Báo cáo khoa học: A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin pot

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A novel 2D-based approach to the discovery of candidatesubstrates for the metalloendopeptidase meprinDaniel Ambort1, Daniel Stalder2, Daniel Lottaz1, Maya Huguenin1, Beatrice Oneda1, Manfred Heller2and Erwin E. Sterchi11 Institute of Biochemistry and Molecular Medicine, University of Berne, Switzerland2 Department of Clinical Research, University Hospital, Berne, SwitzerlandThe astacin-like zinc-dependent metalloendopepti-dase human meprin (hmeprin) (EC 3.4.24.18) wasfirst discovered in 1982 for its ability to hydrolyzeN-benzoyl-l-tyrosyl-p-aminobenzoic acid, a chymo-trypsin substrate used for assessing exocrine pancreasfunction [1]. N-benzoyl-l-tyrosyl-p-aminobenzoic acidhydrolase (PPH) was subsequently purified and charac-terized from human small intestinal mucosa [2]. At thesame time, PPH orthologs, called meprin (metal endo-peptidase from renal tissue) or endopeptidase-2, werefound in mouse and rat kidney, respectively [3,4]. Twosimilar subunits, termed meprina and meprinb, withmolecular masses of 95 and 105 kDa, respectively,were identified. Human meprin cDNA was expressedin Madin–Darby canine kidney (MDCK) cells, a well-established cell system for polarized epithelial cells. Todate, no such thoroughly characterized model systemexists for human epithelial cells. Hmeprina is secretedinto the culture medium of MDCK cells as inactivehomodimers, whereas hmeprinb is primarily mem-brane-bound [5]. Hence, heterodimers of hmeprina ⁄ ballowed for localization of the a-subunit to the plasmamembrane [6]. Inactive zymogens of hmeprina and bare processed by limited proteolysis with trypsin intotheir active forms [5,6]. Hmeprina, but not b, mayalternatively be activated by plasmin [7,8].A first step towards the elucidation of the biologicalfunction of meprin was achieved by testing putativelycleavable polypeptide substrates. A variety of pro-tein and peptide substrates were processed in vitro;Keywordsastacin family; image analysis; Madin–Darbycanine kidney cells; meprin; proteaseproteomicsCorrespondenceE. E. Sterchi, Institute of Biochemistry andMolecular Medicine, University of Berne,Bu¨hlstrasse 28, CH-3012 Berne, SwitzerlandFax: +41 31 631 3737Tel: +41 31 631 4199E-mail: erwin.sterchi@mci.unibe.ch(Received 14 April 2008, revised 8 July2008, accepted 10 July 2008)doi:10.1111/j.1742-4658.2008.06592.xIn the past, protease-substrate finding proved to be rather haphazard andwas executed by in vitro cleavage assays using singly selected targets. In thepresent study, we report the first protease proteomic approach applied tomeprin, an astacin-like metalloendopeptidase, to determine physiologicalsubstrates in a cell-based system of Madin–Darby canine kidney epithelialcells. A simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure wasdesigned to find candidate substrates in conditioned media of Madin–Darby canine kidney cells expressing meprin in zymogen or in active form.The method enabled the discovery of hitherto unkown meprin substrateswith shortened (non-trypsin-generated) N- and C-terminally truncatedcleavage products in peptide fragments upon LC-MS ⁄ MS analysis. Of 22(17 nonredundant) candidate substrates identified, the proteolytic process-ing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysedby means of immunoblotting validation experiments. The classification ofsubstrates into functional groups may propose new functions for meprinsin the regulation of cell homeostasis and the extracellular environment, andin innate immunity, respectively.AbbreviationsADAM, a disintegrin and metalloprotease; BMP-1, bone morphogenetic protein 1; CID, collision-induced dissociation; ECM, extracellularmatrix; hmeprin, human meprin (EC 3.4.24.18); ICAT, isotope-coded affinity tag; MDCK, Madin–Darby canine kidney; MMP, matrixmetalloproteinase; PPH, N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase; TLD, tolloid.4490 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBSbiologically active peptides [2,9], as well as gastrointes-tinal peptides and extracellular matrix (ECM) com-ponents, such as collagen type IV, fibronectin andlaminin-nidogen [10–12]. These findings suggest thatmeprin may be involved in processes such as renal clear-ance of vasoactive peptides from blood plasma, regula-tion of cell movement, secretory activity and growth ofintestinal tract, and tissue remodelling. In addition,marked differences between a- and b-subunits in sub-strate and peptide bond specificity point to distinct func-tions for the two forms [10]. Meprina selects for small(e.g. serine, alanine and threonine) or hydrophobic (e.g.phenylalanine) residues in the P1 and P1¢ sites and pro-line in the P2¢ position. Meprinb prefers acidic aminoacids in the P1 and P1¢ sites and selects against basic res-idues at P2¢ and P3¢. In conclusion, protease-substratediscovery executed by these in vitro cleavage assays wasrather haphazard. Thus, meprin and its substrate reper-toire may be studied in a complex biological context toidentify physiologically relevant substrates.The introduction of protease proteomics enabledidentification of protease and protease-substrate reper-toires on an organism-wide scale by means of proteomictechniques [13]. Using different cell-based systems [14–16] a variety of hitherto unkown substrates were foundin conditioned media for the metzincin metalloendopep-tidases, a disintegrin and metalloprotease (ADAM)-17and matrix metalloproteinase (MMP)-14. Humanplasma was also used to identify substrates for recombi-nant MMP-14 in a cell-free system [17]. Two methodo-logical platforms were successfully applied for proteinseparation: LC-MS ⁄ MS and 2D IEF ⁄ SDS ⁄ PAGE[14–17]. These standard techniques were used in com-bination with lectin-affinity pre-fractionation andquantitative tags such as isotope-coded affinity tags(ICAT) or cyanine dyes for differential in-gel electro-phoresis. From these protease proteomic studies, itbecame obvious that metalloendopeptidases are keymodulators of diverse signalling pathways and notmerely ECM degrading entities [18]. For example, themajor role of the MMP family is the control of cellularresponses critical to homeostatic regulation of the extra-cellular environment and the immune response [19,20].We decided to apply protease proteomics to identifynovel physiologic substrates for meprin, aiming toelucidate its key functions at the cellular level. Forthe above described techniques, some conceptual prob-lems may arise: first, ICAT-based approaches comparepairs of peptides, and therefore it is not possible todiscover cleaved protein fragments with shortened(non-trypsin-generated) N- or C-termini; second,nonglycosylated proteins and fragments escape fromlectin-affinity purification. We thus designed a simple2D IEF ⁄ SDS ⁄ PAGE-based protease proteomic appro-ach that remedied these limitations and circumventedcomplicated quantitative and statistical evaluation.Hmeprina ⁄ b was transfected into MDCK cells andactivated in situ by limited trypsin treatment at conflu-ent cell stage. Conditioned media of meprin activatedand non-activated cells were concentrated with ultrafil-tration and then separated by 2D IEF⁄ SDS ⁄ PAGE. Asimple 2D IEF⁄ SDS ⁄ PAGE-based image analysis pro-cedure allowed for detection of protein spots unique to2D gels produced from conditioned media of meprinactivated cells. LC-MS ⁄ MS analysis of candidatesubstrates confirmed the validity of this protease prote-omic approach for the discovery of shortened (non-trypsin-generated) N- and C-terminally truncatedcleavage products in peptide fragments.ResultsDesign and application of a simple 2D IEF/SDS/PAGE-based protease proteomic approachin substrate findingTraditionally, 2D IEF ⁄ SDS ⁄ PAGE-based image analy-sis is performed on two sets of gels and protein spotsare matched to the same reference gel within one singleanalysis. Statistical tools are then applied to quanti-tatively assess subtle but significant changes in peakvolumes to find up- or down-regulated protein spots.Unfortunately, error-prone matching to wrong refer-ence spots is often underestimated, making quantita-tive statistical information useless. Hence, annotationsof interesting candidate spots to wrong spots in thereference gel leads to misinterpretation of the data setand protein spots unique to only one specific conditionare then not properly displayed in the correspondingreference gel. A remedy to false-positive data interpre-tation is the stepwise reduction in complexity of suchan analysis. Therefore, we designed a simple imageanalysis procedure in which digitized 2D gels were cutinto four parts or quadrant sections. This procedureenabled the performance of four independent imageanalyses in which the gel parts of each correspondingquadrant were used to construct four independent ref-erence gels instead of one. The corresponding quadrantsections were grouped into sets of gels termed level 1match-sets for each condition (activated meprin versusnon-activated meprin) and then into supersets of level1 match-sets (higher-level match-sets) (Fig. 1). Thefour level 1 match-sets are the reference gels of therespective quadrants from the 2D gel sections of eachcondition and the four higher-level match-sets are thereference gels of the two different conditions (activatedD. Ambort et al. Meprin protease proteomicsFEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4491meprin versus non-activated meprin). This procedureallowed for subsequent matching of protein spots firstto reference gels of the same condition and thereafterto reference gels common to both conditions. The step-wise annotation of protein spots to two independentlevels of reference gels allowed for detection of uniquespots in the final higher-level match-sets (Fig. 2). Thesedifferential spots were unique to one specific conditionand absent in the other or vice versa. Applying theabove procedure to conditioned media of MDCKa ⁄ bcells revealed that, among 817 protein spots displayed,35 were unique to media of cells expressing activatedmeprina ⁄ b and 40 to media of cells with non-activatedmeprina ⁄ b (Table 1). These unique protein spots weretherefore absent in the corresponding other condition.Thus, unique spots were indicative of proteins releasedinto or proteolytically cleaved in the extracellularmilieu by hmeprina ⁄ b. We then hypothesized that,upon LC-MS ⁄ MS analysis of candidate substrates, itmay be feasible to find shortened (non-trypsin-gener-ated) N- and C-termini in peptide fragments. Suchpotential N- or C-terminally truncated cleavage prod-ucts can be identified in protein spots unique to condi-tioned media of trypsin activated MDCKa ⁄ b cells, asshown below.Fig. 1. Simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure.The procedure is based on qualitative differences among referencegels (level 1 match-sets) of each group of five gel replicates (threepooled biological gel replicates and two more technical gel repli-cates). Gel replicates of each group (activated meprin versusnon-activated meprin) were cut virtually into four equally spacedquadrants for four independent image analyses. Reference gels ofeach group were then clustered into a new set for higher-level imageanalysis. The spot matching features ofPDQUEST (version 7.3.1)allowed for detection of unique protein spots. The combined higher-level match-set is the final fusion of all annotated unique spots intoone big 2D reference map.Fig. 2. Application of a simple 2D IEF ⁄ SDS ⁄ PAGE-based protease proteomic approach in substrate finding. A representative image analysisof the first quadrant is shown. Two hundred and fifty micrograms of conditioned medium protein from trypsin activated and non-activatedMDCKa ⁄ b cells was separated by IEF in a 24 cm long IPG pH 3–10 NL strip. Vertical separation was according to mass in a 12.5% SDS gel.Optimized Ruthenium staining: for each condition (activated meprin versus non-activated meprin), three pooled biological gel replicates (from18 dishes per pooled sample) and two more technical gel replicates (of one pooled sample) were produced for subsequent image analysis.Unique protein spots are labelled in level 1 and higher-level match-sets with SSP assigned by the image analysis software.Meprin protease proteomics D. Ambort et al.4492 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBSProtein identification by means of LC-MS/MS,PHENYX-based and BLASTP-based protein databasesearchingBy visual inspection, the 35 protein spots unique tomedia of trypsin activated MDCKa ⁄ b cells could bereduced to 33 putative candidates. The redundancy oftwo spots present in more than one quadrant fromeach set of 2D gels analysed prompted correction(Fig. 2; see Fig. S1). On colloidal Coomassie stainedpreparative 2D gels, 24 protein spots of interest weredetectable. These spots could be rematched to putativecandidates found in fluorescence stained analyticalgels (data not shown). Gel plugs were then prepared,in-gel digested with trypsin and peptides thereofseparated ⁄ fragmented by LC-MS ⁄ MS. Collision-induced dissociation (CID) spectra interpretation withphenyx (version 2.1) against the uniprot-SwissProtprotein database (release 48.8) led to 22 (17 nonredun-dant) protein identifications (Fig. 3 and Table 2). Thetaxonomic search space was restricted to Mammalia(40 084 sequence entries). To double-check significanthits, the same spectra were interpreted with the web-based search engine mascot (version 2.1) against thesame database and parameter settings (data notshown) [21]. The identification of nucleophosmin (pro-tein spot SSP 2102; Table 2) was accepted because thepeptide VDNDENEHQLSR and its in-source pro-duced fragment DNDENEHQLSLR were unambigu-ously identified with good scores by phenyx andmascot. In addition, the whole tryptic peptideMSVQPTVSLGGFEITPPVVLR was identified byphenyx and mascot as first ranking identification, butwith scores below the chosen acceptance criteria(Table 2 and data not shown). Beside six positive hitsfor dog, other species (e.g. rat, human, rabbit andmouse) were predominantly represented. The currentrelease (51.3) of the uniprot-SwissProt protein data-base lists 664 sequence entries for dog and thus mayexplain the poor representation in this species.Recently, the dog genome was sequenced to comple-tion [22]. Peptide sequence tags deciphered from ourprevious analysis permitted search with blastp (version2.2.16) against the 33 527 dog RefSeq protein sequenceentries of the NCBI [21]. All top scoring significanthits corresponded to predicted dog protein sequenceentries. Finally, all equivocal uniprot-SwissProt proteindatabase searches were successfully matched to pre-dicted dog protein orthologs (Table 3).Discovery of shortened (non-trypsin-generated)N- and C-terminally truncated cleavage productsin peptide fragmentsphenyx offers the remarkable feature to search fornon-tryptic peptides (i.e. half-cleaved peptides). In-geltryptic digestion of proteins contained within gel plugsproduces peptide fragments terminating C-terminallywith a lysine or arginine residue. Trypsin cleavagespecificity is then fixed to the N- or C-terminus.Table 1. Protein spot matching statistics. 2D IEF ⁄ SDS ⁄ PAGE-based image analysis was performed with PDQUEST (version 7.3.1) on five gelreplicates (three biological replicates, two technical replicates) of conditioned media from trypsin activated and non-activated MDCKa ⁄ b cells.Qualitative spot matching differences among reference gels (level 1 match-sets) are expressed as unique spots (% of each correspondingquadrant section).Condition QuadrantGel replicates Reference gelUniquespots (%)Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5Level 1match-setHigher-levelmatch-setActivated meprin 1 315 313 318 315 316 318 2 (0.6)Non-activated meprin 1 334 333 334 332 332 334 18 (5.4)In total 1 336Activated meprin 2 221 219 221 215 218 222 10 (4.4)Non-activated meprin 2 217 212 218 216 217 218 6 (2.6)In total 2 228Activated meprin 3 106 103 116 115 117 122 12 (9.2)Non-activated meprin 3 107 110 113 103 104 119 9 (6.9)In total 3 131Activated meprin 4 105 107 115 108 105 115 11 (9.0)Non-activated meprin 4 110 108 109 102 104 111 7 (5.7)In total 4 122Activated meprin All 777 35 (4.3)Non-activated meprin All 782 40 (4.9)In total All 817D. Ambort et al. Meprin protease proteomicsFEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4493In silico digestion of the theoretical full-length proteinproduct with trypsin enables the determination of alltryptic peptides terminating with a lysine or arginineresidue. Hence, peptide fragments not featuring alysine or arginine residue in the C-terminal ends ortruncated in the N-termini by some amino acids rela-tive to the preceding in silico-generated tryptic frag-ments are candidates for proteolytically processed(non-trypsin-derived) cleavage products. In a proteaseproteomic approach, this option facilitates the discov-ery of shortened (non-trypsin-generated) N- or C-ter-minally truncated cleavage products defined by meprinprotease activity. To determine new peptide ends otherthan lysine or arginine, peptides must not be identifiedeither C- or N-terminal to the truncated peptide. Weapplied this strategy to all protein database searchesperformed with phenyx. Several shortened half-cleavedpeptides (not full-length tryptic peptides) were detected(Table 2). Half-cleaved peptides may also originatefrom in-source fragmentation of intact tryptic peptidesduring the ionization process. Accordingly, the follow-ing half-cleaved peptides co-eluted with correspondingintact tryptic peptides after chromatographic separa-tion: TDGNSEHLKR and DGNSEHLKR from pro-tein spots SSP 602 ⁄ 9602 and SSP 1602, respectively;PGPVFGSK from protein spot SSP 1602; and DNDE-NEHQLSLR from protein spot SSP 2102. The half-cleaved peptides derived from the sequence stretchingover amino acids 159–182 of clusterin (IDSLLENDR-QQTHALDVMWDSFNR) found in protein spots SSP502 and SSP 1502 were chromatographically separatedand thus may not refer to in-source fragmentationproducts. Those half-cleaved products are most proba-bly related to in-gel digestion artefacts because cleav-age within this protein sequence stretch by meprinmust be excluded due to an overall amino acidsequence coverage of this protein that exceeded aminoacid 182. In addition, the two half-cleaved peptidesDQAVSDTELQEMSTEGSK (residues 23–40) andDTELQEMSTEGSK (residues 28–40) in SSP 502 and1502 were chromatographically separated and were notin-source fragmentation products generated during theionization process. The former peptide represented themature N-terminus of clusterin (aspartate at position23) and hence was not generated by meprin activity.The latter peptide was presumably produced bymeprinb with acidic amino acids preferred in the P1¢position and selecting against basic amino acids in theP2¢ and P3¢ positions [10]. The leguminous lectin-likeVIP36 was present in two different protein spots (SSP1602 and SSP 602⁄ 9602) and also met our criteria forshortened (non-trypsin-generated) C-terminally trun-cated cleavage products in peptide fragments. In bothspots, the truncated peptide LFQLMVEH (residues273–280) was identified with no further peptidestowards the C-terminal end (not ending with a lysineABCFig. 3. Two-dimensional reference maps on protein identifications.Representative 2D gel images of conditioned medium protein fromMDCKa ⁄ b cells. (A) 2D gel of trypsin activated meprin. (B) 2D gelof non-activated meprin. Unique protein spots were labelled withSSP defined by image analysis software. (C) Close-up view of onerepresentative protein spot, namely, SSP 7006. LC-MS ⁄ MS analy-sis of candidate substrates confirmed the validity of this proteaseproteomic approach for the discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products inpeptide fragments (Table 2).Meprin protease proteomics D. Ambort et al.4494 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBSTable 2. LC-MS ⁄ MS analysis of candidate substrates in discovery of shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments.SSPaProteinidentificationbSwissProtaccessionnumberNumberof uniquepeptidescSequenced,eExperimentalm ⁄ z (Th)Theoreticalmass (Da)Match deltam ⁄ z (Th)fPeptidez-scoregPeptideP-valueh8111 ALDOA_HUMAN P04075 5 (60)R ⁄ QLLLTADDR(69) 522.771 1043.561 0.017 10.3 1.20 · 10)19(69)R ⁄ VNPC^IGGVILFHETLYQK(87) 696.365 2087.087 0.338 8.26 2.44 · 10)11(111)K ⁄ GVVPLAGTNGETTTQ#GLDGLSER(134) 758.492 2273.102 0.216 6.71 3.00 · 10)6(153)K ⁄ IGEHTPSALAIM*ENANVLAR(173) 708.385 2122.084 )0.016 8.85 1.51 · 10)13(289)K ⁄ C^PLLKPWALTFSYGR(304) 905.045 1807.944 )0.065 9.57 6.92 · 10)171703 ANXA1_RABIT P51662 2 (58)K ⁄ GVDEATIIDILTK(71) 694.331 1386.76 0.057 12.8 1.54 · 10)32(113)K ⁄ TPAQFDADELR(124) 631.73 1261.593 0.074 10.9 1.56 · 10)222208 CAPG_HUMAN P40121 2 (115)K ⁄ YQEGGVESAFHK(127) 676.259 1350.62 0.059 8.03 1.65 · 10)10(321)Q ⁄ YAPNTQVEILPQGR(335i) 793.287 1584.826 0.134 11.9 6.76 · 10)28502 CLUS_CANFA P25473 11 (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) 985.952 1969.842 )0.023 9.81 6.45 · 10)18(27)S ⁄ DTELQ#EM*STEGSK(40k) 735.824 1470.603 0.485 9.25 1.72 · 10)15(57)K ⁄ TLIEQTNEER(67) 616.842 1231.604 )0.032 11.3 1.82 · 10)24(57)K ⁄ TLIEQTNEERK(68) 680.894 1359.699 )0.037 6.36 1.63 · 10)5(67)R ⁄ KSLLSNLEEAK(78) 616.363 1230.682 )0.014 8.68 3.46 · 10)13(68)K ⁄ SLLSNLEEAKK(79) 616.421 1230.682 )0.072 7.14 8.22 · 10)8(68)K ⁄ SLLSNLEEAK(78) 552.277 1102.587 0.024 11.7 1.79 · 10)26(81)K ⁄ EDALNDTKDSETK(94) 732.427 1464.658 0.91 8.96 5.08 · 10)14(158)R ⁄ IDSLLENDR(167) 537.749 1073.535 0.026 8.04 8.17 · 10)11(167)R ⁄ QQTHALDVM*Q(177) ⁄ Dl593.764 1185.544 0.016 6.31 5.16 · 10)5(182)R ⁄ ASSIM*DELFQDR(194) 714.356 1426.639 )0.029 7.59 2.53 · 10)91405 CLUS_CANFA P25473 3 (57)K ⁄ TLIEQTNEER(67) 616.738 1231.604 0.072 9.69 2.75 · 10)17(68)K ⁄ SLLSNLEEAK(78) 552.23 1102.587 0.071 8.01 2.18 · 10)10(182)R ⁄ ASSIM*DELFQDR(194) 714.245 1426.639 0.082 10.7 4.56 · 10)221502 CLUS_CANFA P25473 12 (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) 985.976 1969.842 )0.047 12.8 9.20 · 10)33(27)S ⁄ DTELQ#EM*STEGSK(40k) 735.838 1470.603 0.471 9.42 3.50 · 10)16(57)K ⁄ TLIEQTNEER(67) 616.818 1231.604 )0.008 8.08 5.51 · 10)11(57)K ⁄ TLIEQTNEERK(68) 680.9 1359.699 )0.043 6.85 6.10 · 10)7(68)K ⁄ SLLSNLEEAK(78) 552.847 1102.587 )0.546 13.4 1.00 · 10)35(81)K ⁄ EDALNDTKDSETK(94) 733.334 1464.658 0.003 6.5 6.00 · 10)6(158)R ⁄ IDSLLENDR(167) 537.774 1073.535 0.001 9.28 1.59 · 10)15(158)R ⁄ IDSLLENDRQQTHAL(173) ⁄ Dl877.014 1751.88 )0.066 7.09 9.27 · 10)8(167)R ⁄ QQTHALDVM*Q(177) ⁄ Dl593.776 1185.544 0.004 7.62 2.11 · 10)9(167)R ⁄ Q#QTHALDVM*QDSFNR(182) 903.468 1805.8 0.44 10.1 2.74 · 10)19(182)R ⁄ ASSIM*DELFQDR(194) 714.365 1426.639 )0.038 8.97 4.82 · 10)14(335)K ⁄ LYDELLQSYQEK(347) 764.85 1527.745 0.03 7.62 3.90 · 10)9D. Ambort et al. Meprin protease proteomicsFEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4495Table 2. ContinuedSSPaProteinidentificationbSwissProtaccessionnumberNumberof uniquepeptidescSequenced,eExperimentalm ⁄ z (Th)Theoreticalmass (Da)Matchdeltam ⁄ z (Th)fPeptidez-scoregPeptideP-valueh4202 CLUS_CANFA P25473 8 (22)G ⁄ DQAVSDTELQEM*STEGSK(40j) 985.781 1969.842 0.148 7.09 8.33 · 10)8(57)K ⁄ TLIEQTNEER(67) 616.785 1231.604 0.025 10.8 5.30 · 10)22(57)K ⁄ TLIEQTNEERK(68) 680.757 1359.699 0.1 8.63 4.85 · 10)13(68)K ⁄ SLLSNLEEAK(78) 552.235 1102.587 0.066 8.81 1.19 · 10)13(158)R ⁄ IDSLLENDR(167) 537.738 1073.535 0.037 8.74 2.17 · 10)13(167)R ⁄ QQTHALDVM*Q#DSFNR(182) 903.272 1805.8 0.636 7.75 6.29 · 10)10(182)R ⁄ ASSIM*DELFQDR(194) 714.215 1426.639 0.112 10.4 1.42 · 10)20(198)R ⁄ EPQDTYHYSPFSLFQR(214) 1007.856 2013.922 0.113 6.16 8.72 · 10)51104 CO5A2_HUMAN P05997 2 (1273)K ⁄ SLSSQIETM*R(1283) 584.217 1166.56 0.071 10.7 1.43 · 10)21(1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) 862.413 2585.197 0.327 7 3.60 · 10)72104 CO5A2_HUMAN P05997 2 (1273)K ⁄ SLSSQIETM*R(1283) 584.201 1166.56 0.087 10.9 1.26 · 10)22(1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) 862.836 2585.197 )0.096 7.27 4.86 · 10)85106 CO5A2_HUMAN P05997 3 (1273)K ⁄ SLSSQIETM*R(1283) 584.212 1166.56 0.076 10.4 2.84 · 10)20(1368)R ⁄ GSQFAYGDHQSPNTAITQM*TFLR(1391) 862.33 2585.197 0.41 6.84 1.10 · 10)6(1406)K ⁄ NSVGYM*DDQAK(1417) 622.16 1242.518 0.107 10 2.68 · 10)187006 EF2_RAT P05197 3 (580)R ⁄ ETVSEESNVLC^LSK(594) 797.785 1593.755 0.1 16.1 1.90 · 10)53(605)K ⁄ ARPFPDGLAEDIDKGEVSAR(625) 714.301 2142.07 0.73 13.6 5.50 · 10)37(727)R ⁄ C^LYASVLTAQPR(739) 689.733 1377.707 0.128 11.9 2.03 · 10)271802 FLNA_MOUSE Q8BTM8 4 (1891)K ⁄ DAGEGGLSLAIEGPSK(1907) 750.424 1499.746 0.457 9.27 1.43 · 10)15(2089)K ⁄ VDINTEDLEDGTC^R(2103) 818.007 1635.704 0.853 8.84 1.46 · 10)13(2264)R ⁄ EAGAGGLAIAVEGPSK(2280) 713.941 1425.746 )0.06 7.27 2.85 · 10)8(2346)K ⁄ VNQPASFAVSLNGAK(2361) 752.41 1501.788 )0.508 10.9 5.55 · 10)23602 ⁄ 9602 LMAN2_CANFA P49256 12 (44)A ⁄ DITDGNSEHLKR(56j) 692.894 1383.674 )0.049 7.81 4.37 · 10)10(46)I ⁄ TDGNSEHLKR(56m) 578.845 1155.563 )0.056 7.5 1.23 · 10)8(47)T ⁄ DGNSEHLKR(56m) 528.275 1054.515 )0.01 7.39 2.74 · 10)8(126)K ⁄ NLHGDGIALWYTR(139) 758.428 1514.763 )0.039 12 2.25 · 10)28(141)R ⁄ LVPGPVFGSK(151) 500.22 999.575 0.575 6.61 7.88 · 10)6(151)K ⁄ DNFHGLAIFLDTYPNDETTER(172) 1235.101 2467.129 )0.529 10.3 5.12 · 10)20(195)R ⁄ WTELAGC^TADFR(207) 713.858 1425.634 )0.033 10.6 3.30 · 10)21(207)R ⁄ NRDHDTFLAVR(218) 448.599 1342.674 )0.034 7.24 5.41 · 10)8(209)R ⁄ DHDTFLAVR(218) 537.316 1072.53 )0.043 11.8 5.80 · 10)27(223)R ⁄ LTVM*TDLEDKNEWK(237) 869.483 1736.829 )0.061 6.41 9.90 · 10)6(246)R ⁄ LPTGYYFGASAGTGDLSDNHDIISM*K(272) 916.184 2745.259 )0.09 8.27 2.00 · 10)11(272)K ⁄ LFQLM*VEH(280) ⁄ Tk516.781 1031.511 )0.018 7.11 1.13 · 10)7Meprin protease proteomics D. Ambort et al.4496 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBSTable 2. ContinuedSSPaProteinidentificationbSwissProtaccessionnumberNumberof uniquepeptidescSequenced,eExperimentalm ⁄ z (Th)Theoreticalmass (Da)Matchdeltam ⁄ z (Th)fPeptidez-scoregPeptideP-valueh1602 LMAN2_CANFA P49256 9 (44)A ⁄ DITDGNSEHLKR(56j) 692.816 1383.674 0.029 8.59 6.59 · 10)13(46)I ⁄ TDGNSEHLKR(56m) 578.799 1155.563 )0.01 6.43 1.04 · 10)5(126)K ⁄ NLHGDGIALWYTR(139) 757.873 1514.763 0.516 13.4 2.85 · 10)36(143)V ⁄ PGPVFGSK(151m) 394.734 787.422 )0.015 9.47 3.28 · 10)16(151)K ⁄ DNFHGLAIFLDTYPNDETTER(172) 1234.648 2467.129 )0.076 9.1 4.37 · 10)15(195)R ⁄ WTELAGC^TADFR(207) 713.829 1425.634 )0.004 7.65 3.25 · 10)9(209)R ⁄ DHDTFLAVR(218) 537.151 1072.53 0.122 8.88 1.30 · 10)13(237)K ⁄ NC^IDITGVR(246) 524.223 1046.517 0.043 9.45 3.29 · 10)16(272)K ⁄ LFQLM*VEH(280) ⁄ Tk516.778 1031.511 )0.015 7.94 4.22 · 10)105101 LMNA_RAT P48679 9 (28)R ⁄ LQEKEDLQELNDR(41) 815.299 1628.8 0.109 9.73 1.57 · 10)17(50)R ⁄ SLETENAGLR(60) 545.274 1088.546 0.007 9.19 8.26 · 10)15(62)R ⁄ ITESEEVVSR(72) 574.713 1147.572 0.081 12.2 2.80 · 10)29(78)K ⁄ AAYEAELGDAR(89) 582.603 1164.541 0.675 9.6 1.52 · 10)16(133)R ⁄ LKDLEALLNSK(144) 622.29 1242.718 0.077 9.89 3.58 · 10)18(144)K ⁄ EAALSTALSEKR(156) 638.275 1274.683 0.074 6.15 6.67 · 10)5(144)K ⁄ EAALSTALSEK(155) 560.2 1118.581 0.098 9.7 2.86 · 10)17(156)R ⁄ TLEGELHDLR(166) 591.74 1181.604 0.07 9.18 3.50 · 10)15(196)R ⁄ LQ#TLKEELDFQK(208) 497.874 1491.782 0.394 7.24 5.12 · 10)83502 LOXL1_HUMAN Q08397 2 (400)K ⁄ C^LASTAYAPEATDYDVR(417) 951.853 1901.846 0.078 9.85 8.28 · 10)18(540)K ⁄ YIVLESDFTNNVVR(554) 834.825 1667.851 0.108 14.1 2.75 · 10)40403 ⁄ 9302 LYOX_RAT P16636 4 (231)R ⁄ C^AAEENC^LASSAYR(245) 801.35 1600.661 )0.012 12.9 3.00 · 10)33(314)K ⁄ ASFC^LEDTSC^DYGYHR(330) 661.002 1979.778 )0.069 7.82 5.07 · 10)10(371)K ⁄ VSVNPSYLVPESDYSNNVVR(391) 1119.092 2237.096 0.464 8.29 6.36 · 10)12(395)R ⁄ YTGHHAYASGC^TISPY(411j) 892.899 1783.762 )0.01 6.27 2.29 · 10)52102 NPM_RAT P13084 3 (32)K ⁄ VDNDENEHQLSLR(45) 784.81 1567.722 0.059 8.1 3.99 · 10)11(33)V ⁄ DNDENEHQLSLR(45m) 735.34 1468.654 )0.005 8.31 7.53 · 10)12(80)K ⁄ M*SVQPTVSLGGFEITPPVVLR(101) 1121.629 2242.203 0.48 6.52 7.52 · 10)68110 SERC_HUMAN Q9Y617 8 (5)R ⁄ QVVNFGPGPAK(16) 557.281 1112.597 0.025 8.24 3.43 · 10)11(61)R ⁄ ELLAVPDNYK(71) 580.824 1160.607 0.487 7.51 1.14 · 10)8(169)K ⁄ GAVLVC^DM*SSNFLSK(184) 821.989 1642.769 0.403 7.3 2.52 · 10)7(169)K ⁄ GAVLVC^DM*SSNFLSKPVDVSK(190) 757.019 2268.113 0.026 8.83 1.93 · 10)13(213)R ⁄ DDLLGFALR(222) 509.705 1018.544 0.575 7.01 4.97 · 10)7(222)R ⁄ EC^PSVLEYK(231) 562.753 1123.522 0.016 7.92 2.03 · 10)10(323)K ⁄ ALELNM*LSLK(333) 573.944 1146.631 0.379 9.29 2.90 · 10)15(342)R ⁄ ASLYNAVTIEDVQK(356) 775.374 1549.798 0.533 9.82 6.97 · 10)184104 STC1_HUMAN P52823 3 (119)R ⁄ M*IAEVQEEC^YSK(131) 751.869 1501.642 )0.04 8.45 2.22 · 10)12(139)K ⁄ RNPEAITEVVQ#LPNHFSNR(158) 741.405 2221.124 )0.023 10.1 1.04 · 10)18(165)R ⁄ SLLEC^DEDTVSTIR(179) 818.872 1636.761 0.516 10.1 2.41 · 10)19D. Ambort et al. Meprin protease proteomicsFEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4497or arginine residue). Additionally, this truncated pep-tide was not generated by in-source fragmentationbecause there was no co-eluting ion trace of the corre-sponding whole tryptic peptide LFQLMVEHTPDEE-NIDWTK. VIP36 was described as a single-pass type Imembrane protein with an extracellular carbohydraterecognition domain exactly terminating at those aminoacids (residues 52–280) [23]. Moreover, the amino acidsequence following the putative cleavage site corre-sponded to cleavage preference for meprina with theamino acids threonine and proline in the P1¢ and P2¢positions [10]. The targeted cleavage by hmeprin afterthis specific domain may indicate protein ectodomainshedding. Nevertheless, the biological consequence ofthis remains to be elucidated.Functional clustering into biological process andmolecular functionNext, the proteins identified by LC-MS ⁄ MS as puta-tive meprin substrates were classified into functionalgroups according to the Human Protein ReferenceDatabase (Table 3) [24]. Ten proteins could beassigned to the biological process of ‘cell growthand ⁄ or maintenance’ and four to ‘immune response’(Fig. 4). The remaining proteins were equally distrib-uted into functional classes such as ‘transport’, ‘cellcommunication; signal transduction’, ‘metabolism;energy pathways’ and ‘protein metabolism’. In conclu-sion, these findings suggest possible functions formeprin in the regulation of cell homeostasis andthe extracellular environment, and in the immuneresponse.Effect of in situ trypsin treatmentZymogen activation by limited trypsin treatment maylead to changes elicited by the trypsin and not bymeprin. To exclude such unspecific side effectscaused by the trypsin treatment rather than by theeffector (membrane-bound hmeprina ⁄ b), wild-type(WT) and meprina ⁄ b MDCK cells were treated inthe same way. Media of trypsin-treated and non-treated cells were prepared and then subjected to 1DSDS ⁄ PAGE and subsequent densitometric imageanalysis with aida software (Fig. 5). We decided toperform a comparison between conditioned media ofWT and meprina ⁄ b MDCK cells on 1D gels. Quan-titative assessment of protein bands revealed nosignificant differences between trypsin-treated andnontreated WT samples, whereas meprina ⁄ b samplesshowed substantial differences upon trypsin activa-tion. Moreover, the protein patterns of WT versusTable 2. ContinuedSSPaProteinidentificationbSwissProtaccessionnumberNumberof uniquepeptidescSequenced,eExperimentalm ⁄ z (Th)Theoreticalmass (Da)Matchdeltam ⁄ z (Th)fPeptidez-scoregPeptideP-valueh4302 TSP1_HUMAN P07996 4 (50)K ⁄ GPDPSSPAFR(60) 515.657 1029.488 0.095 9.93 3.01 · 10)18(60)R ⁄ IEDANLIPPVPDDKFQDLVDAVR(83) 860.853 2578.328 )0.403 8.3 1.43 · 10)11(74)K ⁄ FQDLVDAVR(83) 531.714 1061.55 0.069 8.82 1.02 · 10)13(201)K ⁄ GGVNDNFQGVLQNVR(216) 808.804 1615.806 0.107 8.53 1.08 · 10)127510 VINC_HUMAN P18206 4 (199)K ⁄ ELLPVLISAM*K(210) 614.446 1228.71 0.917 8.52 2.86 · 10)12(464)K ⁄ Q#VATALQNLQTK(476) 657.778 1314.714 0.587 8.2 1.93 · 10)11(570)R ⁄ ALASQLQDSLK(581) 587.247 1172.64 0.081 10.5 8.42 · 10)21(802)K ⁄ AVAGNISDPGLQK(815) 635.247 1268.672 0.097 9.06 1.02 · 10)14aSSP assigned by image analysis software PDQUESt, version 7.3.1.bCID spectra interpretation with public search engine PHENYX (version 2.1) on vital-it.ch against uniprot-SwissProt protein data-base (release 48.8). Taxonomy search space restricted to Mammalia (40 084 sequence entries). CANFA, Canis familiaris,dog;RAT,Rattus norvegicus, rat; HUMAN, Homo sapiens, human;RABIT, Oryctolagus cuniculus, rabbit; MOUSE, Mus musculus,mouse.cFor multiple peptide matches to same primary sequence, the top scoring peptide was listed.dModifications: C^, carb-amidomethylation of cysteine; M*, oxidation of methionine; Q#, deamidation of glutamine.eNumbers in parentheses indicate the P1 positions of cleavages. [Correction added 6 August 2008,after first online publication: in the preceding sentence ‘superscript numbers’ was corrected to ‘numbers in parentheses’.]fMatch delta is the difference between theoretical m ⁄ z of matchedpeptide and observed m ⁄ z of parent ion.gPeptide search criteria were set to a minimum peptide z-score of ‡ 5.hOnly protein identifications consisting of at least two unique peptides reaching aP-value of £ 0.00000001 were accepted.iNormal tryptic peptide (dog protein ortholog with arginine in P1 position instead of glutamine).jN- and C-terminal half-cleaved peptides.kShortened(non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments.lHalf-cleaved peptides generated during in-gel digestion.mIn-source fragmentation products.Meprin protease proteomics D. Ambort et al.4498 FEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBSmeprina ⁄ b differed as well (Fig. 5A) and indicatedthat overexpression of hmeprina ⁄ b per se causes dif-ferences that are independent from zymogen activa-tion. Finally, triplicate image analysis of gel lanesconfirmed these findings (Fig. 5B) but, more impor-tantly, revealed a trend towards the appearanceof low molecular weight proteins in media ofmeprina ⁄ b MDCK cells. Hence, the triplicate assess-ment of data generated unambiguously pointed toreproducible differences triggered by the activationand not by the overexpression of meprina ⁄ b(Fig. 5C). Obviously, activation of meprina ⁄ b resultsin the release of proteins into the culture medium.Validation of direct or indirect effects byimmunoblotting follow-up experimentsProteomics is a very powerful tool for protease-substrate identification, but the data obtained need tobe verified by means of alternative techniques. WesternTable 3. BLASTP-based protein database searching and functional classification. All peptide sequence tags (Table 2) were searched againstthe dog genome database usingBLASTP, version 2.2.16. Database size was 33 527 dog RefSeq protein sequences. The database is hostedat NCBI. Functional classification according to Human Protein Reference Database.Protein description Biological process Molecular functionNCBI accessionnumber SSPaScorebExpectedvaluecPREDICTED: similar toannexin A1Cell communication;signal transductionCalcium ion binding XP_533524 1703 57.1 1.00 · 10)9dClusterin Immune response Complement activity NP_001003370 502 99 9.00 · 10)221405 61.3 7.00 · 10)11d1502 125 1.00 · 10)294202 103 2.00 · 10)23PREDICTED: similar tocollagen alpha 2(V)chain precursorCell growth and ⁄ ormaintenanceExtracellular matrix,structural constituentXP_535998 1104 50.4 3.00 · 10)72104 50.4 3.00 · 10)75106 62.8 6.00 · 10)11PREDICTED: similar toelongation factor 2Protein metabolism Translationregulator activityXP_533949 7006 62 1.00 · 10)10PREDICTED: similar tofilamin A isoform 8Cell growth and ⁄ ormaintenanceCytoskeletalanchoring activityXP_867537 1802 60.8 2.00 · 10)10PREDICTED: similar tofructose-bisphosphatealdolase A isoform 2Metabolism; energypathwaysLyase activity XP_849434 8111 117 3.00 · 10)27PREDICTED: similar tolamin A ⁄ C isoform 5Cell growth and ⁄ ormaintenanceStructuralmolecule activityXP_864487 5101 104 2.00 · 10)23Lectin, mannose-binding 2 Transport Transporter activity NP_001003258 602 ⁄ 9602 219 8.00 · 10)581602 129 7.00 · 10)31PREDICTED: similar tomacrophage capping proteinCell growth and ⁄ ormaintenanceCytoskeletalprotein bindingXP_540197 2208 48.6 4.00 · 10)7dPREDICTED: similar tonucleophosmin 1 isoform 12Protein metabolism Chaperone activity XP_866781 2102 57.8 2.00 · 10)9PREDICTED: similar tophosphoserineaminotransferase isoform 1Metabolism; energypathwaysTransaminase activity XP_533520 8110 72.4 7.00 · 10)14PREDICTED: similar toprotein-lysine 6-oxidaseprecursor isoform 3Cell growth and ⁄ ormaintenanceCatalytic activity XP_859412 403 ⁄ 9302 90.5 3.00 · 10)193502 33.3 0.017dPREDICTED: similar tostanniocalcin-1 precursorCell communication;signal transductionCalcium ion binding XP_543238 4104 79.7 5.00 · 10)16PREDICTED: similar tothrombospondin 1 precursorCell growth and ⁄ ormaintenanceExtracellular matrix,structural constituentXP_544610 4302 72 1.00 · 10)13PREDICTED: similar to vinculin Cell growth and ⁄ ormaintenanceCytoskeletal proteinbindingXP_536395 7510 42.6 7.00 · 10)5daSSP assigned by image analysis software PDQUEST, version 7.3.1.bOnly the top scoring significant hit was accepted.cSearch parameters:word size 3, filter low complexity, expect value 0.01, score matrix BLOSUM62.dFailed searches were repeated with settings for ‘short andnearly exact matches’: word size 2, filter off, expect value 20 000, score matrix PAM30.D. Ambort et al. Meprin protease proteomicsFEBS Journal 275 (2008) 4490–4509 ª 2008 The Authors Journal compilation ª 2008 FEBS 4499[...]... information The following supplementary material is available: Fig S1 Application of a simple 2D IEF ⁄ SDS ⁄ PAGEbased protease proteomic approach in substrate finding 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the second quadrant Fig S2 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the third quadrant Fig S3 2D IEF ⁄ SDS ⁄ PAGE-based image analyses of the fourth quadrant This supplementary material can... methionine and variable deamidation of asparagine and glutamine Parent and fragment mass tolerances were set to 1 Da Up to two missed cleavages and half tryptic peptides were allowed The taxonomic search space was restricted to Mammalia (40 084 sequence entries) Peptide search criteria were set to a minimum peptide z-score of ‡ 5 and a maximum peptide P-value of £ 0.0001 All protein identifications consisting... side) and meprina ⁄ b peaks (right hand side) from three independent analyses Intensity of peak areas (QL) was backgroundcorrected (Bkg) proteins and peptide fragments with lectin affinity pre-fractionation In the present study, we demonstrate the applicability of a simple 2D IEF ⁄ SDS ⁄ PAGE-based image analysis procedure to analyse candidate substrates for meprin in a cell culture system-based approach. .. Babiychuk for the generous gift of vinculin and annexin A1 antibodies, Professor Bernhard Erni for free access to the Fuji Film Fluorescent Image Analyzer FLA-3000R and aida software and Professor Robert Beynon for teaching MS-based techniques This work was funded by the Swiss National Science Foundation (SNSF) (grant 310 0A0 -100772 to E.E.S.) and the European Science Foundation (ESF) Integrated Approaches for. .. 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al tion of protein identifications into functional groups with the Human Protein Reference Database facilitated the interpretation of the data generated (Fig 4 and Table 3) [24] Hence, the metalloendopeptidase meprin may be involved in processes of ‘cell growth and ⁄ or maintenance’ and ‘immune response’ Taken together, the novel strategies and... using the program pdquest, version 7.3.1 (Bio-Rad Laboratories) Data were inverted and images displayed as black spots on white background Each gel replicate was cropped into four quadrants of same image size (2157 · 1682 pixels) Image cropping of same areas among gel replicates was realized by reference to highly conserved landmark spots present in all gels In total, four independent analyses were performed... and applications presented herein may help to understand more precisely the function of a protease in a complex environment Novel roles for hmeprin in homeostasis of cell, cellular environment and in immune response BMP-1, mammalian TLD and hmeprin belong to the same metzincin subfamily of metalloendopeptidases, the astacin family [32] The main functions of BMP1 ⁄ TLD-like metalloendopeptidases are ascribed... 2008 The Authors Journal compilation ª 2008 FEBS D Ambort et al meprin) for subsequent analytical image analysis For analytical gels, 250 lg of concentrated medium protein from trypsin activated and non-activated MDCKa ⁄ b cells was solubilized in 450 lL of buffer containing 7 m urea, 2 m thiourea, 4% (w ⁄ v) Chaps, 1% (w ⁄ v) dithioerythritol and 2% (v ⁄ v) Pharmalyte 3–10 for 1 h on a rotary shaker at... formic acid, glycerol (approximately 87%) and paraffin oil were purchased from Merck (Darmstadt, Germany); ProtoGel acrylamide stock solution was obtained from National Diagnostics (Atlanta, GA, USA); sequencing grade modified trypsin was obtained from Promega (Madison, WI, USA); acetonitrile was from Riedel-de-Haen ⁄ Fluka; SDS ¨ 4504 was obtained from Serva (Heidelberg, Germany); Phenylmethanesulfonyl... overexpression of meprin per se and not by the activity status of meprin Discussion Establishment of a simple 2D IEF/SDS/ PAGE-based protease proteomic approach To date, some MMPs and ADAMs have been characterized on a system-wide level by means of protease proteomics [14–17] Two protease proteomic approaches defined the substrate repertoire of membrane-type 1-MMP (MT1-MMP ⁄ MMP-14) in a cell culture system-based . A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin Daniel Ambort1, Daniel Stalder2, Daniel. metalloendopeptidases in that it acts as a procollagen C protease as well as an activator of lysyloxidase. Therefore, an important role for hmeprina ⁄ bmay be ascribed
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