Within the GO biological process category, GO software assigned 1,659 GO terms to the detected proteins (Fig. 1B), suggesting multifunctional properties of some proteins. Further analysis showed that the IPEC-J2 proteome was abundant in proteins related to metabolic processes (597 proteins), cellular processes (342 proteins), biological regulation (136 proteins), and localization (162 proteins). Furthermore, proteomic analysis identified 49 and 25 proteins related to immune system processes and biological adhesion, respectively, which are important for host–microbe interactions.

Quantification of Proteins in the IPEC-J2 Proteome

The Q Exactive™ Orbitrap MS allowed for absolute quantification of proteins in IPEC-J2 cells and detected 20 highly abundant proteins (with >1.0% relative abundance) among the 1,517 proteins (Fig. 2). The most abundant protein was actin (ActB), with a mean abundance of 6.06% in the IPEC-J2 proteome. The high abundance of ActB suggests its significant role in IPEC-J2 structural integrity [21,29]. ActB has been observed as the core component necessary for structural stability of intestinal epithelial microvilli [38]. In addition, it allows for the detection of stochastic (e.g., thermal) and coherent activation energies (e.g., electromagnetic forces and mechanical stimuli) in the external environment [21]. Therefore, actin cytoskeleton regulation might affect important gut functions, including intestinal absorption, secretion, and cellular adhesion, as well as signal reception and transduction. Other highly abundant proteins that might play structural roles in IPEC-J2 cells include keratin 8 (type II), actinin α-4, tubulin α-1b, keratin type I cytoskeletal 18 partial, myosin-9, keratin 19 type II, and tubulin β-4B class IVb (Fig. 2). Many of these proteins are involved in structural integrity, as well as regulation of cell–cell and host–microbe signaling mechanisms.

Fig. 2.Relative abundance of proteins (n = 1,517) in the global proteome of the IPEC-J2 cell line. Highly abundant proteins (>1.0% relative abundance, n = 20) and less abundant proteins (<1.0% relative abundance, n = 1,497) were detected. The values are the mean relative abundances (%) calculated from the proteomic data from four biological replicates.

The second most abundant protein (4.07% abundance in the IPEC-J2 proteome) was annexin A2 (AnxA2). AnxA2 is associated with important biological functions, including signal transduction, angiogenesis, apoptosis, tumor invasion, and metastasis. It is involved in anti-inflammatory effects, Ca2+-dependent exocytosis, immune responses, and phospholipase A2 regulation. In a recent study, AnxA2 was assessed as a potential biomarker for the early detection of hepatocellular carcinoma and other gastrointestinal disorders [8]. In another study, anxA2-deficient mice were found to be highly susceptible to gram-negative bacteria sepsis with enhanced inflammatory responses [46]. This suggests that AnxA2 is important in host defense against infection and gastrointestinal diseases. In addition, annexin A1 and A8 were detected among the highly abundant proteins related to AnxA2 (Fig. 2), which might be important in controlling gastrointestinal and inflammatory diseases.

The third most abundant protein (2.60% of the IPEC-J2 proteome) was AHNAK nucleoprotein (AHNAK). In a recent study, the expression of the gene encoding AHNAK was significantly associated with aging in human skeletal muscle [40]. The role of AHNAK suggests its potential as a biological marker useful for elucidating the proper functioning of the IPEC-J2 cell line. However, limited studies have been conducted on this highly abundant protein in IPEC-J2 cells, and further research is required to elucidate its specific mechanism in gastrointestinal health and interactions with specific gut microbiota.

Other highly abundant proteins detected in the IPEC-J2 proteome were heat shock protein (HSP) 70 kDa (HSP70), 90kDa (HSP9 0), and 27 kDa (HSP27) (Fig. 2). HSPs are highly conserved molecular chaperones associated with maintaining gut homeostasis and protecting cellular components. In a recent study, HSP27 induction in the intestinal epithelium (IPEC-J2 cells) by Lactobacillus rhamnosus GG, Lactobacillus reuteri strain P43-HUV, and Lactobacillus johnsonii strain P47-HY increased gut barrier function and cellular defenses [24]. Inducing HSPs might be associated with tight junction stability and actin cytoskeleton regulation [25]. Furthermore, high abundances of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and α-enolase suggested the generation of ATP via cellular metabolism, but these proteins could also be involved in cell repair, apoptosis, and barrier functions in the intestinal epithelium [7,41,48]. Quantifying differentially abundant proteins in the IPEC-J2 proteome provided extensive and comprehensive insight into the basic functions and essential processes of this intestinal cell line, including the maintenance of gut barrier structure and integrity, metabolic activities, and immune responses.

Analysis of IPEC-J2 Membrane Proteins and Signaling

As mentioned above, many of the highly abundant proteins in the IPEC-J2 proteome are involved in cellular structure, barrier function, and regulation of metabolic and immune responses. These structural and regulatory functions suggest an intricate and complex network of proteins that allows inter-connection and -communication within the intestinal cell line. First, to determine the network of proteins and pathways in IPEC-J2 cells, all proteins were sorted according to GO annotation for cellular localization (Fig. S2), and proteins with putative secretory and membrane features that might interact with external stimuli (i.e., bacteria, viruses, and stress) were analyzed further. Using the SignalP, SecretomeP, and TMHMM programs, proteins were assessed for the presence of signal peptides, nonclassical secretory features, and transmembrane domains, respectively (Table 1). The proteomic data and bioinformatics analysis confirmed the presence of a number of secreted proteins, including laminins (e.g., Lamc1 and Lamb1), integrins (e.g., Itgb1 and Itgb6), and antigen molecules (e.g., Flrt3) that could serve as glycoproteins, epithelial signaling receptors, and antigen molecules, respectively. However, several of the GO annotations for cellular localization must be confirmed using third-party bioinformatics tools and actual proteomic data. Laminins (Lamc1 and Lamb1) and integrins (Itgb1 and Itgb6) also form an extracellular protein complex providing potential sites for bacterial binding [37]. Laminin–integrin complexes are bound to intracellular signaling mechanics that can influence different pathways related to proliferation, structural properties, and immune response (Fig. 3) [1,23,42]. The external signals induced through the protein complex are generated by an intracellular non-receptor protein tyrosine kinase (Src; proto-oncogene tyrosine-protein kinase Src) or other proteins (i.e., growth factors and apolipoproteins) detected in the IPEC-J2 proteome (Fig. 3), which are likely bound to the laminin–integrin complex and might participate in major signaling pathways [22]. Src relays signals from integrin to Ras homolog protein member A (RhoA) to regulate gene transcription, immune responses, cell adhesion, cell cycle progression, apoptosis, migration, and transformation (Fig. 3) [9,14]. RhoA is mutually antagonistic to other proteins (i.e., Rac1) in cells to enable balance among cellular activities [15].

Table 1.aInitially categorized based on the GO database for their extracellular and membrane localization (GO cellular localization).

Fig. 3.Proposed pathway for the regulation of mucosal immunity, epithelial barrier function, and structural integrity. The network of IPEC-J2 proteins annotated was based on KEGG (http://www.kegg.jp/). The map shows the presence of proteins involved in receiving external stimuli, signal transduction, and regulation of cellular functions in IPEC-J2 cells. F3, tissue factor F3; Src, tyrosine kinase; RhoA, Ras homolog A; ZO-1, tight junction protein 1/zonula occludens; MAPKs, mitogen-activated protein kinases; Rac1, rho family, small GTP binding protein Rac1; NFκB, nuclear factor kappa B complex; ApoD, apolipoprotein.

Other membrane-bound proteins were verified using the TMHMM program, such as basigin and tissue factor F3 (Table 1). Basigin is a transmembrane glycoprotein associated with different functions, including inflammatory and oxidative pathways (Fig. 3) [28], whereas tissue factor F3 initiates the extrinsic coagulation pathway and secretion of coagulation proteases upon contact with the gut microbiota [34]. These potentially secreted and membrane proteins might be responsible for directly relaying signals among the extracellular region, cell basal membrane domain, and basement membrane for cell proliferation, migration, and differentiation [19].

Proposed Innate Immune Pathway in the IPEC-J2 Cell Line

Additional proteins identified in the IPEC-J2 dataset with a potential role in functional pathways were further analyzed using the KEGG pathway mapping tool. The results revealed many proteins involved in a total of 270 pathways. Cell metabolism accounted for the largest proportion (193 proteins), followed by biosynthesis of antibiotics (67 proteins), and ribosomes (51 proteins) (Table S1). Furthermore, approximately 456 proteins are involved in 44 signaling pathways, especially innate immune pathways (Table S1), such as the PI3K/Akt (34 proteins) and mitogen-activated protein kinase (MAPK) (20 proteins) signaling pathways, likely responsible for regulation of metabolism (glycolysis/gluconeogenesis), cell differentiation, proliferation, inflammation (apoptosis), DNA repair, and cell survival (NFκB and p53 system) [5,18,43,47].

Since IPEC-J2 is primarily used to investigate host–microbe interactions, including innate immune responses, we focused on the signaling properties of IPEC-J2 proteins to create an overview of its immune pathways (Fig. 3). The data revealed the presence of the rho family small GTP binding protein Rac1 (Rac1), which mediates regulation of the actin cytoskeleton and multiple signaling pathways (i.e., PI3K/Akt and MAPK) (Fig. 3). Rac1 is an essential signaling protein in the NFκB signaling pathway, cell proliferation, and regeneration of human intestinal cells [26,27]. Other studies suggested that Rac1 is involved in the up-regulation and secretion of interleukin-8 from epithelial cells infected with Salmonella Typhimurium [16] and in gut barrier function via regulation of the expression of the tight junction protein zonula occludens 1 (ZO-1) [33].

Other essential markers for proper immune responses might be involved in NFκB protein complex transcription. Several transcription factors detected in the IPEC-J2 proteome included v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) and the NFκB p105 subunit. These two proteins have been widely used to monitor human, porcine, and murine immune reactions in inflammatory disease and infection studies [10,20,43]. Furthermore, the signaling machinery and regulatory genes in the NFκB complex were used to evaluate the immunomodulatory effects of L. rhamnosus GG in the IPEC-J2 cell line [11]. These proteins might serve as potential biomarkers for innate immune response regulation in IPEC-J2 cells. The characterization of these proteins should lead to a broader understanding of the underlying regulatory networks that can contribute to immunomodulation by host-microbe interactions.

In summary, we conducted a proteomic analysis of the IPEC-J2 cell line for the first time to identify and quantify the total proteins expressed using label-free Q Exactive™ Orbitrap MS. The results showed that some proteins involved in structural integrity, gut barrier function, and homeostasis are highly abundant. Further analysis revealed a protein network related to signaling and regulation of mucosal immunity. In addition, bioinformatics tools confirmed the location of proteins in relation to the extracellular region and membrane, suggesting a possible function as extracellular signaling receptors in the IPEC-J2 cell line. Finally, the label-free proteomic analysis of IPEC-J2 cells resulted in a good coverage and reliable quantification of proteins that enabled the creation of a comprehensive reference dataset. The IPEC-J2 reference proteome will significantly aid in analyses, especially when investigating host-microbe interactions and comparing their effects on immune reaction and inflammatory mechanisms.