Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy
2015; Elsevier BV; Volume: 43; Issue: 9 Linguagem: Inglês
10.1016/j.exphem.2015.04.009
ISSN1873-2399
AutoresXiang Li, Dongliang Li, Xingchen Pang, Ganglong Yang, H. Joachim Deeg, Feng Guan,
Tópico(s)Ubiquitin and proteasome pathways
Resumo•An integrated strategy was used to profile N-glycan and glycogenes in two human bone marrow cells.•Bisecting N-glycans, catalyzed by MGAT3 (β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase), were enhanced in HS5 cells.•The glycan structure of Galβ1,4GlcNAc, catalyzed by β4GalT1 (β4-galactosyltransferase I), was significantly increased in HS27a cells. Altered expression of glycans is associated with cell–cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture. Altered expression of glycans is associated with cell–cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture. The bone marrow micro-environment consists of a specialized population of cells that play essential roles in regulation, self-renewal, and differentiation of adult stem cells. The microenvironment supports maturation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) and their release into the vascular system [1Fliedner T.M. The role of blood stem cells in hematopoietic cell renewal.Stem Cells. 1998; 16: 13-29PubMed Google Scholar]. Mesenchymal/stromal cells that represent integral components of the micro-environment contribute to the regulation and release of HPC adhesion molecules, extracellular matrix (ECM), and soluble factors, including cytokines and chemokines [2Mendez-Ferrer S. Michurina T.V. Ferraro F. et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature. 2010; 466: 829-834Crossref PubMed Scopus (2413) Google Scholar, 3Bhatia R. McGlave P.B. Dewald G.W. Blazar B.R. Verfaillie C.M. Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: Role of malignant stromal macrophages.Blood. 1995; 85: 3636-3645PubMed Google Scholar]. HS5 and HS27a, two bone marrow stroma cell lines, both derived from the same healthy marrow donor [4Roecklein B.A. Torok-Storb B. Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes.Blood. 1995; 85: 997-1005Crossref PubMed Google Scholar], have strikingly different functions [5Graf L. Iwata M. Torok-Storb B. Gene expression profiling of the functionally distinct human bone marrow stromal cell lines HS-5 and HS-27a.Blood. 2002; 100: 1509-1511Crossref PubMed Scopus (59) Google Scholar, 6Iwata M. Sandstrom R.S. Delrow J.J. Stamatoyannopoulos J.A. Torok-Storb B. Functionally and phenotypically distinct subpopulations of marrow stromal cells are fibroblast in origin and induce different fates in peripheral blood monocytes.Stem Cells Dev. 2014; 23: 729-740Crossref PubMed Scopus (17) Google Scholar]. 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Mice with knockout (Fut8−/−) of the gene that encodes the α1,6-fucosyltransferase enzyme exhibited abnormal pro-B cell to pre-B cell transition and reduction of peripheral blood B cells and immunoglobulin production [20Li W. Ishihara K. Yokota T. et al.Reduced α4β1 integrin/VCAM-1 interactions lead to impaired pre-B cell repopulation in alpha 1,6-fucosyltransferase deficient mice.Glycobiology. 2008; 18: 114-124Crossref PubMed Scopus (22) Google Scholar]. Cell surface antigens such as CD133, a 120-kDa glycosylated polypeptide used as an HSC biomarker, are often glycosylated [21Freeze H. Elbein A. Glycosylation precursors.in: Varki A. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009: 66Google Scholar, 22Zhou F. Cui C. Ge Y. et al.α2,3-Sialylation regulates the stability of stem cell marker CD133.J Biochem. 2010; 148: 273-280Crossref PubMed Scopus (37) Google Scholar]. Differences in glycosylation expression between stromal HS5 and HS27a cells have not been addressed in any study to date. We used an integrated strategy that combines genomic, proteomic, and functional glycomic techniques for comparative profiling of glycans, their related genes, and proteins in HS5 and HS27a cells. Expression levels of glycosyltransferases and glycosidases were quantitatively analyzed by the stable isotope labeling by amino acids in cell culture (SILAC) method, and glycans were subjected to lectin microarray analysis. This integrated strategy (summarized in Fig. 1) was useful in revealing global differences in glycan expression between these two cell lines, and can be extended to similar comparative studies of other cell lines.MethodsCell lines and cultureHuman marrow stromal cell lines HS5 and HS27a, originally derived from marrow of a healthy subject and immortalized by transduction with human papilloma virus E6/E7 constructs, were maintained as previously described [23Li X. Marcondes A.M. Gooley T.A. Deeg H.J. The helix–loop–helix transcription factor TWIST is dysregulated in myelodysplastic syndromes.Blood. 2010; 116: 2304-2314Crossref PubMed Scopus (37) Google Scholar]. Multiple aliquots from early passages were cryopreserved for later use.Stable isotope labeling by amino acids in cell culture was performed as described previously [24Ong S.E. Stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics.Mol Cell Proteomics. 2002; 1: 376-386Crossref PubMed Scopus (4514) Google Scholar, 25Yang G. Tan Z. Lu W. et al.Quantitative glycome analysis of N-glycan patterns in bladder cancer vs normal bladder cells using an integrated strategy.J Proteome Res. 2015; 14: 639-653Crossref PubMed Scopus (52) Google Scholar]. SILAC reagents and media were from Thermo Scientific (San Jose, CA); the final concentration of arginine (Arg) and lysine (Lys) was 100 μg/mL. HS27a cells were cultured in SILAC medium containing 13C615N4 Arg and 13C6 Lys (heavy). HS5 cells were cultured in SILAC medium containing 12C614N4 Arg and 12C6 Lys (light). Culture medium was replaced every other day until cells were 70%–80% confluent. Cells were grown for five or six passages. Labeling efficiency was checked to ensure an incorporation rate >95%.Glycogene microarray analysisDifferential gene expression in HS5 and HS27a cells was analyzed by Torok-Storb's group (Fred Hutchinson Cancer Research Center; Seattle, WA) as described previously [5Graf L. Iwata M. Torok-Storb B. Gene expression profiling of the functionally distinct human bone marrow stromal cell lines HS-5 and HS-27a.Blood. 2002; 100: 1509-1511Crossref PubMed Scopus (59) Google Scholar]. Open-access data at http://www.ncbi.nlm.nih.gov/sites/GDSbrowser (GEO Accession No. GSE463) were downloaded. Glycogenes listed on the GlycoV4 oligonucleotide microarray (covering 1,260 human glycogenes) were extracted and analyzed as described [26Tan Z. Lu W. Li X. et al.Altered N-glycan expression profile in epithelial-to-mesenchymal transition of NMuMG cells revealed by an integrated strategy using mass spectrometry and glycogene and lectin microarray analysis.J Proteome Res. 2014; 13: 2783-2795Crossref PubMed Scopus (57) Google Scholar, 27Guan F. Schaffer L. Handa K. Hakomori S.I. Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-β.FASEB J. 2010; 24: 4889-4903Crossref PubMed Scopus (50) Google Scholar]. Raw values were normalized using the robust multichip average (RMA) expression summary. Data were processed using R program software and Bioconductor project (GEOquery 2.23.2, www.r-project.org/). Fold changes were estimated by fitting a linear model for the genes, and linear modeling was performed with the Limma package in the R program software for differential expression analysis. Transcripts differentially expressed in HS5 and HS27a samples were compared using thresholds of fold change >1.5, fold change <0.67, adjusted p value 1.8) was used.Quantitative real-time polymerase chain reactionPrimers were designed using the DNAMAN program, Version 6.0.3 (Lynnon Biosoft, Vaudreuil, Quebec, Canada). Total RNA was converted into cDNA using a ReverTra Ace-α First-Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Quantitative real-time polymerase chain reaction (RT-PCR) was performed by Light Cycler-based SYBR Green I dye detection with UltraSYBR Mixture (CWBiotech). Messenger RNA levels of target genes were normalized to expression of β-actin and quantified using the 2−ΔΔCT method [28Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative pcr and the 2–ΔΔCT method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (119535) Google Scholar].Total protein extraction and Western blot analysisUnlabeled and labeled cells with 75%–90% confluence were lysed with T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Hudson, NH, USA) according to the manufacturer's instructions. In brief, cells were trypsinized, resuspended in 1× phosphate-buffered saline (PBS: 0.01 mol/L phosphate buffer containing 0.15 mol/L NaCl, pH 7.2), added with an appropriate amount of T-PER Reagent containing 0.1% aprotinin, incubated on ice for 30 min, and homogenized. The sample was centrifuged for 15 min at 12,000 rpm (4°C), and the supernatant was collected and stored at −80°C. Protein content was determined with the BCA assay (Beyotime, Shanghai, China).Proteins from each sample were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Bio-Rad, Hercules, CA) with the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were soaked in 5% (w/v) skim milk in TBST (20 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.05% Tween 20, pH 8.0) for 2 hours at 37°C, probed with primary antibodies against β-1,4-mannosyl-glycoprotein 4-β-N- acetylglucosaminyltransferase (MGAT3; Santa Cruz Biotechnology, Santa Cruz, CA) and β4-galactosyltransferase I (β4GalT1; Abcam, Cambridge, MA) overnight at 4°C, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Bands were visualized using the enhanced chemiluminescence detection kit Westar Nova (Cyanagen, Bologna, Italy) with imaging by ChemiDoc XRS+ (Bio-Rad).Quantitative analysis of proteins associated with glycan biosynthesis by SILACCells with various labeling were lysed with T-PER reagent. Proteins were mixed at equivalent ratios, reduced (10 mmol/L dithiothreitol, 45 min), and alkylated (30 mmol/L iodoacetamide, 45 min at room temperature [RT] in the dark). Sequencing grade modified trypsin (Promega, Madison, WI) was added to 1:100 (w/w), and the mixture was incubated at 37°C for 24 hours [29Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat Methods. 2009; 6: 359-362Crossref PubMed Scopus (4865) Google Scholar]. Two-dimensional liquid chromatography–mass spectrometry was performed using the LTQ Orbitrap MS (Thermo Fisher Scientific, Waltham, MA), as described previously [30Olsen J.V. de Godoy L.M. Li G. et al.Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol Cell Proteomics. 2005; 4: 2010-2021Crossref PubMed Scopus (1233) Google Scholar, 31Cox J. Matic I. Hilger M. et al.A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.Nat Protoc. 2009; 4: 698-705Crossref PubMed Scopus (616) Google Scholar] (see Supplementary Data, online only, available at www.exphem.org). Data were analyzed using the MaxQuant software program, Version 1.4.1.2 [31Cox J. Matic I. Hilger M. et al.A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.Nat Protoc. 2009; 4: 698-705Crossref PubMed Scopus (616) Google Scholar, 32Cox J. Mann M. Maxquant enables high peptide identification rates, individualized PPB-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (8849) Google Scholar].Lectin microarray analysis and data analysisLectin microarray analysis was performed as described previously [33Yu H. Zhu M. Qin Y. et al.Analysis of glycan-related genes expression and glycan profiles in mice with liver fibrosis.J Proteome Res. 2012; 11: 5277-5285Crossref PubMed Scopus (50) Google Scholar]. In brief, 37 commercial lectins from Vector Laboratories (Burlingame, CA), Sigma-Aldrich, and Calbiochem Merck (Darmstadt, Germany) were immobilized on a solid support at high spatial density. Glycoprotein samples labeled with the fluorescent dye Cy3 (GE Healthcare; Buckinghamshire, UK) were applied and scanned with a GenePix 4000B confocal scanner (Axon Instruments, Union City, CA). Raw values less than the average background were omitted. The median for each lectin was globally normalized to the sum of the medians of all valid data for the 37 lectins [33Yu H. Zhu M. Qin Y. et al.Analysis of glycan-related genes expression and glycan profiles in mice with liver fibrosis.J Proteome Res. 2012; 11: 5277-5285Crossref PubMed Scopus (50) Google Scholar]. Differences between the two data sets were evaluated with Student's t-test applied to each lectin signal (fold changes >1.5 and <0.67, p value < 0.05).Lectin stainingHS5 and HS27a cells were cultured in 24-well plates with sterilized Glycergel (DakoCytomation; Carpinteria, CA) to 60%–70% confluence. Culture medium was discarded, and 2% fresh paraformaldehyde was added to fix cells. Cells were immobilized for 15 min at RT, washed with 1× PBS, permeabilized with 0.2% Triton X-100 in 1× PBS for 2 min at RT, and blocked with 5% bovine serum albumin (BSA) in 1× PBS overnight at 4°C. Fixed cells were incubated with 15–20 μg/mL Cy3 fluorescein-labeled lectins in 5% BSA for 3 hours in the dark at RT, washed with PBS, stained with 4 μg/mL DAPI (4′,6-diamidino-2-phenylindole) in 1× PBS for 10 min at RT, washed again with PBS, and observed by laser confocal fluorescence microscopy (Eclipse Ti-U, Nikon, Tokyo, Japan).ResultsExpression of glycan-related genes in HS5 and HS27a cellsOf ∼17,000 genes in the micro-array, 130 glycogenes were differentially expressed and were visualized as a "heatmap" using the Cluster and Tree View software program (http://www.eisenlab.org/eisen/?page_id=42) (Fig. 2A). On the basis of DAVID software analysis (http://david.abcc.ncifcrf.gov/), 66 glycogenes were annotated and classified into the following eight groups based on their functions: N-glycan synthesis (12 genes), glycosaminoglycan degradation (5), other glycan degradation (3), cytokine–cytokine receptor interaction (21), amino and nucleotide sugar metabolism (4), galactose metabolism (6), and other functions (26) (Fig. 2B; Supplementary Table E1, online only, available at www.exphem.org).Figure 2Comparative glycogene expression of HS5 and HS27a cells by glycogene micro-array analysis. (A) "Heatmap" representation of differentially expressed genes involved in metabolism of glycosphingolipids, glycoproteins, and glycosaminoglycans. Red = genomic activation; green = genomic inhibition; black = no clear link; gray = missing data. (B) Annotations of genes by DAVID software program were classified, and category distribution numbers are shown as a bar chart. Categories and numbers: N-Glycan biosynthesis, 12; glycosaminoglycan degradation, 5; other glycan degradation, 3; cytokine–cytokine receptor interaction, 21; amino and nucleotide sugar metabolism, 4; galactose metabolism, 6; other, 26. (C) Gene expression of ALG5, ALG6, β4GalT1, DPAGT1, DDOST, FUT4, GLB1, GUSB, HEXA, HPSE, MAN1B1, MAN1A2, MAN2A1, MGAT2, MGTA3, NEU1, and RPN1 was analyzed by real-time polymerase chain reaction, as described under Methods. Experiments were performed in biological triplicate. Relative expression was analyzed by the 2–△△CT method and presented as log2 relative expression for HS5 versus HS27a, with log2(3/2) and log2(2/3) as threshold values. Values above log2(3/2) and below log2(2/3) indicate significant upregulation and downregulation, respectively. (D) Western blot analysis of MGAT3 (β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase) and β4GalT1 (β4-galactosyltransferase I), with β-tubulin expression as protein loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Among the 15 N-glycan-related genes (including glycan degradation and N-glycan synthesis), 12 (ALG5, ALG6, β4GALT1, DPAGT1, DDOST, MAN1A2, MAN1B1, MGAT2, RPN1, GLB1, HEXA, NEU1) exhibited increased expression, and 3 (β4GalT3, MGAT3, MAN2A1), reduced expression, in HS27a compared with HS5. Among the glycosaminoglycan degradation group, 4 genes (GLB1, GUSB, HPSE, HEXA) were upregulated and 1 (HS3ST3B1) was downregulated in HS27a. These findings are summarized in Table 1 (p value < 0.05 for all arrays).Table 1Differential expression of glycan-related genes in HS27a and HS5 cells (fold change = HS27a/HS5)Gene symbolaGroup A = N-glycan biosynthesis-related genes. Group B = glycosaminoglycan degradation-related genes. Identifiers, names, and functional descriptions are from information available in public databases, primarily the National Center for Biotechnology Information (NCBI) UniGene database and GenBank. Average value in gene expression of HS27a (n = 4) was divided by average value in gene expression of HS5 (n = 4).Accession IDp valueFold changeGene descriptionABCALG5NM_0133380.0052.010Dolichyl phosphate β-glucosyltransferaseALG6NM_0133390.0091.545α1,3-GlucosyltransferaseB4GALT1NM_0014970.0002.723βGlcNAc β1,4- galactosyltransferase, polypeptide 1B4GALT3NM_0037790.0320.765βGlcNAc β1,4- galactosyltransferase, polypeptide 3DDOSTNM_0052160.0251.417Dolichyl diphospho-oligosaccharide protein glycosyltransferase subunitDPAGT1NM_0013820.0021.565Dolichyl phosphate N-acetylglucosamine-phosphotransferase 1MAN1A2NM_0066990.0001.605Mannosidase, α, class 1A, member 2MAN1B1NM_0162190.0021.718Mannosidase, α, class 1B, member 1MGAT2NM_0024080.0041.613Monoacylglycerol O-acyltransferase 2MGAT3NM_0024090.0000.262Mannosyl-glycoprotein β-1,4-N-acetyl-glucosaminyltransferaseRPN1NM_0029500.0021.661Ribophorin IMAN2A1NM_0023720.0000.182Mannosidase, α, class 2A, member 1GLB1NM_0004040.0051.492Galactosidase, β1HEXANM_0005200.0062.140Hexosaminidase A (α polypeptide)NEU1NM_0004340.0012.231Sialidase 1 (lysosomal sialidase)GLB1NM_0004040.0051.492Galactosidase, β1GUSBNM_0001810.0002.614GlucuronidaseHS3ST3B1NM_0060410.0000.135Heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1HPSENM_0066650.0093.330HeparanaseHEXANM_0005200.0172.140(α Polypeptide)a Group A = N-glycan biosynthesis-related genes. Group B = glycosaminoglycan degradation-related genes. Identifiers, names, and functional descriptions are from information available in public databases, primarily the National Center for Biotechnology Information (NCBI) UniGene database and GenBank. Average value in gene expression of HS27a (n = 4) was divided by average value in gene expression of HS5 (n = 4). Open table in a new tab The genes listed in Table 1 were defined with significant change of expression, either fold change >1.5 or fold change 1.50) and one (MAN2A1) had reduced expression (ratio < 0.67), consistently with gene micro-array analysis results. Protein expression levels were confirmed by Western blot analysis. The protein level of β4GalT1 in HS27a is illustrated in Figure 2D. Both micro-array and RT-PCR analyses revealed a significant reduction of MGAT3 mRNA level in HS27a (Fig. 2A and C). However, a reduced MGAT3 protein level in HS27a was revealed only by Western blot, not by SILAC (Fig. 2D). Taken together, integrated findings from microarray analysi
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