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Analysis of Low Abundance Membrane-Associated Proteins from Rat Pancreatic Zymogen Granules

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Analysis of Low Abundance Membrane-Associated Proteins from Rat Pancreatic Zymogen Granules
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  Analysis of Low Abundance Membrane-Associated Proteins fromRat Pancreatic Zymogen Granules Heike Borta, †,‡ Miguel Aroso, †,§ Cornelia Rinn, § Maria Gomez-Lazaro, § Rui Vitorino, | Dagmar Zeuschner, ⊥ Markus Grabenbauer, # Francisco Amado, | and Michael Schrader* ,§ Department of Cell Biology and Cell Pathology, Philipps University of Marburg, Robert Koch Strasse 6,35037 Marburg, Germany, Max-Planck-Institute of Molecular Biomedicine, Ro¨ntgenstrasse 20,48149 Mu¨nster, Germany, Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Strasse 11,44227 Dortmund, Germany, Department of Chemistry, and Centre for Cell Biology and Department of Biology,University of Aveiro, Campus Universita´rio de Santiago, 3810-193 Aveiro, Portugal  Received January 20, 2010 Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow thesorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need forfurther understanding of the key processes in regulated secretion to develop new therapeutic optionsin medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a “basic” group (pH range 6.2 - 11) with about46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis - trans isomeraseB (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogengranules and not contaminants of the preparation, we conducted a series of validation experiments.Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like  bona fide   peripheralmembrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructuraland immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase - YFP fusionprotein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins,proteoglycan-binding properties have been reported. The importance of our findings for sorting andpackaging during ZG formation is discussed. Keywords:  organelle biogenesis  •   zymogen granules  •   exocrine pancreas  •   2D gel electrophoresis  •  tandem mass spectrometry  •   membrane-associated proteins Introduction Zymogen granules (ZGs) are specialized storage organelles within the acinar cells of the exocrine pancreas. Their maincargo is constituted by pancreatic digestive enzymes as inactiveprecursors which are released by regulated apical secretion,triggered by an external stimulus. ZG formation is initiated atthe  trans  -Golgi network (TGN) where the regulated secretory ZG proteins coaggregate at the mildly acidic pH and high Ca 2 + levels and condensing vacuoles/immature secretory granulesare formed. 1 - 4 Their maturation involves further concentrationof the cargo proteins with selective removal of components notdestined for regulated secretion, and a reduction in granulesize. 5 - 7 The mature ZGs are transported to the apical domainof the acinar cell, where they are stored until a neuronal orhormonal stimulus (e.g., acetylcholine and cholecystokinin)releases intracellular calcium stores, thus triggering the fusionof ZGs with the plasma membrane and with neighboring granules. Fusion results in exocytosis of digestive enzymes intothe apical lumen and the pancreatic duct system. 8,9 Undernormal conditions, the digestive enzymes are finally activatedby enterokinase via proteolytic cleavage of trypsinogen in thesmall intestine. Once in the intestinal tract, granule proteinsare also supposed to fulfill regulatory and protective functions,e. g. in host defense. 10,11  Although the ZG has long been amodel for the understanding of secretory granule biogenesisand functions, the molecular mechanisms required for ZGformation at the TGN, for packaging and sorting of cargoproteins, as well as for granule fusion and exocytosis are stillpoorly defined. 6,9,12  According to recent models, part of themolecular machinery required for digestive enzyme sorting,granule trafficking and exocytosis is supposed to be associated with the granule membrane (ZGM). In addition to basicresearch interests, ZG play important roles in pancreatic injury and disease. 13 Thus, there is currently great interest in theidentification and characterization of ZG and ZGM components * To whom correspondence should be addressed. Michael Schrader,Centre for Cell Biology & Dept. of Biology, University of Aveiro, CampusUniversita´rio de Santiago, 3810-193 Aveiro, Portugal. Tel.: + 351-234 370 200ext. 22789. Fax:  +  351-234 372 587. E-mail: mschrader@ua.pt. † These authors contributed equally to this work. ‡ Philipps University of Marburg. § Centre for Cell Biology & Department of Biology, University of Aveiro. | Department of Chemistry, University of Aveiro. ⊥ Max-Planck-Institute of Molecular Biomedicine. # Max-Planck-Institute for Molecular Physiology. 10.1021/pr100052q  ©  2010 American Chemical Society  Journal of Proteome Research  2010, 9, 4927–4939   4927 Published on Web 08/14/2010  by conducting antibody screens, raft analyses and proteomicstudies. 14 - 19 In this study, we have focused on the identification andcharacterization of a “basic” group of peripheral granulemembrane proteins from rat exocrine pancreas. A 2D-gelapproach combined with tandem mass spectrometry led to theidentification of membrane-associated proteins including clas-sical ZG content proteins, lipid binding proteins as well aspreviously unknown low abundant proteins such as chymase(RMCP-1), a serine protease previously described in mast cellgranules, and peptidyl-prolyl cis - trans isomerase B (PpiB), aknown ER-resident enzyme. We performed a series of validationexperiments and for the first time demonstrated that chymaseand PpiB are  bona fide   peripheral membrane proteins of ZG. As we identified several peripheral ZGM proteins with pro-teoglycan-binding properties, our findings may help to unveilthe possible role of proteoglycans in the sorting and packaging of zymogens. Experimental Section  Antibodies and cDNA.  Antibodies were used as follows:rabbit polyclonal antibodies to carboxypeptidase A (RocklandImmunochemicals, Gilbertsville, PA), cyclophilin B (PpiB)(Abcam, Cambridge, U.K.), RNase A (Sigma-Aldrich, St. Louis,MO), PDI (kindly provided by H. D. So¨ling, MPI for Biophysi-ological Chemistry, Go¨ttingen, Germany), Calnexin (Stressgen, Ann Arbor, MI) and ZG16p, 20 mouse monoclonal antibodiesdirected to GP2 (kindly provided by A. Lowe, Stanford Univer-sity School of Medicine, Palo Alto, CA), Myc epitope 9E10 (SantaCruz Biotechnology, USA), p115 (BD Transduction Laborato-ries), BiP (BD Diagnostics, NJ), R  -amylase and R  -tubulin DM1 R  (Sigma, St. Louis, MO). A sheep polyclonal antibody to RMCP-1/chymase was kindly provided by H. R. Miller, University of Edinburgh, U.K.; goat polyclonal antibody to RMCP-1/chymase was kindly provided by L. B. Schwartz, Commonwealth Uni-versity, Richmond, VA; goat anti-mouse-tryptase   - 1 wasobtained from R&D systems (Minneapolis, MN). A polyclonalantibody to recombinant carboxyl ester lipase from rat (CEL) was raised in chicken (Eurogentec, Belgium) and isolated fromegg yolk as described. 21 Species-specific IgG antibodies con- jugated to HRP were obtained from BioRad (Richmond, CA),Molecular Probes Europe (Leiden, The Netherlands) and Sigma- Aldrich (St. Louis, MO). Species-specific anti-IgG antibodiesconjugated to the fluorophores TRITC and Alexa 488 wereobtained from Jackson ImmunoResearch (West Grove, PA), andInvitrogen (Carlsbad, CA). Concanavalin A conjugated withTetramethylrhodamine (TRITC) was obtained from MolecularProbes Europe (Leiden, The Netherlands).The following primer sequences were used to amplify thecoding sequence of rat chymase (NM_017145.1) from a ratpancreas cDNA library (Clontech, Heidelberg, Germany): 5 ′ -TTG GAT CCA TGC AGG CCC TAC TAT TCC-3 ′  (forward) and5 ′ -TTG AAT TCC TAG CTT GGA GAC TCT GAC-3 ′  (reverse).Using the restriction sites for  Bam  HI and  Eco  RI at the ends of the PCR products (underlined), the cDNA was cloned in frameinto the pcDNA3 vector (Invitrogen). Fusion of YFP to theC-terminus of chymase was achieved by insertion into pEYFP-N1 vector (Clontech, Saint-Germain-en-Laye, France) using therestriction sites for  Eco  RI and  Bam  HI after amplification withthe primer sequences 5 ′ -TTG AAT TCC CAT GCA GGC CCT ACT ATT CC-3 ′  (forward) and 5 ′ -TTG GAT CCC CGC TTG GAG ACTCTG ACT CG-3 ′  (reverse). In frame insertion of all constructs was verified by sequencing (MWG). Plasmid YFP-ER was kindly provided by R. Jacob (University of Marburg, Germany) andplasmid GFP-Sec61    was a kind gift from W. A. Prinz (NationalInstitute of Diabetes and Digestive and Kidney Diseases, NIH,Bethesda, MD). Isolation of Zymogen Granules.  ZGs were isolated from thepancreas of male Wistar rats (200 - 230 g) (Charles River,Sulzfeld, Germany) which were fasted overnight. Animals werehandled according to the German law for the protection of animals, with the permit of the local authorities. Tissuehomogenization was performed in the following buffer: 0.25M sucrose, 5 mM 2- N  -morpholino-ethanesulfonic acid (MES),pH 6.25, 0.1 mM MgSO 4 , 10  µ M Foy 305 (Sanol Schwarz), 2.5mM Trasylol (Bayer, Leverkusen, Germany), and 0.1 mMphenylmethylsulfonyl fluoride (PMSF). 4 Pancreata were ho-mogenized on ice using a brendle type homogenizer (Yellow line, OST20 Digital). The homogenate was centrifuged for 10min, 4  ° C at 500 × g  , and the resulting post nuclear supernatant was further centrifuged for 10 min, 4  ° C at 2,000 × g   to sedimentZG. The brownish layer of mitochondria on top of the pellet was removed. The white zymogen granules were collected inhomogenization buffer and centrifuged for 20 min, 4  ° C at2000 ×  g  . Tissue homogenization was repeated 2 - 3 times.Purified granules were resuspended in 50 mM Hepes, pH 8.0,carefully lysed by freezing and thawing and separated intozymogen granule content (ZGC) and membrane (ZGM) frac-tions by centrifugation at 100 000 × g   for 30 min in a swinging-bucket rotor (Beckman SW50.1). The membranes were rinsedand resuspended in Hepes buffer and treated with 150 mMNa 2 CO 3 , pH 11.5 for 2 h on ice. Treated membranes (ZGM carb ) were recovered by centrifugation at 100 000 × g   for 30 min andthereby separated from peripheral membrane proteins (wash). Alternatively, purified ZG were resuspended in 50 mM Hepes,pH 8.0, 80 mM KCl, carefully lysed by freezing and thawing and centrifuged through a 0.3/1 M sucrose step gradient for1 h, 4  ° C at 200 000 ×  g   (Beckman 80 Ti rotor). ZGM wererecovered at the interface and washed twice in 50 mM Hepes,pH 8.0 or in 100 mM NaHCO 3 , pH 8.1. After each washing step,ZGM were recovered by centrifugation for 1 h, 4  ° C at 150 000 × g   (Beckman 80 Ti rotor). Finally, membranes were rinsed andresuspended in 50 mM Hepes, pH 8.0. A microsome-enrichedfraction was obtained from a pancreas homogenate aftersubcellular fractionation and ultracentrifugation (100 000 ×  g  for 1 h, 4  ° C; Beckman SW50.1). A postnuclear supernatant of rat tongue homogenate was obtained by centrifugation at 500 × g  , 4  ° C for 10 min. Protein concentrations were determinedusing the Bradford assay. Assays were run with a recording spectrophotometer (Ultraspec 100 pro, Amersham Biosciences,Uppsala, Sweden). 1D and 2D-Gel Electrophoresis and Immunoblotting.  Pro-tein samples were separated by SDS-PAGE on 12.5% polyacryl-amide mini gels, transferred to nitrocellulose (Schleicher andSchu¨ll, Dassel, Germany) via a semidry apparatus (Trans-BlotSD, Biorad) and analyzed by immunoblotting using horseradishperoxidase-conjugated secondary antibodies and enhancedchemiluminescence reagents (Amersham Bioscience, ArlingtonHeights, IL). For quantification, immunoblots were scannedand processed using “Gel Pro Analyzer” software.The first dimension of 2D-gel electrophoresis was performedin a horizontal apparatus (Ettan IPGphor, GE Healthcare, SanFrancisco, USA). Granule subfractions were precipitated with20% TCA (ratio 1:1). Protein samples (300  µ g) were solubilizedfor 30 min at 30  ° C in rehydration buffer. 22 The samples werethen applied onto IPG strips (11 cm, pH 3 - 11) and isoelectric research articles  Borta et al. 4928 Journal of Proteome Research  •  Vol. 9, No. 10, 2010  focusing was conducted at 20  ° C with 50  µ  A, for a minimumof 10 h at 50 V, 1 h at 500 V, 1 h at 1000 V and 110 min at 8000 V. The strips were afterward incubated for 15 min in equilibra-tion buffer containing 6 M urea, 75 mM Tris-HCL pH 8.8, 34.5%Glycerol (87%), 2% SDS, 0.002% bromophenol blue and 1.5 mMDTT and then applied on top of a SDS-PAGE gel (14 cm by 14cm, 15%). Proteins were separated according to molecular weight in a Hoefer 600 SE RUBY chamber (GE Healthcare).Silver staining of gels was performed according to. 23 For trypticdigestion the SDS-PAGE gels were stained using colloidalCoomassie blue and silver staining. TrypticDigestionandMassSpectrometry. The protein spots were excised from the gels and washed three times with 25 mMammonium bicarbonate/50% acetonitrile (ACN), one time with ACN and dried. Twenty-five  µ L of 10  µ g/mL sequence grademodified porcine trypsin (Promega, Madison, WI) in 25 mMammonium bicarbonate were added to the dried gel pieces andthe samples were incubated overnight at 37  ° C. Extraction of tryptic peptides was performed by adding 10% formic acid(FA)/50% ACN (3 × ) being lyophilized in a SpeedVac (ThermoFisher Scientific, Asheville, NC). Tryptic peptides were resus-pended in 13  µ L of a 50% acetonitrile/0.1% formic acid solution. Aliquots of samples (0.5  µ L) were spotted onto the MALDIsample target plate and mixed (1:1) with a matrix consisting of a saturated solution of  R  -cyano-4-hydroxycinnamic acid (5mg/mL) prepared in 50% acetonitrile/0.1% formic acid. Peptidemass spectra were obtained on a MALDI-TOF/TOF massspectrometer (4800 Proteomics Analyzer, Applied Biosystems,Europe) in the positive ion reflector mode. Spectra wereobtained in the mass range between 800 and 4500 Da with ca.1500 laser shots. For each sample spot, a data dependentacquisition method was created to select the six most intensepeaks, excluding those from the matrix, trypsin autolysis, oracrylamide peaks, for subsequent MS/MS data acquisition.Trypsin autolysis peaks were used for internal calibration of the mass spectra, allowing a routine mass accuracy of betterthan 20 ppm. Spectra were processed and analyzed by theGlobal Protein Server Workstation (Applied Biosystems, FosterCity, CA), which uses internal Mascot (Matrix Science Inc.,Boston, MA) software for searching the peptide mass finger-prints and MS/MS data. Searches were performed against theNCBI nonredundant protein database. Cell Culture and Transfection Experiments.  AR42J cells(ATCC no.CRL-1492) were cultured as described. 19 Briefly, cells were maintained in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal calf serum and penicillin (100U/mL)/streptomycin (100  µ g/mL) (PAA Laboratories GmbH,Linz, Austria) at 37  ° C in a 5% CO 2 -humidified incubator. Toimprove cell adherence, the culture dishes were coated withan extract of Engelbreth-Holm-Swarm tumor. 24 To inducedifferentiation and zymogen granule formation, cells wereincubated with 10 nM dexamethasone for 2 - 3 days. Cells weretransfected with DNA constructs by electroporation. 19 Briefly,cells grown to 90% confluency were harvested by trypsination,resuspended in 0.5 mL complete cell culture medium andtransferred to a sterile 0.4 cm gap electroporation cuvettecontaining 10  µ g of DNA. Electroporation was performed withan ECM 630 Electro Cell Manipulator (BTX Harvard Apparatus,Holliston, MA) at 250 V, 1500  µ F and 125  Ω . After electropo-ration, cells were immediately resuspended in complete me-dium, plated on coverslips, and dexamethasone was added 24 hlater. To knock down the expression of rat PpiB (Acc. No.NM_022536) by RNA interference, predesigned 21-nucleotidesmall interfering RNAs (siRNA) (sense strands: 5 ′ -GCAAGUUC-CAUCGUGUCAUtt-3 ′ ; 5 ′ -GGAUGUGAUCAUUGUAGACtt-3 ′ ; 5 ′ -CGAUAAGAAGAAGGGACCUtt-3 ′ ) (Ambion, Austin, TX) weretransfected into the cells using electroporation. As a controlcells were transfected with siRNA duplexes targeting luciferase(Dharmacon, Lafayette, CO). Dexamethasone (10 nM) wasadded 24 h after transfection and cells were assayed forsilencing and organelle morphology after 2 - 3 days. Immunofluorescence and Microscopy.  Cryostat sections of rat pancreas and AR42J cells grown on glass coverslips werefixed with 4% paraformaldehyde in PBS, pH 7.4. The samples were permeabilized with 0.2% Triton X-100, blocked with 1%BSA and incubated with primary and secondary antibodies asdescribed. 25 Samples were examined using an Olympus BX-61microscope (Olympus Optical Co. GmbH, Hamburg, Germany)equipped with the appropriate filter combinations and a 100 × objective (Olympus Plan-Neofluar; numerical aperture, 1.35).Fluorescence images were acquired with an F-view II CCDcamera (Soft Imaging System GmbH, Mu¨nster, Germany)driven by Soft imaging software. Confocal images were acquiredon a Zeiss LSM 510 confocal microscope (Carl Zeiss, Oberkochen,Germany) using a Plan-Apochromat 63 ×  or 100  ×  /1.4 oilobjective. Images were processed and quantified using LSM-510 software (Carl Zeiss MicroImaging, Inc.). Background noise was minimal when optimal gain/offset settings for the detectors were used. Digital images were optimized for contrast andbrightness using Adobe Photoshop (Adobe Systems, San Jose,CA). For quantification of granule formation and morphology in AR42J cells, usually 3 - 5 slides per preparation were analyzed,and 3 - 5 independent experiments were performed. Immunoelectron Microscopy.  Tissue from rat pancreas andrat tongue was fixed in 0.1 M cacodylate buffer, pH 7.35containing 2% paraformaldehyde and 0.1% glutaraldehyde(Serva, Heidelberg, Germany). The samples were dehydratedin a graded series of alcohol, embedded in Lowicryl K4M(Polysciences Ltd., Eppenheim, Germany) and polymerized at - 20  ° C and UV light (360 nm) for 48 - 72 h. Thin sections (70nm) were incubated with polyclonal antibodies directed tochymase (1:200 - 500) and visualized using a 10 nm Protein A-Gold solution (J. Slot, University of Utrecht, The Netherlands)at a dilution of 1:60 or 1:70, both in 0.5% BSA in PBS. Sections were stained with uranyl acetate/lead citrate and analyzedusing a Zeiss EM 109 electron microscope (Carl Zeiss,Oberkochen, Germany). The labeling density (gold particles/  µ m 2 ) on zymogen granules and control regions was determinedmanually on images with the same magnification. For cryo-sectioning and PpiB-labeling, rat exocrine pancreas got initially fixed in 2% paraformaldehyde, 0.1% glutaraldehyde, 2% sucrosein 0.1 M phosphate buffer, pH 7.4. Samples were washedextensively in 0.1 M phosphate buffer, pH 7.4 and infiltratedin 2.3 M sucrose for cryoprotection. Ultrathin-cryosectioning and immunogold-labeling was performed as described. 26 ForHM20 embedding, fixed samples were dehydrated by theprogressive lowering of temperature (PLT) method, 27 polym-erized at - 35  ° C with subsequent immunolabeling of ultrathinsections at room temperature. Samples were analyzed at 80kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven,Netherlands) or at 120 kV on a JEM-1400 (JEOL Germany,Eching, Germany) and selected areas were documented withimaging plates (Ditabis, Pforzheim, Germany) or via CCDcamera (F-214, TVIPS, Gauting, Germany). Isolation of RNA, Reverse Transcription, and PCR.  RT-PCR was performed to amplify parts of the coding sequence of rat Peripheral Membrane Proteins of Zymogen Granules   research articles Journal of Proteome Research  •  Vol. 9, No. 10, 2010  4929  chymase (NM_017145.1), tryptase   1 (NM_019322), amylase(NM_031502), PpiB (BC061971) and GAPDH (BC087069) frommRNA isolated from pancreas of fasted or Foy-treated rats, andfrom tongue tissue. Foy 305 (Sanol Schwarz, Monheim, Ger-many) is a low-molecular-weight serine protease inhibitor. 28 Total RNA was isolated using the RNeasy Protect Mini Kit(Qiagen, Hilden, Germany), and reverse transcribed using oligo(dT) primer and M-MuLV reverse transcriptase (Stratagene Amsterdam, The Netherlands) at 42  ° C. PCRwasperformedwith100 ng of template using the following primer pairs: 5 ′ -GTT TCTTGT GAC CCG CCA ATT-3 ′  (forward chymase); 5 ′ -TTA ATC CAGGGC ACA TAT GGG-3 ′  (reverse chymase); 5 ′ -GAA TAA GGC TGA CCCCAACA-3 ′ (forwardtryptase   1);5 ′ -CTTGGGGACATAGCGGTA GA-3 ′  (reverse tryptase   1); 5 ′ - GCC TTC TGG ATC TTG CACTC-3 ′  (forward amylase); 5 ′ -AGT GCT TGA CAA AGC CCA GT-3 ′ (reverseamylase);5 ′ -TCCGTTGTCTTCCTTTTGCT-3 ′ (forwardPpiB); 5 ′ -GTT CTC CAC CTT CCG TAC CA-3 ′  (reverse PpiB); 5 ′ - ACG ACC CCT TCA TTG ACC-3 ′  (forward GAPDH); 5 ′ -CCA GTG AGC TTC CCG TTC AGC-3 ′  (reverse GAPDH). GAPDH was usedas a loading control. Samples were analyzed by 1% agarose gelelectrophoresis. Results Identification of Peripheral Membrane Proteins of Zymogen Granules.  To understand the biogenesis and functionof pancreatic zymogen granules as well as their role in diseasea profound analysis of their membrane and content compo-nents is required. We have special interest in the analysis of membrane-associated ZG proteins, and s together with our co- workers s have contributed to the further understanding of thecomposition and architecture of the ZGM. 16,17,19,29 - 32 In thisstudy, we have applied a suborganellar proteomics approachto identify peripheral membrane-associated ZG proteins (Figure1, Supplementary Figure 1, Supporting Information). Zymogengranules were isolated from rat pancreas according to astandardized protocol. 4,16,19 The purity of the isolated ZGfractions ( g 90%) was controlled by electron microscopy andimmunoblotting as shown recently. 19 Intact granules weregently lysed and further separated in a membrane (ZGM) andcontent fraction (ZGC). The isolated membranes (ZGM) weretreated with carbonate to liberate membrane-associated pro-teins and separated in a pellet (ZGM carb ) and supernatantfraction (wash) (Figures 2, 3). The distribution of distinctgranule marker proteins (e.g., amylase, GP2, ZG16p) among thegranule subfractions was controlled by immunoblotting (Figure3). Usually, a contamination with ER or mitochondrial markerproteins was not detected in the ZG subfractions (see Figure 3and ref 19). To characterize the ZG peripheral membrane-associated proteins, 2D gel maps were generated for thesupernatant fraction (wash) of Na 2 CO 3 -washed ZGM. Forcomparison typical 2D gel patterns of the wash and the contentsubfractions (ZGC) separated under equal conditions are shown Figure 1.  Separation of rat ZG content and ZGM wash subfractions by two-dimensional IEF/SDS-PAGE. The whole complement of ZGC(A) and the supernatant fraction (wash) (B) of carbonate-treated ZGM were subjected to 2D-PAGE followed by Coomassie staining. ForIEF, 300  µ g of protein were separated on 11 cm IPG strips (pH 3 - 11NL) and on 15% polyacrylamide gels in the 2nd dimension. Notethe differences in the spot pattern of ZGC (A) and the wash fraction (B). The boxed areas in (B, C) highlight basic protein spots (chymase,PpiB, RNase A) which have been selected for further analysis and are verified by immunoblotting (C) using specific antibodies to ratmast cell chymase, PpiB, and RNase A. Figure 2.  (A) Proteins identified from the “basic group” of a ZGMcarbonate wash fraction. Functional annotation and organelleassignments were made using the UniprotKB database, ad-ditional annotation was incorporated from literature search. Extrainformation supporting the identification of the potential ZGMproteins is summarized in Supplementary Table 1 (SupportingInformation). (B) Diagram illustrating the intracellular distributionof the identified proteins of the wash fraction. On the basis of published data, annotations in databases or predictions basedon similarity to related proteins, the identified proteins aregrouped in a pie chart according to their subcellular distributionand function. research articles  Borta et al. 4930 Journal of Proteome Research  •  Vol. 9, No. 10, 2010  in Figure 1. Note that the representative gels exhibit uniquespot patterns for the two different subfractions. In a represen-tative gel for the wash fraction about 104 spots were repro-ducibly visualized, which represent an acidic and more basicgroup of ZG protein spots (Supplementary Figure 1, Supporting Information). The identification of the “acidic” group, whichappears to contain several proteins from the cytosolic side of the ZG membrane is currently under investigation. In thepresent study, we focused on the analysis of the spots of the“basic” group (pH range 6.2 - 11), which appears to be com-posed mainly of luminal ZG proteins. Two previously identifiednovel peripheral ZGM proteins, ZG16p and syncollin, are as well proteins with a p I   of 9.17 and 8.62, respectively. 20,29,32,33 In this region, 46 spots were visualized among which 44 wereidentified by tandem mass spectrometry (Supplementary Figure 1, Supplementary Table 1, Supporting Information).These spots corresponded to 16 unique proteins (Figure 2A), which were categorized in 6 groups based on their knownsubcellular localization: ZGC ( n   )  6; 38%) and ZGM ( n   )  6;38%) proteins, mast cell proteins ( n   )  1; 6%), ER residentproteins ( n   )  1; 6%), and proteins with other localizations ( n  )  2; 12%) (Figure 2B). Based on their predicted biologicalfunctions we identified 12 enzymes including 9 digestiveenzymes (usually attributed to the ZGC), 2 matrix (ZG16p,syncollin) and 3 slightly acidic glycoproteins (CEL, pancreaticlipase related protein 1 and 2). Based on literature and the Figure 3.  Chymase and PpiB represent peripheral membrane proteins of rat ZG. (A) Lysed granules were separated into a content(ZGC) and membrane fraction (ZGM). In addition, isolated membranes were treated with Na 2 CO 3  at pH 11.5 and separated into pellet(ZGM carb ) and supernatant (Wash) fractions. Equal amounts of protein (20  µ g) were run on 12.5% acrylamide gels, blotted ontonitrocellulose membranes and incubated with antibodies to amylase (ZGC marker protein), GP2 (ZGM marker protein), ZG16p (peripheralZGM marker protein), chymase, tryptase   1 (mast cell control), carboxyl ester lipase (CEL), RNase A, BiP, PDI and calnexin (ER control)and PpiB. (B) Densitometric quantification of immunoblots shown in (A). The distribution of the labeling density to ZGC and ZGM (%labeling density of total ZGC + ZGM) as well as the distribution of the labeling density to ZGM carb  and wash (% labeling density of totalZGM) is depicted (see also Supplementary Table 2, Supporting Information). Note that the overall distribution of chymase and PpiBresembles that of ZG16p, a peripheral ZGM marker protein. A lysate from rat tongue and a microsome-enriched fraction (Micros)served as controls for the detection of mast cell proteins and ER resident proteins, respectively. Antibodies to BiP, PDI, and PpiB havebeen incubated successively on the same immunoblot. Peripheral Membrane Proteins of Zymogen Granules   research articles Journal of Proteome Research  •  Vol. 9, No. 10, 2010  4931
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