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Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and… Jordans, Silvia; Jenko-Kokalj, Saša; Kühl, Nicole M; Tedelind, Sofia; Sendt, Wolfgang; Brömme, Dieter; Turk, Dušan; Brix, Klaudia Sep 22, 2009

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ralssBioMed CentBMC BiochemistryOpen AcceResearch articleMonitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditionsSilvia Jordans1, Saša Jenko-Kokalj2, Nicole M Kühl1, Sofia Tedelind1, Wolfgang Sendt3, Dieter Brömme4, Dušan Turk2 and Klaudia Brix*1Address: 1School of Engineering and Science, Jacobs University Bremen, Campus Ring 6, Research II, 28759 Bremen, Germany, 2Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, 3Krankenhaus St. Joseph-Stift Bremen, Schwachhauser Heerstrasse 54, 28209 Bremen, Germany and 4Department of Oral and Biological Sciences, Faculty of Dentistry, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, CanadaEmail: Silvia Jordans - sjordans@jacobs-alumni.de; Saša Jenko-Kokalj - sasa.jenko@ijs.si; Nicole M Kühl - nicolekuehl@hotmail.com; Sofia Tedelind - s.tedelind@jacobs-university.de; Wolfgang Sendt - wsendt@sjs-bremen.de; Dieter Brömme - dbromme@interchange.ubc.ca; Dušan Turk - dusan.turk@ijs.si; Klaudia Brix* - k.brix@jacobs-university.de* Corresponding author    AbstractBackground: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditionswithin endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing andneutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key tounderstand physiological protease functions, redox conditions have only rarely been considered in routine enzymeactivity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteinecathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular spacein comparison to those within endo-lysosomes of mammalian cells.Results: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed underconditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, theprecursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteinecathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances,starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that allcathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these arereportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin atextracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellularconditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions.Conclusion: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specificprocessing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substratethyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our studyemphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes butinclude pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fineadjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activityPublished: 22 September 2009BMC Biochemistry 2009, 10:23 doi:10.1186/1471-2091-10-23Received: 20 July 2009Accepted: 22 September 2009This article is available from: http://www.biomedcentral.com/1471-2091/10/23© 2009 Jordans et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 15(page number not for citation purposes)assays.BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23BackgroundProteolytic processes are of vital importance due to theirreversibility of peptide bond cleavage. Spatio-temporalregulation of proteolysis is therefore of highest signifi-cance in determining the fate of cells. To date more than66.500 gene sequences have been annotated as coding forproteolytic enzymes. The diversity of proteases is furtherreflected in broad tissue-, compartment- and substratespecificities. Most of all protease genes encode for serinepeptidases (~25.000 sequences), followed by metallo(~23.000) and cysteine peptidases (~10.400) [1,2].Because of this variety, one key issue of degradomics is toinvestigate networks of proteases in order to explain dis-tinct proteolytic functions in the spatio-temporal contextof substrate cleavage [3]. It is common practice to employproteolytic activity assays to achieve this goal.In this report we focused on cysteine cathepsins B, K, Land S, and analyzed their processing patterns using a nat-ural substrate, thyroglobulin (Tg), while taking intoaccount the suspected redox- and pH values of the physi-ological cleavage environments.It has been shown before that the cysteine cathepsins B, K,L and S are localized to the endo-lysosomal system of thy-roid epithelial cells. Moreover, they are present at extracel-lular locations such as the apical plasma membrane andwithin the follicular lumen of the thyroid gland [4-9].Generally, cysteine cathepsins of the papain superfamilyare well known to engage in bulk protein turnover at theincreasingly acidic pH values of endocytic compartments[10]. Nevertheless, the proteolytic potency of cysteinecathepsins towards peptidic or natural substrates has beendemonstrated to expand over a wide range including neu-tral pH values [4,11,12]. In fact, cysteine cathepsins wereoften accredited as extracellularly acting enzymes in vivo(for reviews, see [10,13]). Diverse protocols are availablethat describe methods to perform activity assays ofcysteine cathepsins at neutral conditions, usually afteractivation of the proteases by incubation with reducingagents [14-19]. In reality, however, the extracellular spaceis not only a neutral but also an oxidizing environment, asmuch as endosomes and lysosomes exhibit not onlyacidic but also reducing conditions.In this study, we have chosen redox-values of -220 mV tomimic the reducing environment of the cysteine-richendosomes and lysosomes [20-22]. Moreover, endo-lyso-somes are characterized by increasingly acidic conditions[23-25], and we considered pH 6.0 and pH 5.0 as closelyreflecting the endosomal and lysosomal milieu, respec-tively. On the other hand, the thyroid follicle lumen rep-resents a secluded compartment in which the tightbetween thyrocytes [26,27]. The follicle lumen is assumedto be neutral in pH thereby rendering Tg proteolysis byextracellular hydrolases unlikely (for reviews, see [26-28]). However, more recently we provided evidence for Tgproteolysis mediated by cysteine cathepsins within theextracellular thyroid follicle lumen before its endocytosis[6,7]. Extracellularly stored Tg of the human thyroid folli-cle lumen is covalently cross-linked by disulfide bonds[29] which can be formed in a self-assisted fashion via theCXXC-boxes within Tg [30], thereby attesting an oxidizingmilieu within the thyroid follicle lumen. Furthermore, thedirect pericellular environment adjacent to the apicalplasma membrane of thyroid epithelial cells is oxidizingdue to the presence of an H2O2 generating system whichenables thyroid peroxidase to iodinate Tg at neutral pH[31,32]. We have chosen a value of -150 mV to mimic theextracellular space which is slightly more oxidizing thanthe range of -160 mV to -170 mV that was reported for thelumen of the endoplasmic reticulum [33].As yet, there are only very few studies which considerboth, pH values and redox potentials of the cleavage envi-ronment [21,34]. The assay described herein thereforeemploys buffers accounting for the conditions in the fol-licle lumen equivalent to the extracellular space or withinendo-lysosomal compartments. We assessed the suitabil-ity of such buffers to promote proteolytic cleavage of Tg byspecific cysteine cathepsins in order to simulate proteoly-sis within thyroid follicles, i.e. within an extracellularcompartment, or within the endo-lysosomes of thyrocytes(Figure 1). Processing of Tg in the thyroid serves as anexcellent model system for our purposes, because it hasbeen demonstrated that (i) the cysteine cathepsins B, K, Land S cleave Tg in vitro [4,35-38], (ii) Tg is located in allcompartments where cysteine cathepsins principally meettheir substrates, i.e. endosomes, lysosomes, and in theextracellular space [[4,6-9], and this study], (iii) cysteinecathepsins and Tg represent a naturally occurring andphysiologically relevant enzyme-substrate couple in vivo[6,7]. Under physiological conditions, proteolysis of Tgleading to thyroid hormone liberation is accomplished bycysteine cathepsins in a sequential process starting withthe solubilization of Tg from its covalently cross-linkedmacromolecular storage forms that fill the extracellularfollicle lumen. The initial substrate cleavage results in theproduction of smaller protein fragments that are internal-ized by thyroid epithelial cells together with partiallyprocessed Tg [6,7]. Accordingly, liberation of the thyroidhormones T3 and T4, which are essential for the regulationof mammalian development and metabolism beginsextracellularly and is perpetuated within endocytic com-partments of thyroid epithelial cells.Page 2 of 15(page number not for citation purposes)junctions between thyroid epithelial cells tie up the extra-cellular storage space for Tg from the intercellular spacesThis study aims at elucidating crucial roles of selectedcysteine cathepsins in this sequence of prohormoneBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23Figure 1Schematic overview representing the organization of a thyroid follicle. The thyroid follicle lumen displays a pro-tected extracellular compartment which is enclosed by a monolayer of thyroid epithelial cells interconnected by tight junctions.The apical plasma membrane of thyrocytes faces the follicle lumen in which compacted and covalently cross-linked thyroglobu-lin (Tg) is stored in globules reaching up to 150 m in diameter. Tg-globules are far too large to be engulfed by single thyro-cytes on the whole, thus partial degradation in the extracellular follicle lumen must precede endocytosis. By limiteddegradation, mediated by cysteine cathepsins within the pericellular space, Tg fragments are liberated and are subsequentlyendocytosed for degradation in endosomes and lysosomes. Thyroxine liberation by processing of the prohormone Tg startsextracellularly and is continued intracellularly after Tg-fragment internalization. Compartments in which proteolysis of Tg prin-cipally takes place are highlighted and the chosen redox- and pH-conditions were used as indicated in the associated boxes (forfurther details please refer to Background section).processing. The development of an in vitro assay systemmimicking the in situ pH and redox conditions enabledanalysis of the potency of cathepsins B, K, L and S to cleavetheir natural substrate Tg and to liberate thyroid hor-mones under conditions simulating favorable (endo-lyso-somal) versus non-favorable (extracellular)compartments.ResultsLocalization of cysteine cathepsins in the human thyroid glandThe subcellular localization of cysteine cathepsins hasbeen studied in detail in almost all tissues but not to thesame extent in the human thyroid gland. Therefore and inorder to analyze the distribution patterns of particularcysteine cathepsins in the compartments of Tg processing,human thyroid tissue was immunolabeled with antibod-ies against cathepsins B, K, L, and S in single and doublelabeling experiments. Cathepsins B, K, and L were co-Page 3 of 15(page number not for citation purposes)BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23Figure 2 (see legend on next page)Page 4 of 15(page number not for citation purposes)BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23localized within the same intracellular vesicles (Figure 2Band 2D, yellow signals). Cathepsin S appeared to be alsolocalized to distinct vesicular compartments, because co-localization with cathepsins K and L was not as prominent(Figure 2A and 2C). Some cathepsin L-positive vesicleswere even lacking cathepsin S (Figure 2A, inset, arrow-heads). Likewise, cathepsin K-positive structures wereoften negative for cathepsin S (Figure 2C, inset, arrow-heads). Consequently, cathepsin S-positive vesicles lack-ing cathepsins K or L were frequently detected (Figure 2Aand 2C, insets, arrows). Moreover, all cathepsins analyzedwere detected in the extracellular space, i.e. within the fol-licle lumen, indicating their secretion (Figure 2, arrows inlumina labeled with asterisks). Note, that the extracellularfollicle lumen (Figure 2, asterisks) represents a storagecompartment for Tg, hence, all cathepsins are found inclose proximity to their natural substrate already here.Cathepsin S was prominently present in the extracellularfollicle lumen (Figure 2A and 2C, arrows in luminalabeled with asterisks). Cathepsins K and L were addition-ally found pericellularly in close association with the api-cal plasma membrane (Figure 2B and 2C, arrowheads),where thyroid hormone liberation by Tg processing isbelieved to start. We conclude that trafficking of proteasesin thyroid epithelial cells results in a distinct proteasecomposition within vesicles of the endocytic pathway inaddition to their secretion into the extracellular space.Hence, it is important to analyze Tg processing with differ-ent combinations of cysteine cathepsins and to simulatesubstrate cleavage at the conditions of the respective com-partments, i.e. extracellular follicle space, endosomes andlysosomes.Establishment of buffers with stable redox potentials at distinct pH valuesBuffers representative for the extracellular space, the endo-somal compartment and for lysosomes were establishedthrough the use of a two-buffer system of citric acid,sodium phosphate, and L-cysteine. In order to verifybuffer stability, the actual pH and redox values were deter-redox potential of -150 mV and neutral pH 7.2 was shownto be stable over 24 hours (Figure 3A). Although additionof Tg marginally lowered redox and pH values, the overallstability under extracellular conditions was more stringentin the presence of substrate (Figure 3B). Buffers mimick-ing endosomes and lysosomes were adjusted to -220 mVand differed from one another in their pH values, only.The endosomal-representative buffer was set to pH 6.0and the lysosomal to pH 5.0. The stability of these bufferswith respect to redox potential and pH value was verifiedover a 4 h time period (Figure 3C and 3D) as these bufferswere stable from the very beginning.Determination of cleavage sites within TgIsolated Tg was incubated with cathepsins in buffers sim-ulating extracellular conditions and the extent of substratecleavage was compared to that achieved under conditionscharacteristic for intracellular proteolysis within endo-lys-osomal compartments. Cathepsins B, K, L and S or com-binations thereof were incubated with Tg in the respectivebuffers and proteolysis was allowed to occur for 2 hours.Preparations were then heated in LDS sample buffer with-out any further addition of reducing agents. This proce-dure ensured consistent behavior of all samples from thesame conditions upon separation by PAGE since theywere run on separate gels. Fragmentation patterns wereanalyzed by SDS-PAGE followed by immunoblottingusing antibodies specific for Tg (Figure 4) and by N-termi-nal sequencing of selected Tg-fragments (Figure 5). Note,that for all incubations an identical amount of Tg was sub-jected to cysteine cathepsin degradation, whereby the par-ticular amount of distinct enzymes was kept constant inall incubations. Therefore, the ratios of total enzyme tosubstrate differed between single and combinatorial incu-bations (see also Materials and Methods). We were espe-cially decided about such conditions, because we considerthis setup to best reflect Tg cleavage in situ. As we haveshown in the localization studies (see Figure 1), vesiclesmay contain only one type of enzyme or combinations ofdifferent enzymes. For the extracellular follicle lumen,Localization of cysteine cathepsins in human thyroid tissueFigure 2 (see previous page)Localization of cysteine cathepsins in human thyroid tissue. Multi-channel fluorescence (A-D and insets), single chan-nel and corresponding phase contrast micrographs (black and white) of cryo-sections taken from human thyroid tissue that were immunolabeled with antibodies directed against cysteine cathepsins B, K, L, or S in different combinations, as indicated. Co-localization is displayed by yellow signals as a result of overlapping red and green signals for either antibody labeling. Cathe-psins B and K as well as K and L were mainly co-localized to the same intracellular compartments (B and D, insets, arrows), whereas cathepsin S was found in structures that were lacking other cysteine cathepsins (A and C, insets, arrows). Likewise, vesicles containing only cathepsin L or K (A and C, insets, arrowheads) were also prominent. All cysteine cathepsins were present within the Tg-containing extracellular follicle lumen (asterisks, Tg) where no co-localization of either cysteine cathep-sin with one another was detectable. Cathepsins K and L were further detected in association with the apical plasma mem-brane of thyrocytes (B and C, insets, arrowheads). Bars represent 50 m.mined for a minimum time period of 24 hours. The buffer similar approximations might be relevant, since wePage 5 of 15(page number not for citation purposes)simulating extracellular conditions with an oxidizing observed almost no co-localization of the distinct cysteineBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23cathepsins in the extracellular space, but differentenzymes were detectable and can therefore, in principle,cleave luminal Tg. Hence, the observed degradation pat-terns must be interpreted as resulting from different com-binations of enzymes acting sequentially and/or at thesame time on Tg.Under neutral and oxidizing conditions mimicking theextracellular thyroid follicle lumen full-length Tg ofapproximately 330 kDa was cleaved by each of theaction of cysteine cathepsins (Figure 4A). The effective-ness of substrate-cleavage was obvious from the appear-ance of two fragments of approximately 250 and 200 kDa(Figure 4A, arrowheads), which were also detected whenthe assay buffers reflected more favorable endosomalcleavage conditions, i.e. a reducing and slightly acidicenvironment (Figure 4B, arrowheads). In addition to thehigh molecular mass fragments, all samples containingcathepsin S displayed a specific 70 kDa fragment underextracellular conditions (Figure 4A, arrow). The same-Stability of assay buffersFigure 3Stability of assay buffers. Redox potentials [mV] (open squares) and pH values (filled circles) were measured for the indi-cated time intervals. A two-buffer system of citric acid and sodium phosphate was used to adjust the desired pH values while the redox values were adjusted by the addition of L-cysteine. Timepoint [t = 0] reflects the initially adjusted value; the setpoint of the redox potential is given as dashed line. Redox values above the setpoint are considered more oxidizing, whereas changes towards the negative potential are more reducing. Buffers representative for the extracellular space, i.e. mimicking an oxidizing (-150 mV) and neutral (pH 7.2) compartment, were measured in the absence (A) or presence (B) of the substrate Tg. Buffers mimicking the endosomal compartment were characterized by reducing (-220 mV) and slightly acidic (pH 6.0) conditions (C). Buffers representing the lysosomes displayed the same reducing redox-conditions as endosomes (c.f. C), but were more acidic (pH 5.0) (D). Values are given as mean +/- SD.Page 6 of 15(page number not for citation purposes)cysteine cathepsins tested, even though these buffer con-ditions are considered as 'non-favorable' for proteolyticsized fragment was generated at conditions characteristicfor endosomes and lysosomes by several combinations ofBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23Figure 4Cleavage efficiency of cysteine cathepsins at distinct subcellular conditions. Single cathepsins or combinationsthereof were incubated with thyroglobulin (Tg) in vitro in buffers representative of extracellular conditions (A), endosomes (B)or lysosomes (C) for 2 hours at 40°C. As a control, purified Tg was incubated in the respective buffers without proteases (A-C, last lanes). Samples were PAGE-separated and Tg fragments were visualized by immunoblotting. Efficiency of cleavage atneutral and oxidizing conditions was indicated by the appearance of a 250 kDa and a 200 kDa fragment (A, arrowheads). Simi-lar fragments were observed under endosome-representative conditions (B, arrowheads). Most effective substrate degrada-tion, discernible by numerous low molecular weight fragments, was observed under reducing, acidic conditions mimickinglysosomes (C). Cathepsin L can be considered most effective in Tg-processing within the lysosomal compartment, becausesamples containing this protease (C, asterisks) featured numerous low molecular weight fragments (arrows) and almost lackedintact, full-length Tg (dashed arrow). Note, that under neutral and oxidizing conditions, cathepsin S qualified to be most effec-tive in Tg-processing. All samples including this protease featured a specific 70 kDa fragment (A, arrow) additionally to highermolecular mass products. In contrast to extracellular or lysosomal conditions, all peptidases tested performed equally well atendosome-representative conditions.cysteine cathepsins but notably not by cathepsin K aloneor in combination with cathepsins L and S. The analysis ofdegradation patterns using assay conditions mimickinglysosomal cleavage revealed a number of low molecularmass fragments of Tg as generated by all cathepsin combi-nations (Figure 4C, arrows). Samples containing cathep-70 kDa (Figure 4C, asterisks). Note that all preparationscontained equal amounts of Tg at the start of incubationwith cysteine cathepsins under different conditions. Thelanes were loaded after the incubation period with identi-cally sized aliquots from each preparation. Hence, theabsence of signal in certain lanes of the immunoblots isPage 7 of 15(page number not for citation purposes)sin L lacked the intact, full-length Tg (Figure 4C, dashedarrow), but contained numerous smaller fragments of 40-representative of extensive Tg degradation rather thanbeing the result of unequal loading of the gels. Accord-BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23Figure 5 (see legend on next page)K~S SK~SB~K~SBSK~SL~S*QQGEP*FGFE*LFLQ*NIFEYSN III VI VII VIII IX XICXXC CXXCCXXCIII IV V XT4 T4 T4T4T3CBIII VI VII VIII IX XINCXXC CXXCCXXCIII IV V XT4 T4DBBLB D*NIFE*SFGF*SLLEL*SGNF*FSPVCBD D LLLLT4T4T3*LIGS*LREP*SQDD*IRSG*KVPT*RAVL*VSGPB~L~SB*EGEF*NIFEY*EAGQQ*QELES SNCIII VI VII VIII IX XICXXC CXXCCXXCIII IV V XT4 T4 T4T4T3CDMALVLEIFTLLASICWVSANIFEYQVDAQPLRPCELQRETAFLKQADYVPQCAEDGSFQTVQCQNDGRSCWCVGANGSEVLGSRQPGRPVACLSFCQLQK 100QQILLSGYINSTDTSYLPQCQDSGDYAPVQCDVQQVQCWCVDAEGMEVYGTRQLGRPKRCPRSCEIRNRRLLHGVGDKSPPQCSAEGEFMPVQCKFVNTT 200DMMIFDLVHSYNRFPDAFVTFSSFQRRFPEVSGYCHCADSQGRELAETGLELLLDEIYDTIFAGLDLPSTFTETTLYRILQRRFLAVQSVISGRFRCPTK 300CEVERFTATSFGHPYVPSCRRNGDYQAVQCQTEGPCWCVDAQGKEMHGTRQQGEPPSCAEGQSCASERQQALSRLYFGTSGYFSQHDLFSSPEKRWASPR 400VARFATSCPPTIKELFVDSGLLRPMVEGQSQQFSVSENLLKEAIRAIFPSRGLARLALQFTTNPKRLQQNLFGGKFLVNVGQFNLSGALGTRGTFNFSQF 500FQQLGLASFLNGGRQEDLAKPLSVGLDSNSSTGTPEAAKKDGTMNKPTVGSFGFEINLQENQNALKFLASLLELPEFLLFLQHAISVPEDVARDLGDVME 600TVLSSQTCEQTPERLFVPSCTTEGSYEDVQCFSGECWCVNSWGKELPGSRVRGGQPRCPTDCEKQRARMQSLMGSQPAGSTLFVPACTSEGHFLPVQCFN 700SECYCVDAEGQAIPGTRSAIGKPKKCPTPCQLQSEQAFLRTVQALLSNSSMLPTLSDTYIPQCSTDGQWRQVQCNGPPEQVFELYQRWEAQNKGQDLTPA 800KLLVKIMSYREAASGNFSLFIQSLYEAGQQDVFPVLSQYPSLQDVPLAALEGKRPQPRENILLEPYLFWQILNGQLSQYPGSYSDFSTPLAHFDLRNCWC 900VDEAGQELEGMRSEPSKLPTCPGSCEEAKLRVLQFIRETEEIVSASNSSRFPLGESFLVAKGIRLRNEDLGLPPLFPPREAFAEQFLRGSDYAIRLAAQS 1000TLSFYQRRRFSPDDSAGASALLRSGPYMPQCDAFGSWEPVQCHAGTGHCWCVDEKGGFIPGSLTARSLQIPQCPTTCEKSRTSGLLSSWKQARSQENPSP 1100KDLFVPACLETGEYARLQASGAGTWCVDPASGEELRPGSSSSAQCPSLCNVLKSGVLSRRVSPGYVPACRAEDGGFSPVQCDQAQGSCWCVMDSGEEVPG 1200TRVTGGQPACESPRCPLPFNASEVVGGTILCETISGPTGSAMQQCQLLCRQGSWSVFPPGPLICSLESGRWESQLPQPRACQRPQLWQTIQTQGHFQLQL 1300PPGKMCSADYAGLLQTFQVFILDELTARGFCQIQVKTFGTLVSIPVCNNSSVQVGCLTRERLGVNVTWKSRLEDIPVASLPDLHDIERALVGKDLLGRFT 1400DLIQSGSFQLHLDSKTFPAETIRFLQGDHFGTSPRTWFGCSEGFYQVLTSEASQDGLGCVKCPEGSYSQDEECIPCPVGFYQEQAGSLACVPCPVGRTTI 1500SAGAFSQTHCVTDCQRNEAGLQCDQNGQYRASQKDRGSGKAFCVDGEGRRLPWWETEAPLEDSQCLMMQKFEKVPESKVIFDANAPVAVRSKVPDSEFPV 1600MQCLTDCTEDEACSFFTVSTTEPEISCDFYAWTSDNVACMTSDQKRDALGNSKATSFGSLRCQVKVRSHGQDSPAVYLKKGQGSTTTLQKRFEPTGFQNM 1700LSGLYNPIVFSASGANLTDAHLFCLLACDRDLCCDGFVLTQVQGGAIICGLLSSPSVLLCNVKDWMDPSEAWANATCPGVTYDQESHQVILRLGDQEFIK 1800SLTPLEGTQDTFTNFQQVYLWKDSDMGSRPESMGCRKDTVPRPASPTEAGLTTELFSPVDLNQVIVNGNQSLSSQKHWLFKHLFSAQQANLWCLSRCVQE 1900HSFCQLAEITESASLYFTCTLYPEAQVCDDIMESNAQGCRLILPQMPKALFRKKVILEDKVKNFYTRLPFQKLMGISIRNKVPMSEKSISNGFFECERRC 2000DADPCCTGFGFLNVSQLKGGEVTCLTLNSLGIQMCSEENGGAWRILDCGSPDIEVHTYPFGWYQKPIAQNNAPSFCPLVVLPSLTEKVSLDSWQSLALSS 2100VVVDPSIRHFDVAHVSTAATSNFSAVRDLCLSECSQHEACLITTLQTQPGAVRCMFYADTQSCTHSLQGQNCRLLLREEATHIYRKPGISLLSYEASVPS 2200VPISTHGRLLGRSQAIQVGTSWKQVDQFLGVPYAAPPLAERRFQAPEPLNWTGSWDASKPRASCWQPGTRTSTSPGVSEDCLYLNVFIPQNVAPNASVLV 2300FFHNTMDREESEGWPAIDGSFLAAVGNLIVVTASYRVGVFGFLSSGSGEVSGNWGLLDQVAALTWVQTHIRGFGGDPRRVSLAADRGGADVASIHLLTAR 2400ATNSQLFRRA VLMGGSALSPAAVISHERAQQQAIALAKEVSCPMSSSQEVVSCLRQKPAN VLNDAQTKLLAVSGPFHYWGPVIDGHFLREPPARALKRSL 2500WVEVDLLIGSSQDDGLINRAKAVKQFEESRGRTSSKTAFYQALQNSLGGEDSDARVEAAATWYYSLEHSTDDYASFSRALENATRDYFIICPIIDMASAW 2600AKRARGNVFMYHAPENYGHGSLELLADVQFALGLPFYPAYEGQFSLEEKSLSLKIMQYFSHFIRSGNPNYPYEFSRKVPTFATPWPDFVPRAGGENYKEF 2700SELLPNRQGLKKADCSFWSKYISSLKTSADGAKGGQSAESEEEELTAGSGLREDLLSLQE PGSKTYSK 2768Asignal peptide extracellularendosomal lysosomalYYhormogenic site (T4)hormogenic site (T3)Adopted from Dunn et al. [Ref. 36-37]Page 8 of 15(page number not for citation purposes)BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23ingly, blank or weakly labeled lanes indicate completedegradation of Tg by the respective cysteine cathepsin/s atthese conditions. As a control of its stability, Tg was incu-bated in the respective buffers, but without any proteaseadded (Figure 4A-C, last lanes).In order to determine the cleavage sites that were used bycysteine cathepsins, characteristic Tg fragments were N-terminally sequenced (Figure 5). A band with a molecularmass of approximately 330 kDa exhibited the same N-ter-minus as unprocessed Tg in all instances, thus represent-ing intact, full-length protein. Tg-fragments originatingfrom proteolytic processing by cathepsin S under extracel-lular conditions had molecular masses of approximately200 kDa, 170 kDa and 70 kDa. Two of these fragmentsexhibited newly generated N-termini, while the 70 kDafragment showed the same N-terminus as unprocessed Tg.Arrowheads indicate relative positions of identified cleav-age sites as deduced from N-terminal sequencing of extra-cellular- (Figure 5B) or endosomal-like Tg degradation(Figure 5C). In Figure 5D a scheme of lysosomal cleavagesites is depicted, which was determined by Dunn and col-leagues [36,37] under near-physiological conditions sincelysosomal extracts were used to degrade Tg. The schematicdrawings in Figure 5 illustrate that the cleavage sites variedwithin Tg depending on the conditions under whichcleavage took place.Analysis of physiological significance of Tg cleavageDetermination of cathepsin cleavage sites revealed thatthe N-terminal half of Tg was prone to extracellular andendosomal cleavage, whereas the thyroid hormone-richC-terminal half is, according to [36,37], best cleavedunder conditions of lysosomal Tg processing. In order toimplement a physiologically relevant read-out for effi-ciency of substrate cleavage, we analyzed the in vitro-deg-fT4 was only detected in samples that contained cathepsinS alone or in combinations with other cathepsins (Figure6A). Under endosomal conditions, the overall efficiencyof fT4 liberation was considerable, about 70 fold higherthan compared to extracellular conditions with values offT4 ranging from approximately 35 to 55 pM (Figure 6B).Yet, another augmentation in the amount of liberated T4was observed under conditions simulating lysosomeswhere cathepsins B, K and L performed equally well byliberating T4 in a range from 75 to 100 pM (Figure 6C).DiscussionOne important mechanism to control protease activity isgiven by endogenous inhibitors, another is represented bythe dependency of proteolytic enzymes on the pH andredox potential of the cleavage environment. Whereas pHsensitivity has been studied quite extensively (for review,see [39]), much less is known about their sensitivity to theredox potential of the environment. Endo-lysosomalcysteine proteases like cathepsins L and B are consideredhighly instable at neutral pH values with half lives of 1.3and 15 minutes, respectively [40,41]. The inactivationappears to be irreversible and is associated with a loss ofnative 3D structure. However, a few other cysteine cathe-psins of the papain superfamily clan C1A display pro-longed stability and activity at neutral pH values,cathepsins K and S are the most prominent examples[4,11,18]. In contrast, papain as well as cruzain exhibitstability at broad ranges of pH values and withstand neu-tral to even basic pH-conditions [42,43]. On the otherhand, other plant proteases are particularly prone to turninstable at pH values differing from their acidic optimum[43]. These pronounced differences in the pH-dependentstability indicate that papain-like enzymes have evolved[44,45] to play roles in a variety of biological processes,bringing proteolysis into milieus outside acidic endo-lys-Preferential cleavage sites for cysteine cathepsins within the thyroglobulin sequence at distinct redox- and pH-conditionsFigu  5 (see previou  page)Preferential cleavage sites for cysteine cathepsins within the thyroglobulin sequence at distinct redox- and pH-conditions. Characteristic Tg-fragments obtained by in vitro degradation at extracellular or intra-endo-lysosomal conditions were N-terminally sequenced. Newly generated N-termini are highlighted (A); box colors represent the respective condition at which fragments were obtained (c.f. Figure 1). Hormogenic sites, i.e. pre-formed thyroid hormones tri-iodothyronine (T3) and thyroxine (T4), are tagged in blue and green; the signal peptide is highlighted in grey. (B-D) Schematic drawings sketch the Tg molecule that comprises Tg Type I domains (green), Type II (blue), Type III A (light grey), Type III B repeats (dark grey), Thyropins (red), and CXXC-motifs (see [6] for details). Arrowheads indicate relative positions of identified cleavage sites uti-lized in extracellular- (B) or endosomal-like Tg processing (C). Cleavage sites of cathepsins B, D, and L were determined by N-terminal sequencing of Tg fragments derived from incubation of rabbit Tg with lysosomal extracts or cathepsins isolated from human thyrocytes (D; according to [36,37]). This scheme (D) is shown for comparison, only, and shaded to indicate that it is adopted (see [6]) from the results of studies performed by Dunn and co-workers [36,37]. Corresponding cysteine cathepsins or combinations thereof are given below the arrowheads; the respective neo-N-termini are specified above the representative schematic drawing. Arrows in B and D indicate similar cleavage sites under extracellular conditions and within lysosomes, respectively.radation samples for their content of liberated, free osomal compartments. Therefore, mammalian cysteinePage 9 of 15(page number not for citation purposes)thyroxine (fT4) by ELISA. Under extracellular conditions, cathepsins might be the "specialists" in acidic environ-BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23ments, although they are still suited to perform theirtions of the extracellular space that are nowadays consid-ered non-favorable or even "extreme" [5,10,46].In fact, proteolytic activity of lysosomal cysteine pepti-dases in an environment exhibiting conditions oppositeto the reducing and acidic milieu of endo-lysosomal com-partments was not long ago considered to be a missionimpossible. Oxidizing conditions enforce the formationof disulfide bonds by reactive thiol groups which makesthe active site cysteine of cathepsins highly sensitive tooxidation. Thus, under reducing conditions, like it isassumed for the endo-lysosomal compartment, the activ-ity of cysteine cathepsins should be considerably higherthan in other compartments exhibiting oxidizing condi-tions, like e.g. the extracellular space. In the thyroid, how-ever, the extracellular follicle lumen is the scene of actionfor the initial steps of thyroid hormone utilization, i.e. thesolubilization of covalently cross-linked Tg and thyroxineliberation by proteolytic processing mediated primarilythrough cysteine cathepsins B, K, L and S [7].Up to now, the hypothesis that cysteine peptidases holdproteolytic activity in oxidizing environments was rarelychallenged experimentally due to the lack of suitableactivity assays with conditions mimicking the in vivo situ-ation in the most life-like fashion, i.e. at realistic pH andredox conditions in physiological settings. In situ, redoxconditions in cellular compartments or in the extracellu-lar space are achieved and maintained by redox pairs ofwhich cysteine(Cys)-cystine(CySS) constitutes the promi-nent and natural redox-pair of the extracellular space andendo-lysosomal compartments [47]. In keeping with thisnotion, exact values describing redox potentials are aston-Figure 6Cysteine cathepsins efficiency to liberate thyroxine (T4) in vitroFigure 6Cysteine cathepsins efficiency to liberate thyroxine (T4) in vitro. Samples taken after 2h-in-vitro-degradation of Tg were assayed for their content in free thyroxine (fT4) by ELISA. Efficiency of hormone liberation by distinct cysteine cathepsins or combinations thereof is directly correlated to the amount of fT4. Under conditions mimicking the extracel-lular space, exclusively cathepsin S proved to be efficient in thyroxine liberation (A), whereas under endosomal-repre-sentative conditions all cysteine cathepsins performed equally well (B). The same holds true for conditions mimicking the lysosomal compartment, although cathepsin S was less active as reflected by lower thyroxine liberation potency as com-pared with e.g. cathepsin B (C). Highest amounts of fT4 were measured for lysosomal-mimicking environments, followed by those performed under conditions simulating endosomes and the extracellular space (A-C). Values are given as mean values +/- SD.actions, at least temporarily, under the neutral pH-condi- ishingly scarce and have been reported for the cytosol (-Page 10 of 15(page number not for citation purposes)221 mV to -236 mV) and the ER lumen (-160 mV to -170BMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23mV) [21,22,33]. The latter represents an oxidizing intrac-ellular compartment of protein folding in which disulfidebridges are formed and maintained, whereas the cytosol isclearly reducing. For the choice of our buffer system weused the (Cys)-(CySS) redox-pair to adjust the redoxpotential and we considered a redox potential of -220 mVsuitable to simulate reducing conditions of endosomesand lysosomes. A redox value of -150 mV was adjusted formimicking the extracellular space, i.e. we assumed aslightly more oxidizing milieu for the extracellular spacethan that observed for the ER lumen. Through the use ofa two-buffer system with citric acid and sodium phos-phate and through the addition of L-cysteine, we haveestablished buffers with stable redox potentials at distinctpH values for in vitro analyses of proteolytic substrateprocessing potency.Another critical factor in proteolytic profiling is the choiceof substrate. Commonly, small synthetic substrates areused for standard protease activity assays. More recently itbecame mandatory to use natural substrates in in vitroassays to collect more appropriate data; especially consid-ering that protease-substrate interaction may also affectenzyme activity [3]. The cleavage of Tg by cathepsins is anaturally occurring process of high physiological impor-tance [7], which makes Tg an excellent substrate in ourproteolytic assays. The partial degradation of extracellu-larly stored full-length Tg within the thyroid folliclelumen is of great importance in vivo. It represents the nec-essary first step to produce smaller fragments of the pro-hormone that can easily be endocytosed to ensure endo-lysosomal processing and efficient thyroid hormone liber-ation. In the in vitro assay presented herein, partial prote-olytic processing of full length Tg was achieved by allcathepsins, resulting in the generation of three distinctfragments even at neutral and oxidizing conditions. Theabundance of low molecular mass Tg-fragments in lyso-somal-mimicking conditions of this assay must be consid-ered representative of an extensive overall cleavage of thesubstrate. It can therefore be deduced that all cysteinecathepsins tested exhibited highest proteolytic activities,indeed, under acidic and reducing conditions. This notionis in line with the catabolic function of cysteine cathepsinsin situ which is mainly conducted within the endo-lyso-somal compartment. We consider cathepsin L as the mostpotent peptidase to catabolize Tg within the lysosomes,because the respective samples lacked full-length, intactTg almost entirely, but contained numerous smaller frag-ments of 40-70 kDa.The application of several cathepsins at once proved to beefficient in well-directed substrate cleavage furtherstrengthening results from our previous in vivo studies inpresented herein further support the conclusion that Tg-processing and thyroid hormone liberation is achieved byproteases acting consecutively towards efficient pro-hor-mone utilization. Cathepsin S proved to be the best-suitedcysteine cathepsin to extracellularly liberate T4 from Tg,i.e. to conduct hormone liberation before the pro-hor-mone eventually reached endosomes and lysosomes.Cathepsin S was also able to liberate T4 under endosomaland lysosomal conditions, but then, it was not any moreefficient than the other cysteine cathepsins tested. In fact,it has been shown that cathepsin S is the only cysteinecathepsin which is truly stable at neutral pH when ana-lyzed under reducing conditions, whereas cathepsins Kand L are unstable and cathepsin B exhibits limited stabil-ity at reducing and neutral pH conditions[11,17,18,40,41,48]. Interestingly, an additional cleavagesite for the combination of cathepsins K and S was deter-mined within the N-terminal half of Tg at extracellularconditions as compared to cleavage by cathepsin S alone(Figure 5B). This might explain why liberation of T4 underthese conditions was less effective when both, cathepsinsK and S were used in contrast to combinations of cathep-sin S with cathepsins B or L, in which the cathepsin S abil-ity to utilize T4 from Tg clearly dominated (Figure 6A).However, it should be kept in mind that the assay condi-tions as presented herein differ significantly from those ofprevious studies in that we mimicked physiological con-ditions by testing cysteine cathepsins under oxidizing andneutral pH conditions. Therefore, our results gained bystudying cleavage of their natural substrate Tg under suchcomparatively "extreme" non-favorable conditions clearlydiffer from the standard assay conditions in which theread-out is not physiological, but instead cleavage of syn-thetic substrates is monitored. Hence, we believe that thedata presented here has to be interpreted on the back-ground of physiological protein processing with the bio-logically significant read-out, that is, utilization of T4 byprocessing of the precursor Tg.None of the cleavage sites used by any of the cysteinecathepsins tested was identical in both, extracellular andendosomal conditions, i.e. in the compartments of well-directed substrate processing. Thus, we conclude that pref-erential cleavage is largely up to the compartment inwhich substrate processing takes place. The presence ofdifferent sets of proteolytic enzymes in such compart-ments may allow for a combinatorial and sequential sub-strate cleavage. This is to say that specific cleavage sites areused in distinct environments, which provide stable, bio-chemically defined conditions for specific sets ofenzymes, but do not necessarily exhibit the most favora-ble conditions for bulk proteolytic processing. This notionis further supported by comparative analysis of endo-Page 11 of 15(page number not for citation purposes)which it was shown that a concerted action of cathepsinsK and L is needed for T4 liberation in vivo [7]. The resultssomal-typical cleavage with respect of lysosomal-typicalcleavage sites, which completely lack analogies as hasBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23been determined by us (this study) and by Dunn and co-workers using isolated cathepsins or extracts of thyroidlysosomes [36,37]. Even though the cysteine cathepsins Band L were included in both studies, only one cleavage-site (Figure 5B and 5D, arrows) was found to be alike inextracellular conditions (this study) and in Tg fragmentsidentified in lysosomal extracts (studies by Dunn and col-leagues [36,37]). Interestingly, this particular cleavage sitewas not reproduced by identical cathepsins in differentenvironments, but the extracellular-representative cleav-age of Tg by cathepsin S (this study) generated an almostidentical neo-N-terminus as the cathepsin B-mediatedcleavage determined by Dunn and co-workers [36,37].Thus, although the amino acid sequence of the substrateitself predominantly dictates its cleavage, the specificity ofsubstrate cleavage is additionally directed by the biochem-ical environment of cleavage and by the set of actuallycleaving enzymes.It is important to keep in mind that the extracellularly act-ing cysteine cathepsins in the thyroid follicle are derivedfrom their secretion into the follicle lumen after beingrecruited out of endo-lysosomal compartments [9]. It isgenerally accepted that the proteolytic activity of suchalready activated cysteine peptidases can be maintainedunder oxidizing conditions in the extracellular space forlonger time intervals [10,34,49]. Since the proteolyticenzymes cleaving Tg in the thyroid follicle lumen haveexperienced a reducing and acidic environment for properprocessing and activation before being secreted, they mayas well contribute significantly to physiological thyroidhormone liberation by extracellular proteolysis of Tg.ConclusionOur study shows that a specific subcellular location is thekey figure in the nature of proteolytic processing, i.e. tim-ing and location of peptidase activities determine whetherbulk protein turnover or, instead, specific substrateprocessing takes place. Importantly enough, the onset ofmany pathological processes like tumor cell metastasis,rheumatoid arthritis, pancreatitis or multiple sclerosisgoes along with alterations in the patterns of cysteinecathepsin localization and with pericellular acidificationwhich may well be suited to prolong the extracellularactions of these proteases (for reviews see [10,13,50]).The routine to analyze cysteine cathepsin-mediatedprocessing of Tg presented herein will be easily adaptableto investigate any protease's proteolytic potential towardsany natural or artificial substrate. Hence, our approach isapplicable in order to study the degradome in a physio-logical context at simulated in vivo conditions. By adapt-ing our assay system to other proteases, we anticipate aing in the determination of 'vital' or 'fatal' changes incellular destiny. Comprehensive knowledge of proteolysisin physiological or pathological conditions, in turn, iscritical for the development of effective treatments andnovel therapeutics.MethodsDepletion of non-covalently associated T4 from isolated human TgPurified human Tg [29,51] was mixed with 200 g/mL 8-anillino-1-naphtalene-sulfonic acid ammonium salt(ANS, Calbiochem/Merck, Darmstadt, Germany) andincubated on an end-over-end-rotator (14 rpm) for 1 h inorder to dissociate non-covalently bound T4 from its pre-cursor that is known to eventually function as a T4-bind-ing protein. The mixture was cleared of free T4 by twosubsequent incubations with an anion exchange resin,DOWEX 1 (1 × 8 chloride form, 200-400 mesh; 50 mg/mL; Sigma, Munich, Germany). The crude and fine resinparts were removed by two centrifugation steps at 1,000and 30,000 × g for 10 and 20 minutes, respectively. Super-natants were used for protein determination according toNeuhoff [52].Preparation of assay buffersA two-component buffer system of (A) 200 mM citric acidand (B) 200 mM dibasic sodium phosphate was used.Specific volumes of (A) and (B) were mixed as indicatedbelow and buffers were pre-warmed to 40°C (accordingto [14]) while constantly stirring. Redox potentials wereadjusted by the addition of L-cysteine (Roth, Karlsruhe,Germany) in the indicated amounts. Adequacy and stabil-ity of pH and redox values were constantly monitoredusing a pH electrode (SenTix 81) and an electrode forredox control (SenTix ORP), both connected to an InoLabpH Level 1 (all WTW, Weilheim, Germany). The pH of 7.2in the buffer simulating the extracellular space wasachieved by mixing 6.9 mL of buffer A and 87.1 mL ofbuffer B. Upon the addition of 9.58 mg L-cysteine theredox potential of the buffer shifted to -150 mV. The buff-ers mimicking slightly acidic endosomal compartments(pH 6.0, -220 mV) and acidic lysosomal compartments(pH 5.0, -220 mV) were prepared by mixing 19.7 mL ofbuffer A and 68.3 mL of buffer B with 190 mg L-cysteine,or 28 mL of buffer A with 65.5 mL of buffer B and 2.2 g L-cysteine, respectively. Buffers were equilibrated at assaytemperature before use.In vitro Tg degradation assayCathepsins B and L, purified from human liver, were pur-chased from Calbiochem (Darmstadt, Germany), whereashuman cathepsins K and S were recombinant enzymes[11,18]. Human Tg and single cathepsins or combina-Page 12 of 15(page number not for citation purposes)better understanding of the interplay and fine-adjust-ments of protease-networks that control substrate process-tions thereof were mixed in 100 L of buffers mimickingextracellular, endosomal or lysosomal conditions to aBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23final concentration of 9.5 pM for Tg, and 4.75 pM ofeither of the cathepsins. Therefore, the substrate to pepti-dase ratios varied from 1:0.5 up to 1:2 in single, double,triple, and quadruple cathepsin combinations. Sampleswere prepared in LowBind protein tubes (Eppendorf,Hamburg, Germany), spun down and incubated on anend-over-end rotator (40 rpm) for 2 hours at 40°C in aheated incubator. Then, samples were briefly centrifugedand processing of Tg was stopped either by freezing 50 Laliquots of the samples at -20°C or by direct preparationof the samples for subsequent SDS-PAGE analysis. Sam-ples were mixed with lithium dodecyl sulfate (LDS) sam-ple buffer, heated to 70°C for 10 minutes, and 50 L ofeach individual sample was loaded per lane. Gel-electro-phoresis was performed in an XCell SureLock Mini-Cellusing pre-cast 3-8% NuPAGE Novex Tris-Acetate gels andNuPAGE Tris-Acetate SDS running buffer (all Invitrogen,Karlsruhe, Germany) at 150 V for 75 minutes at RT. Toensure consistent running behavior, gels were loaded withsamples from either of the three conditions so that reduc-ing and non-reducing samples would not affect each otherduring separation. Subsequently, Tg and fragments wereblotted onto polyvinylidenfluoride (PVDF) membranes at30 V for 60 minutes using the XCell II Blotting Moduleand NuPAGE transfer buffer (all Invitrogen) according tothe manufacturer's instructions.Immunodetection of Tg and its degradation fragmentsMembranes were blocked using 5% non-fat milk powderin PBS-Tween (0.34 M NaCl, 0.316 M Na2HPO4, 58.49NaH2PO4, pH 7.2, supplemented with 0.3% Tween-20).Primary antibodies directed against bovine Tg [51] werediluted 1:1,000 in 2.5% non-fat milk powder and mem-branes were incubated 90 minutes at RT. Secondary,horseradish peroxidase (HRP) -coupled antibodies wereallowed to bind for 60 minutes at RT. Immunoreactionswere visualized by enhanced chemiluminescence (ECL)and imaged on CL-XPosure Clear Blue X-ray films (bothPierce, PerbioScience, Bonn, Germany) which werescanned using a transmitted light scanner device (Optic-Pro ST48, Plustek, Norderstedt, Germany).N-terminal sequencing of separated Tg fragmentsFor sequencing, the degradation assays, gel-electrophore-sis, and blotting were performed as above. The absolutecontent of cathepsins and Tg, however, was increasedfour-fold. PVDF membranes were briefly washed inddH2O and stained overnight with 0.1% Coomassie R250in 1% acetic acid and 40% methanol. Background wasremoved by washing in 50% methanol until bandsbecame clearly visible. Selected protein bands showingadequate staining were cut and stored at -20°C until fur-ther analyzed by automated sequence analyses on a Pro-Determination of thyroid hormone liberation by fT4-ELISATo assess the amount of liberated thyroxine, 50 L of thein vitro degradation samples were analyzed using a com-mercially available solid phase-based competitive immu-noassay (DRG Diagnostics, Marburg, Germany). Serumsamples that were included in the ELISA kit and containeddefined fT4-amounts were used as standards. Serumstandards and in vitro degradation samples were assayedin duplicates and treated equally in all respects. The assaywas conducted according to the manufacturer's recom-mendation with the performance of optional washes (10× 5 minutes) with ddH2O. Immediately after addition ofthe color reactant the absorbance at 450 nm was read in amicroplate reader (MRX Microplate Reader, DynatechLaboratories Inc., Chantilly, VA, USA). The fT4 amountcalculated for blank values was subtracted from individualsamples to accomplish normalization. Data presented arerepresentative for n = 4 (for endosomal conditions) or n =3 (for extracellular and lysosomal conditions); errors aregiven as standard deviations. Calculations were per-formed using Origin 7.0 (OriginLab Corporation, North-ampton, MA, USA).Immunolocalization of cysteine cathepsins in thyroid tissueBiopsies of human thyroid tissue were taken from thenon-disease affected regions after thyroidectomy. The useof human tissue was in compliance with the Helsinki Dec-laration and was approved to be conducted by WS and KBby the Ethics Commission of the Ärztekammer Bremen(Study 120 as of April 18th, 2005) in Germany. Tissue wasfixed in 8% paraformaldehyde in 200 mM Hepes buffer,pH 7.4, and mounted in tissue freezing medium after sev-eral buffer exchanges. Cryo-sections were taken with a CM1900 cryostat (Leica, Bensheim, Germany), and immu-nolabeled following routine procedures [4,7] with sheepanti-human cathepsin B (RD Laboratorien, Diessen, Ger-many), mouse anti-human cathepsin K (IM 55, Oncogenethrough Merck Biosciences, Bad Soden, Germany), sheepanti-human cathepsin L (RD Laboratorien), and rabbitanti-human cathepsin S (kindly provided by Dr. E. Weber,Halle, Germany) as primary antibodies, followed by incu-bation with Alexa 488- or Alexa 546-labeled secondaryantibodies (Molecular Probes through Invitrogen, Karl-sruhe, Germany). Analysis of mounted tissue was per-formed by means of confocal laser scanning microscopy(LSM 510 Meta, Zeiss, Oberkochen, Germany).Authors' contributionsSJ developed the assay buffer system, performed activityassays, immunoblotting, ELISAs and statistical analyses,analyzed the localization studies, and drafted the manu-script. SJK performed N-terminal sequencing and ana-lyzed cleavage site sequences. NMK participated in thePage 13 of 15(page number not for citation purposes)cise pulsed-liquid protein sequencer (Applied Biosystems,Foster City, CA, USA).development of the activity assay buffer system. WS con-tributed to the cathepsin localization study and providedBMC Biochemistry 2009, 10:23 http://www.biomedcentral.com/1471-2091/10/23thyroid tissue. DB purified human cathepsins K and S andprovided advice in data interpretation. DT and ST dis-cussed experimental setups and contributed in draftingthe manuscript. KB devised the study and its design, anddrafted the manuscript together with SJ. All authors readand approved the final manuscript.AcknowledgementsThe authors are indebted to Dr. E. Weber (Halle, Germany) for the kind gift of rabbit anti-human cathepsin S antibodies. The authors would like to thank Adrijana Leonardi (Toxicology Group, Jožef Stefan Institute) for help in the determination of Tg cleavage sites and recognize her outstanding expertise in N-terminal sequencing. Meike Klepsch is acknowledged for excellent technical assistance, in particular cryo-sectioning and immunola-beling. This work was supported by Jacobs University Bremen, then Inter-national University Bremen (to KB) and a Ph.D. student fellowship of the same University (to SJ) [both grant number 2140-90033], by an EMBO short term fellowship (to SJK) [grant number ASTF 168.00-03], by The Foundation BLANCEFLOR (to ST), by Slovenian Research Agency (to DT) [grant number P1-0048], and by NIH (to DB) [grant number AR 46182].References1. Puente XS, Sanchez LM, Overall CM, Lopez-Otin C: Human andmouse proteases: a comparative genomic approach.  Nat RevGenet 2003, 4(7):544-558.2. Rawlings ND, Morton FR, Barrett AJ: MEROPS: the peptidasedatabase.  Nucleic Acids Res 2006:D270-D272.3. Overall CM, Blobel CP: In search of partners: linking extracel-lular proteases to substrates.  Nat Rev Mol Cell Biol 2007,8(3):245-257.4. 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