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Pesu Vesa P. Hytonen, Matti Nykter and Marko Uusimaki, Mika Ramet, Mataleena Parikka, Kivinen, Sampo Kukkurainen, Annemari Hannu Turpeinen, Anna Oksanen, Virpi 1a) β (TGF of Transforming Growth Factor Beta 1a Zebrafish Development and Bioavailability Type 7 (PCSK7) Is Essential for the Proprotein Convertase Subtilisin/Kexin Developmental Biology: published online October 31, 2013 J. Biol. Chem. 10.1074/jbc.M113.453183 Access the most updated version of this article at doi: . JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on the Alerts: When a correction for this article is posted When this article is cited to choose from all of JBC's e-mail alerts Click here Supplemental material: http://www.jbc.org/content/suppl/2013/10/31/M113.453183.DC1.html http://www.jbc.org/content/early/2013/10/31/jbc.M113.453183.full.html#ref-list-1 This article cites 0 references, 0 of which can be accessed free at at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from at FINELIB - Tampere Univ on November 6, 2013 http://www.jbc.org/ Downloaded from

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PesuVesa P. Hytonen, Matti Nykter and Marko Uusimaki, Mika Ramet, Mataleena Parikka,Kivinen, Sampo Kukkurainen, Annemari Hannu Turpeinen, Anna Oksanen, Virpi 

1a)β(TGFof Transforming Growth Factor Beta 1a Zebrafish Development and BioavailabilityType 7 (PCSK7) Is Essential for the Proprotein Convertase Subtilisin/KexinDevelopmental Biology:

published online October 31, 2013J. Biol. Chem. 

  10.1074/jbc.M113.453183Access the most updated version of this article at doi:

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2013/10/31/M113.453183.DC1.html

  http://www.jbc.org/content/early/2013/10/31/jbc.M113.453183.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from at FINELIB - Tampere Univ on November 6, 2013http://www.jbc.org/Downloaded from

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Proprotein Convertase Subtilisin/Kexin Type 7 (PCSK7) Is Essential for the Zebrafish Development andBioavailability of Transforming Growth Factor Beta 1a (TGF 1a)*

Hannu Turpeinen1*, Anna Oksanen1*, Virpi Kivinen2, Sampo Kukkurainen3, Annemari Uusimäki1,Mika Rämet4,5,6, Mataleena Parikka4, Vesa P. Hytönen3,7, Matti Nykter2, Marko Pesu1,7

* equal contribution

1 Immunoregulation, Institute of Biomedical Technology, University of Tampere, and BioMediTech,Tampere, Finland

2 Department of Signal Processing, Tampere University of Technology, Tampere, Finland3 Protein Dynamics, Institute of Biomedical Technology, University of Tampere, and BioMediTech,

Tampere, Finland4 Experimental Immunology, Institute of Biomedical Technology, University of Tampere, and

BioMediTech, Tampere, Finland5 Department of Pediatrics, Tampere University Hospital, Tampere, Finland

6 Department of Children and Adolescents, Oulu University Hospital, and Department of Pediatrics,Medical Research Center Oulu, University of Oulu, Finland7 Fimlab laboratories, Pirkanmaa Hospital District, Finland

*Running title: PCSK7 in zebrafish

Word count: capsule 60, summary 206, body 5540To whom correspondence should be addressed: Hannu Turpeinen, Institute of Biomedical Technology,BioMediTech, University of Tampere, FinnMedi 2, 5th floor, Biokatu 8, FI-33520 Tampere, Finland,email [email protected]

Keywords: proprotein convertase, PCSK7, PC7, zebrafish, otolith, TGF 1a, development, protease, geneexpression, homology modeling

The abbreviations used are: PCSK, proprotein convertase subtilisin/kexin; MO, morpholino; RC, randomcontrol morpholino; ISH, in situ hybridization; TGF , transforming growth factor beta; dpf, days postfertilization; hpf, hours post fertilization; GO, gene ontology

Background: The in vivo importance of PCSK7 inthe vertebrates is currently poorly understood.Results: Inhibiting PCSK7 in zebrafish results invarious developmental defects and dysregulationof gene expressions.Conclusion: PCSK7 is essential for zebrafishdevelopment and regulates the expression andproteolytic cleavage of TGF 1a.Significance: PCSK inhibitors are consideredfuture therapeutics for human diseases;understanding the biological role of PCSK7 istherefore critical.

SUMMARY

Proprotein convertase subtilisin/kexin (PCSK)enzymes convert proproteins into bioactive end-products. While other PCSK enzymes areknown to be essential for biological processesranging from cholesterol metabolism to host-defense, the in vivo importance of theevolutionary ancient PCSK7 has remainedenigmatic. Here, we quantified the expressionsof all PCSKs during the first week of fishdevelopment and in several tissues. PCSK7expression was ubiquitous and evident alreadyduring the early development. To comparemammalian and zebrafish PCSK7 we preparedhomology models, which demonstratedremarkable structural conservation. When the

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.453183The latest version is at JBC Papers in Press. Published on October 31, 2013 as Manuscript M113.453183

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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PCSK7 function in developing larvae wasinhibited we found that PCSK7 deficient fishhave defects in various organs, including thebrain, eye and otic vesicle, and this results inmortality within 7 days post fertilization. Agenome-wide analysis of PCSK7 dependentgene expression showed that, in addition todevelopmental processes, also several immunerelated pathways are regulated by PCSK7.Specifically, the PCSK7 contributed to themRNA expression and proteolytic cleavage ofthe cytokine TGF 1a. Consequently, TGF 1amorphant fish displayed phenotypicalsimilarities with PCSK7 morphants, whichunderscores the importance of this cytokine inthe zebrafish development. Targeting PCSKactivity has emerged as a strategy for treatinghuman diseases. Our results suggest thatinhibiting PCSK7 might interfere with normalvertebrate development.

Seven subtilisin/kexin-like proprotein convertaseenzymes (PCSK1, PCSK2, furin, PCSK4-PCSK7)modulate the biological activity of immatureproproteins by catalyzing limited proteolysis atsites containing a stretch of basic amino acidresidues. Consequently, PCSK enzymes areimportant regulators of a multitude of biologicalevents, including development, host-defense andhormone function (1). While the archetype PCSKspossess closely related, even redundant,biochemical properties in vitro, and often sharedsubstrate molecules, studies with geneticallytargeted animals and humans carrying inactivePCSK alleles argue for substrate specificity. Furin(2), PCSK5 (3) and PCSK6 (4) are essential fornormal mammalian development, whereas theinactivation of PCSK1 (5), PCSK2 (6) and PCSK4(7) result in tissue restricted phenotypes that rangefrom infertility to defects in the neuro-endocrinesystem. Genetic inactivation has also demonstrateda specific function for the more recently identifiedand biochemically unique PCSK family membersMBTPS1 (8) and PCSK9 (9) in cholesterol andlipid metabolism. In contrast, the biologicalfunction of the evolutionarily most ancient enzymePCSK7 has remained enigmatic. Scatteredreferences in the literature postulate that PCSK7deficient mice have little, if any, phenotypicabnormalities, but a comprehensive analysis ofthese animals has not yet been published (10-12).

It has been demonstrated that similarly to furin,PCSK5 and PCSK6, the expression of PCSK7 isubiquitous, and that it exerts its function mainly inthe trans-Golgi network and on the cell surface(13, 14). Importantly, previous biochemical studiesindicate that PCSK7 operates often redundantlywith other PCSKs, especially furin. For example,the activity of PCSK7 can be replaced in the site-specific cleavage of proBMP4 (15), proPDGF(16), proNGF (17, 18) and proVEGF-C (19).However, PCSK7 seems to be solely responsiblefor rescuing an unstable MHC I in post-ERcompartments (20) and for the proteolysis ofproEGF (21). In addition, a genome wideassociation study recently revealed that a SNP(rs236918) in the PCSK7 gene region is associatedwith an increase in the serum level of solubletransferrin receptor, thus implying that PCSK7could have a role in iron metabolism in humans(22). This observation was later confirmed byshowing that PCSK7 is the only convertase thatsheds transferrin receptor into the medium inseveral cell lines (23).

Most mammalian proteins and genes haveorthologs in the zebrafish, which makes them afeasible model for studying the role of PCSKenzymes in vertebrate biology in vivo. Previouslythe functions of furin (24), PCSK5 (25), MBTPS1(26) and PCSK9 (27) have been assessed indeveloping zebrafish. Inactivation of furin andMBTPS1 in zebrafish results in defective skeletaland cartilage formation, respectively, whereasPCSK5 and PCSK9 are important for neurologicaldevelopment. To decode the poorly definedPCSK7 function in vertebrate biology in vivo weinhibited PCSK7 in zebrafish using morpholinos(MO). The phenotypes of the PCSK7 morphantsclearly demonstrate that PCSK7 has a critical andnon-redundant role in early zebrafish development.

EXPERIMENTAL PROCEDURESGenes studiedBlast searches were used to look for zebrafishorthologies for human PCSK genes. The followingzebrafish PCSK genes were found:PCSK1: ENSDARG00000002600PCSK2: ENSDARG00000019451furinA: ENSDARG00000062909furinB: ENSDARG00000070971

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PCSK5a: ENSDARG00000067537PCSK5b: ENSDARG00000060518PCSK7: ENSDARG00000069968MBTPS1: ENSDARG00000014634PCSK9: ENSDARG00000074185

Zebrafish linesEmbryos used in all experiments were obtainedthrough natural crosses of wild-type AB strainindividuals. Adult fish used in QRT-PCRexpression analyses as well as in MO experimentswere also of the wild-type AB strain. All fish usedin the experiments were maintained under standardconditions at 28.5ºC. The care and analyses of theanimals were in accordance with the FinnishLaboratory Animal Welfare Act 62/2006, theLaboratory Animal Welfare Ordinance 36/2006and authorization LSLH-2007-7254/Ym-23 by thenational Animal Experiment Board.

QRT-PCR expression analysesExpression of PCSK genes was measured both invarious adult zebrafish tissues (female / malegonads, liver, kidney, intestine, eye, gill, brain,skin, tail) as well as in whole developing embryosof different ages (1-7 days post fertilization, dpf).Conventional quantitative real-time PCR with areverse-transcribed cDNA template from totalRNA was used. EF1A gene(ENSDARG00000020850) was utilized as ahousekeeping gene (28). Primers used in the QRT-PCR analyses are presented in Table 1.

MorpholinosPrior to morpholino design eight to ten individualfish of the AB strain were sequenced for each genein order to verify the sequence identity with thepublished sequence and to find genomic regionshomomorphic enough for MO design. The furinAand furinB MOs were designed to hit ATG-siteswhile the PCSK7 MOs target exon-intronboundaries in exon 3 (PCSK7 e3 MO) and exon 8(PCSK7 e8 MO). These exons contain codons foramino acids of the catalytic triad of the PCSK7enzyme. The morpholino for p53 wascommercially predesigned and the random control(RC) morpholino is a random base mixture atevery position intended for use as a negativecontrol.The sequences for the gene specific MOs usedwere as follows:

furinA:TCAATGAGGCAAGCCTGAGATCCATfurinB:ACAGCAGGATCAAGCGGCCCTCCATPCSK7e3:AGGACTCTGGAAAACACACAGGTTTPCSK7e8:CTTTATGGTTTGTGGATGTACCTGTp53:GCGCCATTGCTTTGCAAGAATTGTGF 1a:TCAGCACCAAGCAAACCAACCTCATMorpholinos were designed and synthesized byGeneTools (Philomath, OR, USA) and storeddissolved in distilled water (-20 °C) at a 1 mMconcentration.

Morpholino and RNA injectionsFor injections, 0.25 – 1.0 pmols of each MO wasused. A rhodamine dextran tracer was used tocontrol the injections and unsuccessfully injectedembryos were removed at 1 dpf. All injections (1-2nl) were administered into the yolk sac of a 1-4cell stage zebrafish embryo. 0.2 M KCl was usedas buffer in the injection solutions. The RNA usedin the RNA rescue experiments was transcribedfrom PCSK7 cDNA with the SP6 mMESSAGEmMACHINE Kit (Ambion, Austin, TX, USA)according to the manufacturer’s instructions(imaGenes, Berlin, Germany). All MO injectionexperiments were controlled with embryos injectedwith RC MO (GeneTools) as well as with non-injected embryos. Injections were carried out usinga PV830 Pneumatic Pico Pump (PWI) and a NikonSM7645 microscope. For visual analysis and livefish pictures, a SteREO Lumar V12 microscopewith the AxioVision Rel. 4.8 program (Carl Zeiss)were used.

PCSK7 histological stainingThe larvae were fixed in 4% paraformaldehyde-PBS -solution and embedded in 2% agarose. Next,the samples were dehydrated in an alcohol series(70%, 96%, absolute ethanol and xylene, 1 hour ineach). The dehydrated samples were embedded inparaffin and 5 µm transverse sections were cut.These were then fixed on glass slides at 58°C anddeparaffinised in xylene (2x4 min) and in absolute,96% and 70% ethanol (each 1-2x3 min) and rinsedwith dH20. The hematoxylin-eosin staining wasperformed as follows: Mayer’s hematoxylin 2 min,running tap water 2 min, water 1.5 min, 70%ethanol 15 s, eosin Y 15 s, 96% ethanol 30 s twice,absolute ethanol 1 min twice and xylene 1-4 min.

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Thereafter, the slides were mounted and pictureswere taken using an Olympus SZX16 microscopeand a Color View Soft Imaging System camera.

PCSK7 in situ hybridizationWhole mount in situ hybridization (ISH) wasperformed for PCSK7 as described in detail inThisse&Thisse 2008 (29) to analyze the detailedexpression pattern of the gene. 2 dpf embryos wereused. An antisense probe was used for specificdetection of the PCSK7 mRNA and a sense probeto control for unspecific background staining.

RNA microarray100 ng of total RNA (triplicate samples pooledfrom 18-35 embryos / group; two time points of 6hpf and 24 hpf) isolated from fish injected with RC(0.5 pmol) or PCSK7 e3 (0.5 pmol) + p53 (0.75pmol) MO was amplified and Cy3-labeled.Altogether, 1.65 µg of sample was then hybridizedon the arrays (Agilent 4x44K Zebrafish GE v3,65ºC, o/n). Chips were scanned using an AgilentTechnologies Scanner G2565CA and numeric datawere obtained from the Agilent Feature Extractionsoftware v10.7.1 (The Finnish Microarray andSequencing Centre, Turku, Finland). Only featureshaving corresponding Entrez ID available wereretained. Expression value for each gene has beencalculated in three phases: 1) If the same probeappears in the raw data file more than once, therow from the data file that has the highest averageexpression across all samples is used to representthat probe. 2) If the same gene appears in the rawdata file more than once, the probe that has thehighest average expression across all samples isused to represent that gene. 3) The resulting geneexpression values are quantile normalized acrosssamples. Genes with a median expression > 10 ona linear scale in either the PCSK7 morphant orcontrol samples were included in further analyses.A two-sample t-test was used to test thedifferential expression of each gene between themorphant and control samples using the log2 geneexpression values. To control for false discoveryrate, the resulting p-values from the t-test wereused to calculate q-values for each gene asdescribed in (30). Genes with a q-value < 0.05were retained. A log2 ratio between the median ofPCSK7 morphant samples and controls wascalculated for each gene, and genes with anabsolute log2 ratio 2 were considered

differentially expressed. A Gene Ontologyenrichment analysis was conducted usinghypergeometric distribution testing. Here, wetested whether the number of differentiallyexpressed genes annotated to a GO term was largerthan could be expected by chance. The resulting p-values serve as an indication of the possibleenrichment of each GO term.To summarize the GO categories, each enrichedterm was annotated to high level GO term. Highlevel GO term refers to terms that are directlylinked to root terms biological process, molecularfunction or cellular component. Finally the numberof terms under each high level GO term wascalculated. Here, we included 15 most enrichedterms into the analysis.The original raw data of the microarray is availablein the Gene Expression Omnibus database(http://www.ncbi.nlm.nih.gov/geo/).

Cloning, cDNA constructs and western blottingFurin-deficient RPE.40 cells (a kind gift from Prof.J. Creemers, KU Leuven, Belgium) were grown inHam´s F-12 medium supplemented with 10% fetalbovine serum and antibiotics. The cDNA sequenceencoding the zebrafish TGF 1a gene wasamplified from the wild-type AB zebrafish cDNA(7 dpf) by PCR (forward oligo 5 ´GGAGAATTCGCCATGAGGTTGGTTTGCTTGGTGCTG, and reverse oligo 5´-CATGGTGGTGAGGAACTGCAAGTGCAGTGGTACCGGA). The insert was subcloned intopcDNA3.1-MycHis plasmid and validated bysequencing. Human TGF 1 (ATCC) subclonedinto pcDNA3.1-MycHis, pSVL-hfurin (ATCC),pcDNA3-hPCSK7-FLAG (gift from Prof. J.Creemers), pME18S-FL3-zfFurinA, pME18S-FL3-zfFurinB or pCMV-SPORT6.1-zfPCSK7(imaGenes, Germany) were transfected in RPE.40cells using FuGENE® 6 transfection reagent(Promega). 48 hours after transfectionssupernatans were collected and cells lysed inTriton-X lysis buffer (20 mM Tris HCl pH 8.0,300 mM NaCl, 20 % glycerol, 0.1 % Triton x 100,1 mM EDTA, 50 mM NaF, 1 mM TCEP)supplemented with protease inhibitors (CompleteMini, Roche). Equal amounts of proteins wereseparated by SDS–PAGE and immunodetectionwas performed using anti-myc (M5546 Sigma)primary antibody and anti-mouse HRP secondaryantibody (HAF007 R&D Systems). Visualization

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was done using the ECL™ Western BlottingDetection –kit (GE Healthcare) and AGFACP1000 imaging system. Signal intensities wereanalyzed using ImageJ software (NIH, US).

RESULTSQuantification of PCSK gene expression inzebrafishThe zebrafish genome contains orthologs for mostmammalian genes. We blasted the human PCSKprotein sequences against zebrafish genomedatabases and found unambiguous orthologs for allbut two members (PCSK4 and PCSK6) of themammalian PCSK family. Two isoforms for bothhuman furin (furinA and furinB) and PCSK5(PCSK5a and PCSK5b) were present in thezebrafish genome, which probably arose from agenome duplication that occurred early in theevolution of teleost fish (31).

We first analyzed the expression of PCSK genes inthe whole developing zebrafish using a QRT-PCRapproach (1-7 dpf). In these experiments differentPCSKs showed variable expression patterns. Forexample, the PCSK family members with reportedimplications in neuronal development (PCSK1,PCSK2, PCSK5a and PCSK9) were significantlyupregulated during the early development (Figure1A). In contrast, the expression of other PCSKsremained relatively constant during the first sevendays of life. However, a subtle, but significantupregulation of the furinB, PCSK5b and PCSK7mRNA could be observed at 2.5 dpf. In accordancewith previous studies in mammals, ourexperiments assessing tissue specific PCSKexpression in adult fish showed particularly highexpression of PCSK1 and PCSK2 in eye and brain(32, 33) (Figure 1B). Apart from the lack ofPCSK2 in gill tissue, variable degrees ofexpression of all proprotein convertase enzymescould be detected with QRT-PCR in the testedtissues. Interestingly, two orthologs of furin andPCSK5 showed, to some extent, complementarytissue expression profiles. The PCSK7 expressionwas highest in female gonads, brain and eye inadult zebrafish. The magnitude of PCSK5a/b andPCSK7 expression levels were generally lowerthan those of the furin genes. These datacorroborate that PCSK7 is present in bothdeveloping larvae and multiple adult zebrafishtissues together with other convertase enzymes.

This allows the reliable assessment of the specificrole of the PCSK7 in vertebrate biology.

Homology modeling of PCSK7To assess putative structural and electrostaticdifferences between mammalian and zebrafishPCSK7 we prepared a homology model of theprotein with Modeller 9v10 (34) using publishedcrystallographic structures of mouse furin andyeast kexin as templates. Our modeling effortsshowed evident structural conservation incatalytic and P domains of PCSK7 between thehuman and zebrafish (Figure 2). We then analyzedthe binding of a substrate to PCSK7 bysuperimposing the crystallographic structure ofsubtilisin (PDB: 1CSE (35)) with the PCSK7models. These data demonstrated that most of thepotential substrate-binding residues are alsoconserved between human and zebrafish. Thealignment of the PCSK7 catalytic and P domainsof several species further demonstrated that theactive sites are close to identical, except for a non-conserved loop preceding the catalytic histidine(Figure 3). The human sequence VENG in thisloop is replaced by GPSD in zebrafish, whichpotentially affects substrate specificity at the P2 orP1’ site (Figures 2 and 3). Notably also, a littlefurther away from the active site, the differencesin the charge distribution can be observed (Figure2, lower part and Figure 3). In conclusion,PCSK7s in less developed organisms displayremarkable structural similarities, but subtleelectrostatic differences with mammaliancounterparts. This indicates that studying thezebrafish PCSK7 function can give insights alsoon other vertebrate homologues.

Inactivation of PCSK7 in zebrafish larvaeresults in developmental lethalityPrevious in vitro experimental data show that furincan often replace PCSK7 (19, 36, 37). To compareand contrast how these enzymes regulate zebrafishdevelopment we first interfered with furinA andfurinB translation using morpholino technology. Inaccordance with a previous report (24) thesimultaneous blocking of furinA and furinB withMOs resulted in a reduction in ventral jawstructures and consequently an open-mouthphenotype (data not shown). Disruption offurinA/B translation also reduced the survival offish larvae; only 30.6% (22/72 fish) of MO-

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injected fish were alive on 7 dpf. We next designedtwo distinct morpholinos that block the pre-messenger-RNA splicing at exon-intronboundaries around the catalytic site containingexons 3 and 8 in the zebrafish PCSK7 gene (e3 ande8 MOs). Injecting the PCSK7 targeting MOsalone or in combination resulted in numerousdefects that reduced the survival of fish larvaedramatically, and resulted in 100% mortality by 7dpf (Figure 4). The survival rate and grossphenotypes observed were MO dose-dependent,and the development of several organs, includingthe brain, eye, heart, otolith and tail was severelyaffected.

Because p53-dependent off-target neural toxicityhas been estimated to affect 15-20% of allmorpholino injections (38, 39), we co-injected ap53 MO together with the PCSK7 silencing MOs.The phenotypes and survival of morphant fishremained similar compared to fish not injectedwith the p53 MO (Figure 4). This indicates that theobserved lethality of the PCSK7 morphant was notdue to p53 mediated toxicity. To further validatethe specificity of the PCSK7 MO-phenotypes weset up an RNA rescue experiment. When in vitrotranscribed PCSK7 RNA was co-injected togetherwith e3 MO into the developing larvae the severityof the MO phenotypes was reduced and thesurvival improved (p<0.0001, log rank chi square38.94, df=1) (Figure 5A-B). To verify that thePCSK7 MOs specifically disrupt the splicing ofthe PCSK7 pre-mRNA molecules we amplifiedand sequenced the affected exon regions from themorphant fish (Figure 6A-B and data not shown).Our results demonstrated that injecting the e3 MOresults in three mRNA products with differentlengths, whereas the e8 MO deletes a 58 bpfragment at the end of exon 8. These data showthat PCSK7 morpholinos target PCSK7 pre-mRNA and that PCSK7 has a critical and non-redundant role in zebrafish development.

PCSK7 regulates brain, otolith and eyedevelopmentOur expression analyses implicated PCSK7 asprominently present in adult zebrafish eye andbrain tissues. To investigate the PCSK7 expressionpattern in larvae we employed RNA in situhybridization (ISH) on wildtype fish at two dayspost fertilization. These experiments demonstrated

that PCSK7 is most abundant in the entire cranialarea and the eye already in developing wildtypezebrafish (Figure 7A-B).

The observed gross anatomical differences andlocation of early PCSK7 expression prompted usto analyze, in more detail, the head regionphenotypes seen in the developing zebrafish. Adramatic underdevelopment of the entire brain andeyes was observed in histological sections (Figure7C-D). The cranium of PCSK7 morphantscontained mostly a non-cellular, mesh-likesubstance, while the brain remainedunderdeveloped and was located abnormallydownward between the eyes. In addition, thecellular structure in the eyes of MO-injected fishwas unorganized, and photoreceptor, outer andinner plexiform, inner nuclear and ganglion celllayers were undefined. However, the retinalpigment epithelium and lens structures wereevident also in morphant fish. The zebrafish with anon-functional PCSK7 also had an abnormalnumber of otoliths. Injection of the e3 MO resultedin a reduction in otolith number (1 otolith per earin >92% of fish, p=7.9e-63), while the fish injectedwith the e8 MO in contrast had an increasedamount of otoliths per ear (3 otoliths per ear in>26% of fish, p=2.4e-11). All evaluated 149control fish had the normal two otoliths per ear(Figure 8A-E). These data show that in accordancewith its spatio-temporal expression PCSK7 isimportant for several developmental processes ofthe vertebrate cranial organs.

Analysis of PCSK7 dependent gene networksGenome-wide expression analyses can beemployed to clarify the biological relevance ofgene-specific studies. Therefore, to comprehendthe gene networks, which are affected in thePCSK7 morphant fish, we performed a genome-wide mRNA expression study in whole fish larvaeafter PCSK7 inhibition. We chose to evaluate geneexpression at two different time points, 6 and 24hpf. At the gastrula stage (6 hpf), most of thezygotic genes have commenced transcriptaccumulation and often peak in their expression. Incontrast, after 24 hpf the genome-wide geneexpression profile remains relatively steady, aswas demonstrated in a previous analysis withwildtype zebrafish (40).

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Conventional PCSK enzymes are highly redundantin substrate processing. Therefore, it is importantto evaluate whether disrupting PCSK7 functioncauses any significant changes in the expression ofother proprotein convertases (41), which mightcontribute to or even compensate for the observedphenotype. At both 6 and 24 hpf the otherconventional PCSK genes were not found to besignificantly upregulated in PCSK7 morphant fish.PCSK7 expression, however, was significantlyreduced in the morphant zebrafish (logratio of -1.95 / p=5.77e-6 at 24 hpf). The decrease in thePCSK7 mRNA level is presumably due to thecellular deletion of the defectively spliced PCSK7pre-mRNA molecule.

To understand which major biological, molecular,and cellular processes are dependent on the intactPCSK7 enzyme we first performed a GeneOntology (GO) enrichment analysis usinghypergeometric distribution testing. At 6 hpf, asexpected from the observed morphant phenotypes,terms related to development (for example oticplacode formation) and transcription regulationwere abundantly enriched. Consequently, at 24 hpfgenes related to metabolism were also commonlyupregulated. Strikingly, at both 6 hpf and 24 hpfgenome-wide mRNA expression samples showeda clear enrichment in immune system relatedterms, immune system being the most enriched(p=0.0004) term at 24 hpf. Several cytokinesignaling affiliated terms could also be foundamong the most significantly enriched terms atboth time points (Supplementary Table S1, Figure9).

A more detailed analysis of the PCSK7 dependentgenes in the microarray revealed the dysregulationof several immune, neurological and otolith / oticvesicle related genes (Supplementary Table S2,Figure 9). For example, the expression levels ofimportant regulators of both the adaptive andinnate immunity STAT4, TGF 1a and CSFra weregreatly reduced at 6 hpf in the PCSK7 morphants,while otolith/ear related FOXI1 (downregulated at6 hpf) and PAX2A, MSXC and FSTA(upregulated at 6 hpf) were also dysregulated. Inaddition, a multitude of differentially expressedneurological genes, including severalprotocadherin and fibroblast growth factor genes,

were found to be dependent on a functionalPCSK7.

We have previously demonstrated that anotherPCSK family member, furin, is a key regulator ofboth T helper (Th) 1 type immune responses and Tregulatory cell mediated peripheral immunetolerance (42, 43). These crucial events of adaptiveimmunity are dependent on transcription factorSTAT4 function and cytokine TGF 1 signaling,respectively. We noticed that in our microarrayexperiment both of the aforementioned genes weresignificantly repressed when PCSK7 expressionwas blocked. To confirm this finding weperformed a QRT-PCR analysis that revealed adownregulation of both TGF 1a and STAT4 by11.7 and 5.1-fold, respectively, at 6 hours postfertilization in RC versus PCSK7+p53 comparison(three biological replicates, one-tailed p=0.037 forboth genes in Student’s t-test with Welchcorrection, data not shown). These findings implythat in addition to furin, also PCSK7 may have animportant regulatory role in developing adaptiveimmune responses.

PCSK7 regulates the expression of TGF 1a inzebrafishIn mammals, furin is the major proproteinconvertase enzyme that regulates thebioavailability of the anti-inflammatory TGF 1cytokine (43, 44). From previous experiments it isalso noteworthy that TGF 1 can directlyupregulate the expression of furin (45). However,PCSK7 can also proteolytically process the TGFsuperfamily cytokines. Mammalian PCSK7 issuggested to cleave proBMP4 in adevelopmentally regulated fashion and it possessesup to 1/3 of the capacity of furin in proTGF 1processing in vitro (15, 44). Furthermore, themRNA expression correlation of human PCSK7with TGF 1 is very strong, which may indicate aphysiological role for PCSK7 in proTGF 1processing (46). As described above, we observedthat TGF 1a (ENSDARG00000041502), thezebrafish counterpart for the mammalian TGF 1,was markedly downregulated in the microarrayanalysis at 6 hpf in PCSK7 morphant fish(Supplementary Table S2, Figure 9). Thiscoordinated expression of PCSK7 and TGF 1aprompted us to address the importance of PCSK7

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for TGF 1a expression and activation, and assesswhether the lack of TGF 1a could contribute tothe PCSK7 dependent phenotype.

We first wanted to investigate if zfPCSK7 candirectly proteolytically process zfproTGF 1a. Tothis end the cDNA encoding TGF 1a wasamplified from wild-type zebrafish and subclonedinto MycHis tagged expression plasmid. Afterverification by sequencing (data not shown) wethen co-expressed TGF 1a together with zebrafishfurinA, furinB or PCSK7 in furin deficient RPE.40cells. For comparison, we also co-expressedhuman TGF 1 together with human furin andPCSK7 cDNAs. Our results clearly demonstratethat, in addition to furin both zebrafish and humanPCSK7s are able to process and promote therelease of bioactive TGF 1 (zebrafish 16 kDa,human 14 kDa) into cell culture supernatants(Figure 10A). In line with previous reports, thePCSK7s possessed approximately one third of theactivity of furin in the TGF 1 maturation (44).

We then assessed if the TGF 1a mRNAexpression remains downregulated in PCSK7morphant fish also in a later developmental stageand found that TGF 1a gene expression issignificantly repressed also at 48 hpf (p=0.0052,Figure 10B). To investigate how the lack ofTGF 1a contributes to the PCSK7 morphantphenotype we injected zebrafish with a TGF 1ablocking MO. In these experiments we observedclear similarities between the TGF 1a and PCSK7morphants; both showed tail abnormalities,pericardial swelling and an abnormal number ofotholiths (24.4% of TGF 1a-morphants (21/86)had three otoliths / ear (p=2.16e-9 in Fisher 2x2test, Figure 10C-D)). Taking these observationstogether our data suggest that in zebrafish theintact PCSK7 enzyme is important for theexpression and bioavailability of mature TGF 1aand that a defect in this contributes to the observedphenotype of the PCSK7 morphant fish.

DISCUSSIONRegardless of extensive studies on proproteinconvertase enzymes in vertebrate biology thefunction and significance of the evolutionaryancient PCSK7 has remained largely unclear. In aneffort to fill this gap we studied the function of

PCSK7 in zebrafish and observed that it is criticalfor the development of zebrafish larvae. ThePCSK7 morphant fish display severedevelopmental defects that lead to 100% mortalitywithin the first seven days of life. The lack offunctional PCSK7 enzyme interferes with theorganogenesis of several key elements includingthe brain, eyes and otic vesicles. In addition, ourgenome-wide gene expression and biochemicalanalyses demonstrate that PCSK7 regulates genesimportant for organogenesis and immunology andthat it is specifically capable of contributing to thefunction of cytokine TGF a in developing fishlarvae.

To first validate the feasibility of zebrafish as amodel for the analysis of the PCSK7 function invertebrate biology we surveyed the expression ofall identifiable proprotein convertases. Inaccordance with previous reports in mammals, thezebrafish PCSK enzymes also show variation intheir developmental and tissue specific expression.Zebrafish homologues for the neuro-endocrinesystem specific PCSK1 and PCSK2 wereparticularly highly expressed in the fish neuraltissues and the homologues of previously reportedubiquitous enzymes, such as furin, PCSK5 andPCSK7 were found to be relatively widelyexpressed, similarly to earlier reports usingmammals (17, 18, 47). PCSK7 shares severalcommon substrate molecules with PCSK5 andfurin at least in in vitro analyses (48). We foundthat PCSK7 was co-expressed in different tissuesand developing fish together with other PCSKenzymes that have been reported to compensate itsbiological function. Taken together, ourquantitative PCSK expression data demonstratedobvious similarities between the mammalian andfish PCSK expression. Consequently, deleting thePCSK7 function in developing fish can giveimportant insights into the specific biologicalfunction of this poorly defined proproteinconvertase also in other vertebrates.

Others and we have previously reported notableevolutional conservation of the catalytic and Pdomains in the PCSK enzymes (46, 49). Toaddress specifically structural and electrostaticproperties in the PCSK7 enzymes we generatedhomology models of mammalian and fish PCSK7,and investigated the PCSK7 sequences in several

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species. In conclusion, our modeling data supportthe idea that catalytic and P domains in zebrafishPCSK7 share most structural features with thehuman counterpart. Therefore again, investigatingthe fish PCSK7 function is likely to generate novelinsights on the PCSK7 function in othervertebrates, too.

The crucial role of many of the PCSK enzymes invertebrate development is indisputable. Forexample, furin deficient mouse embryos showdefective ventral closure and axial rotation and dieduring the second week of embryonic development(2). Also, a mutation in furinA causes significantembryonic lethality in zebrafish, despite theduplication of the furin gene (24). Thesefundamental phenotypes can be explained by alack of processing of PCSK substrate molecules; inearly mouse development the significance of theproper activation of the TGF family cytokines,such as BMPs and Nodal (50) is particularlyemphasized. In contrast, a thorough functionalanalysis of a mammalian model for PCSK7 is notavailable in the literature. A few scatteredreferences to PCSK7 knockout mice, however,suggest either complete redundancy or at leastnon-critical functions in mammalian development(10-12). Our analysis of the PCSK7 morphant fishand a recent publication by Senturker et al using aXenopus model system (51) demonstrate thatPCSK7 is indispensable at least in lowervertebrates. Both studies come to the conclusionthat a lack of PCSK7 function leads to severedefects especially in the neural system and eye. Inzebrafish these defects lead to 100% mortalitywithin the first week post fertilization.

A complete knock-out or an inactive mutation ofthe gene would give the definitive answer for thebiological relevance of PCSK7 in zebrafish.However, to our knowledge, PCSK7-null zebrafishhave not been produced. To overcome thislimitation we analyzed how the lack of an activePCSK7 protein affects the larvae development.Using morpholinos to assess the biologicalfunction of a protein has provoked somecontroversy. In some cases morpholinos can causeunspecific phenotypes due to p53-dependent off-target neural toxicity. However, in our experimentssilencing the p53 pathway did not alter theconclusive role of PCSK7. In addition, sequencing

the MO-truncated PCSK7 mRNA showed that thee8 morpholino efficiently removes the end of exon8 (58bp) and shifts the reading frame in thecatalytic domain. This replaces roughly 50 C-terminal residues of the catalytic domain by 23residues of non-native sequence followed by a stopcodon. In contrast, the e3 morpholino results in thecomplete deletion of exon 3 of PCSK7. Thisremoves a codon encoding one of the amino acidsof the catalytic triad. In addition, the correctreading frame is lost for the remaining protein. Asa consequence, PCSK7 proteins translated in bothmorphants lack the entire P domain, which isneeded for correct folding of the enzyme (52). APCSK7 without P domain is unlikely to traverse tothe secretory pathway and it might be eventuallydegraded in cell.

Co-injecting PCSK7 mRNA with morpholinosignificantly improved the survival of morphantlarvae and partially restored the defectedphenotypes, but a complete rescue could not bereached. The partial improvement of phenotype byRNA rescue has also been reported by others (53).Therefore, it is probable that a more natural spatio-temporal RNA expression of PCSK7 than whatcould be achieved using RNA injections into theyolk sac would be needed to cancel out all theeffects of PCSK7 MO.

Little information has been available on PCSK7dependent biological processes at the genomiclevel. Our GO enrichment analysis revealedseveral biological, molecular and cellular functionsthat were significantly altered if PCSK7 functionwas inhibited. These findings corroborated theobserved gross phenotype of the PCSK7morphants by highlighting the enrichment ofseveral development-related GO terms.Noteworthy, the enriched GO terms also includedthe otic placode formation, which is a novelfinding and suggests, together with the abnormalamount of otoliths in the PCSK7 morphants, aspecific and non-redundant role for PCSK7 in eardevelopment. In the future, it will be interesting toassess if genetic alterations in human PCSK7 (22)play a role in hearing or balance relatedphenotypes.

One of the few specific PCSK7 functions in vitrois the rescue of an unstable MHC I – peptide

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complex (20). Analyses of microarray data on ourPCSK7 morphant fish revealed that PCSK7, inaddition to having a role in several developmentalpathways, is indeed linked strongly toimmunological processes. Differentially expressedgenes included several genes that are keyregulators of both the adaptive and innateimmunity, such as TGF 1a, STAT4, CSF1a andCCR7 and various MHC genes. The host-defenselinked GO terms containing these genes wereamong the most enriched categories already at 6hpf, and remained significantly overrepresenteduntil 24 hpf. It has been shown earlier that TGF 1upregulates the convertase furin, which is themajor proteolytic activator of this central anti-inflammatory cytokine (44). Interestingly, ourgenome-wide expression analysis demonstratedthat when functional PCSK7 is not availableduring early development TGF 1a was among themost downregulated genes at 6 hpf. The strengthof downregulation was weaker at later phases ofdevelopment, but TGF 1a expression remainedsignificantly downregulated up to two days postfertilization.

Importantly, TGF 1a and PCSK7 morphant fishshared several phenotypic similarities, whichfurther suggests that PCSK7 has a role in theregulation of the TGF 1a cytokine in developingzebrafish. The detailed mechanisms behind thePCSK7-dependent regulation of the TGF 1abioavailability are not fully understood. MatureTGF 1 cytokine is known to promote its ownfunction by upregulating the expressions of its ownmRNA and the converting enzyme furin.Accordingly, because others and we have shownthat also PCSK7 can process and activateproTGF 1 at a 1/3 of furin proteolytic capacity,the reduced PCSK7 expression can interfere thisfeed-forward loop and result in reduced TGF 1aexpression (44). However, it is equally probablethat in vivo PCSK7 promotes the TGF 1a functionin an indirect manner that does not involve directproteolysis (21).

In addition to its fundamental inhibitory role inimmunity (54, 55) TGF 1 directly controls celldifferentiation and proliferation. It is noteworthythat TGF 1 deficiency in mice causes significantintrauterine lethality (56). Our experiments concur

with this multifunctionality of TGF 1 and suggestthat in zebrafish TGF 1a plays a role in variousdevelopmental processes including otolithformation.

Proprotein convertases have a fundamental role inboth health and disease. Interfering with PCSKactivity holds a promise for future treatment of aplethora of diseases ranging from infections toatherosclerosis. These efforts are oftencompromised by the lack of specificity ofinhibitors of PCSK family members. If theinhibitors are considered for clinical use, it is ofutmost importance to understand also thebiological significance of PCSK7. Our datapresented here underscores the importance ofPCSK7 in zebrafish neural development, and morespecifically genomic processes associated withimmunity. These can also be important factors totake into account when extrapolating the unwantedeffects of general PCSK inhibitors in diseasesettings.

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36. McColl, B.K., Paavonen, K., Karnezis, T., Harris, N.C., Davydova, N., Rothacker, J., Nice, E.C.,Harder, K.W., Roufail, S., Hibbs, M.L., Rogers, P.A., Alitalo, K., Stacker, S.A., and Achen, M.G. (2007)Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptorVEGFR-2. FASEB J. 21, 1088-1098

37. Basak, A., Zhong, M., Munzer, J.S., Chretien, M., and Seidah, N.G. (2001) Implication of theproprotein convertases furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebolaand respiratory syncytial viruses: a comparative analysis with fluorogenic peptides. Biochem.J. 353, 537-545

38. Bedell, V.M., Westcot, S.E., and Ekker, S.C. (2011) Lessons from morpholino-based screening inzebrafish. Brief Funct.Genomics. 10, 181-188

39. Eisen, J.S., and Smith, J.C. (2008) Controlling morpholino experiments: don't stop making antisense.Development. 135, 1735-1743

40. Mathavan, S., Lee, S.G., Mak, A., Miller, L.D., Murthy, K.R., Govindarajan, K.R., Tong, Y., Wu,Y.L., Lam, S.H., Yang, H., Ruan, Y., Korzh, V., Gong, Z., Liu, E.T., and Lufkin, T. (2005) Transcriptomeanalysis of zebrafish embryogenesis using microarrays. PLoS Genet. 1, 260-276

41. Kim, W., Essalmani, R., Szumska, D., Creemers, J.W., Roebroek, A.J., D'Orleans-Juste, P.,Bhattacharya, S., Seidah, N.G., and Prat, A. (2012) Loss of endothelial furin leads to cardiac malformationand early postnatal death. Mol.Cell.Biol. 32, 3382-3391

42. Pesu, M., Muul, L., Kanno, Y., and O'Shea, J.J. (2006) Proprotein convertase furin is preferentiallyexpressed in T helper 1 cells and regulates interferon gamma. Blood. 108, 983-985

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43. Pesu, M., Watford, W.T., Wei, L., Xu, L., Fuss, I., Strober, W., Andersson, J., Shevach, E.M.,Quezado, M., Bouladoux, N., Roebroek, A., Belkaid, Y., Creemers, J., and O'Shea, J.J. (2008) T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance.Nature. 455, 246-250

44. Dubois, C.M., Blanchette, F., Laprise, M.H., Leduc, R., Grondin, F., and Seidah, N.G. (2001)Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme. Am.J.Pathol.158, 305-316

45. Blanchette, F., Day, R., Dong, W., Laprise, M.H., and Dubois, C.M. (1997) TGFbeta1 regulates geneexpression of its own converting enzyme furin. J.Clin.Invest. 99, 1974-1983

46. Turpeinen, H., Kukkurainen, S., Pulkkinen, K., Kauppila, T., Ojala, K., Hytonen, V.P., and Pesu, M.(2011) Identification of proprotein convertase substrates using genome-wide expression correlationanalysis. BMC Genomics. 12, 618

47. Pearton, D.J., Nirunsuksiri, W., Rehemtulla, A., Lewis, S.P., Presland, R.B., and Dale, B.A. (2001)Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates.Exp.Dermatol. 10, 193-203

48. Remacle, A.G., Shiryaev, S.A., Oh, E.S., Cieplak, P., Srinivasan, A., Wei, G., Liddington, R.C.,Ratnikov, B.I., Parent, A., Desjardins, R., Day, R., Smith, J.W., Lebl, M., and Strongin, A.Y. (2008)Substrate cleavage analysis of furin and related proprotein convertases. A comparative study. J.Biol.Chem.283, 20897-20906

49. Henrich, S., Lindberg, I., Bode, W., and Than, M.E. (2005) Proprotein convertase models based on thecrystal structures of furin and kexin: explanation of their specificity. J.Mol.Biol. 345, 211-227

50. Mesnard, D., Donnison, M., Fuerer, C., Pfeffer, P.L., and Constam, D.B. (2011) Themicroenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolyticactivities. Genes Dev. 25, 1871-1880

51. Senturker, S., Thomas, J.T., Mateshaytis, J., and Moos, M.,Jr (2012) A homolog of subtilisin-likeproprotein convertase 7 is essential to anterior neural development in Xenopus. PLoS One. 7, e39380

52. Zhu, X., Muller, L., Mains, R.E., and Lindberg, I. (1998) Structural elements of PC2 required forinteraction with its helper protein 7B2. J.Biol.Chem. 273, 1158-1164

53. Shu, X., Zeng, Z., Gautier, P., Lennon, A., Gakovic, M., Cheetham, M.E., Patton, E.E., and Wright,A.F. (2011) Knockdown of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene results inretinal degeneration. Invest.Ophthalmol.Vis.Sci. 52, 2960-2966

54. Shull, M.M., Ormsby, I., Kier, A.B., Pawlowski, S., Diebold, R.J., Yin, M., Allen, R., Sidman, C.,Proetzel, G., and Calvin, D. (1992) Targeted disruption of the mouse transforming growth factor-beta 1gene results in multifocal inflammatory disease. Nature. 359, 693-699

55. Prud'homme, G.J., and Piccirillo, C.A. (2000) The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases. J.Autoimmun. 14, 23-42

56. Kulkarni, A.B., Huh, C.G., Becker, D., Geiser, A., Lyght, M., Flanders, K.C., Roberts, A.B., Sporn,M.B., Ward, J.M., and Karlsson, S. (1993) Transforming growth factor beta 1 null mutation in micecauses excessive inflammatory response and early death. Proc.Natl.Acad.Sci.U.S.A. 90, 770-774

57. Baker, N.A., Sept, D., Joseph, S., Holst, M.J., and McCammon, J.A. (2001) Electrostatics ofnanosystems: application to microtubules and the ribosome. Proc.Natl.Acad.Sci.U.S.A. 98, 10037-10041

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58. Henrich, S., Cameron, A., Bourenkov, G.P., Kiefersauer, R., Huber, R., Lindberg, I., Bode, W., andThan, M.E. (2003) The crystal structure of the proprotein processing proteinase furin explains its stringentspecificity. Nat.Struct.Biol. 10, 520-526

59. Wheatley, J.L., and Holyoak, T. (2007) Differential P1 arginine and lysine recognition in theprototypical proprotein convertase Kex2. Proc.Natl.Acad.Sci.U.S.A. 104, 6626-6631

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AcknowledgementThis study was financially supported by the Academy of Finland (projects 128623, 135980 (MP), 121003(MaP), 140978 (VH), 132877 (MN)), a Marie Curie International Reintegration Grant within the7th European Community Framework Programme (MP), Emil Aaltonen Foundation (MP), Sigrid JuséliusFoundation (MP), Tampere Tuberculosis Foundation (MP), Tampere Graduate Program in Biomedicineand Biotechnology (TGPBB) (AO, SK), Finnish Cultural Foundation (AO, VK), Finnish Foundation forTechnology Promotion (VK), Laboratoriolääketieteen edistämissäätiö (AO) and Competitive ResearchFunding of the Tampere University Hospital (grants 9M080, 9N056 (MP)).CSC (IT Center for Science Ltd) is acknowledged for the computational resources and the FinnishMicroarray and Sequencing Centre, Turku, Finland, for the analysis service. The zebrafish work wascarried out at the Tampere Zebrafish Core Facility funded by the Biocenter Finland, TampereTuberculosis Foundation and Emil Aaltonen Foundation. Leena Mäkinen, Matilda Martikainen andHannaleena Piippo are acknowledged for technical assistance and advice in zebrafish work. TimoKauppila is acknowledged for the technical analyses of the PCSK genes and fruitful discussions.

Competing Financial Interests: none

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Figure legends

Table 1. Primers used in QRT-PCR analyses.

Figure 1. Expression of PCSK genes in zebrafish. QRT-PCR analysis was used to analyze the relativeexpression of the PCSK genes in A) developing zebrafish larvae (1-7 dpf) and B) various adult zebrafishtissues. A) PCSK gene expression levels were normalized to the housekeeping gene EF1A and thenormalized gene expression on 1 dpf was given a value of 1. Other time points are shown as relative tothis. Asterisks denote statistical significance for differences in comparisons between 1 dpf and other timepoints, * p<0.05, ** p<0.01, *** p<0.001 in Welch-corrected two-tailed Student’s t-tests. Experiments in1 A) were performed with three biological replicates (each sample consisted of 15 to 30 individual larvaedepending on the age of larvae). B) In adult tissue analyses gene expression levels related to that of EF1Aare shown. Experiments in 1 B) were performed twice in technical replicates with essentially similarresults. Error bars represent standard deviation. Note: diverse scales on the Y axis.

Figure 2. Homology models of human and zebrafish PCSK7. Upper part: yellow: catalytic domain,green: P-domain. The catalytic site is marked with an oval, and the surface of the catalytic serine ishighlighted in red. Amino acid differences close to the conserved substrate binding site are shown in stickpresentation. Lower part: Electrostatic potentials (kT/e) for the models were calculated using APBS 1.3(57) and visualized in PyMOL 2.7. Red-white-blue color indicates the ± 10 kT/e electrostatic potentialplotted on the protein surface.

Figure 3. ClustalW alignment of PCSK7 homologs. Human (Homo sapiens, UniProt: Q16549),zebrafish (Danio rerio, RefSeq: NP_001076494.1), mouse (Mus musculus, UniProt: Q61139), frog(Xenopus laevis, RefSeq: NP_001090019.1), pufferfish (Tetraodon nigroviridis, Ensembl:ENSTNIP00000017367), vase tunicate (Ciona intestinalis, RefSeq: XP_002125956.1), and starlet seaanemone (Nematostella vectensis, RefSeq: XP_001638665.1) PCSK7 catalytic and P-domains werealigned with those of mouse furin (PDB: 1P8J (58) and yeast kexin (Saccharomyces cerevisiae, PDB:2ID4 (59). The catalytic triad is marked with stars, and potential substrate-binding residues in dark gray.Residues showing differences in Figure 2 are highlighted with rectangles.

Figure 4. Phenotypes and survival of PCSK7 morphant fish. Zebrafish larvae (2 dpf) A) non-injectedor injected with B) random control (RC) MO (0.5pmol), C) PCSK7e3 MO (0.25pmol), D) PCSK7e8 MO(0.25pmol), E) PCSK7e3 MO (0.5pmol), F) PCSK7e8 MO (0.5pmol), G) PCSK7e3 + p53 MO(0.5+0.75pmol) or H) PCSK7e3+e8 MO (0.25+0.25pmol). A)-H) 35x magnification. I) Survival of fishinjected with PCSK7 e3 MO (0.5 pmol), PCSK7 e3 + e8 MOs (0.25 + 0.25 pmol) and PCSK7 e3 + p53MOs (0.5 + 0.75 pmol) was significantly lower than that of RC morphant (0.5 pmol) or uninjected fish(p<1e-51 for all comparisons by log-rank test). PCSK7 e8 MO reduced the survival of larvae similarly toe3 MO: all larvae die by 6 dpf (n=98 larvae, e8 MO 0.5 pmol, data not shown).

Figure 5. PCSK7 mRNA improves the survival and reduces the severity of the PCSK7 morphantphenotype. A) Survival of zebrafish uninjected or injected with PCSK7 e3 MO alone or together with invitro transcribed PCSK7 mRNA was monitored daily. Data are pooled from two independent experiments.B) 2 dpf zebrafish larvae co-injected with PCSK7e3 MO (0.5pmol) and PCSK7 mRNA (100pg).

Figure 6. PCSK7 MO injections result in erratic splicing of the pre-mRNA. A) Total RNA wasisolated from the control (RC MO) and different PCSK7 morphant fish and reverse-transcribed intocDNA, which was amplified by PCR and run on an agarose gel. Lanes from the left: 1. 100 bp molecularweight marker, 2. PCSK7 e3 MO, 3. control (=RC MO with PCSK7 e3 primers), 4. PCSK7 e8 MO, 5.control (=RC MO with PCSK7 e8 primers), 6. 100 bp molecular weight marker. Sequencing showed that345 bp and 288 bp DNA fragments represent intact, wildtype PCSK7 mRNA that can be detected in RCMO samples with the e3 (lane 3) and e8 primers (lane 5), respectively. The PCSK7 e3 MO injectionresulted in three differentially sized fragments: i) a fragment corresponding to the completely deleted exon3 (the shortest band in lane 2), ii) a fragment with a 58 bp deletion from the start of exon 3 followed by a49 bp polymorphic region either from exon 3 or from exon 4 and further supplemented with the untouchedend of exon 3 (the middle band in lane 2) and iii) a faint band of wild-type exon 3 (the longest band in

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lane 2). The PCSK7 e8 MO injection deleted a 58 bp fragment from the end of exon 8 (lane 4) resulting ina truncated PCSK7 mRNA molecule. B) Sequences for the primers used for the sequencing described inA).

Figure 7. PCSK7 is expressed in the head region and PCSK7 morphant fish have cranialdevelopmental defects. PCSK7 expression (dark blue) was analyzed with RNA in situ hybridization inwildtype zebrafish larvae at 2 dpf. A) PCSK7 antisense probe, B) negative control (PCSK7 sense probe).Right-hand panels show hematoxylin-eosin stained transverse sections of zebrafish cranial region of C)random control (RC) and D) PCSK7 e3 morphant (3 dpf).

Figure 8. PCSK7 morphant fish have an abnormal number of otoliths. Zebrafish were injected withA) random control (RC) MO (0.5 pmol), B) PCSK7 e3 + p53 MO (0.5 pmol + 0.75 pmol), C) PCSK7 e3MO (0.5 pmol) or D) PCSK7 e8 MO (0.5 pmol) and otoliths were visualized on 3 dpf. Quantification ofotoliths is presented in E).

Figure 9. Heatmap of the most differentially expressed genes and high level summary of geneontology enrichments between control and PCSK7 morphant fish. Samples were prepared and dataanalyzed as described in the methods section. A) Zebrafish genes that have an identifiable humanhomolog are shown in the heatmaps. B) Summaries from the enriched gene ontology terms. Pie chartsshow enriched high level categories for cellular component, biological process and molecular function at 6hpf and 24 hpf. In each chart the size of the wedge corresponds to the number of terms enriched under thegiven high level category.

Figure 10. PCSK7 regulates TGF 1a, which affects the zebrafish larvae development and otolithformation. A) Furin-deficient RPE.40 cells were transiently transfected with zebrafish TGF 1a-myc orhuman TGF 1-myc together with zfFurinA, zfFurinB, zfPCSK7, huFurin or, huPCSK7. ProTGF 1 (45kDa) and mature TGF 1 (16 kDa in zebrafish, 14 kDa in human) expressions were detected with westernblotting. Mature/pro TGF 1 ratios were quantified using ImageJ software and ratios in cells transfectedonly with TGF 1 cDNAs were given an arbitrary value 1. Equal loading of cell lysates and supernatantswere verified by Ponceau S staining (data not shown). Experiment was repeated twice with similar results.B) TGF 1a mRNA expression was measured by QRT-PCR from PCSK7e3+p53 and RC morphantembryos at 48 hpf (p=0.0052, two-tailed Student’s t test). C) and D) depict the phenotypes (4 dpf) ofzebrafish injected with TGF 1a+p53 MOs (TGF 1a MO 1.0 pmol, p53 MO 1.5 pmol). TGF 1amorphants had incorrect otolith number: TGF 1a MO: 65 fish with 2 otoliths / ear, 21 fish with 3 otoliths/ ear, RC MO: 120 fish with 2 otoliths / ear, 0 fish with 3 otoliths / ear, p=2.16e-9 in Fisher 2x2 test.

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Table 1.

gene sequence 5'-3'PCSK1 F CGGGAAAAGGAGTGGTCAT

R GGTGGAGTCGTATCTGGGPCSK2 F CGGATCTGTATGGAAACTGC

R GCCGGACTGTATTTTATGAATGfurinA F AGCATGTTCAAGCGCAG

R CCAGTCATTGAAGCCCTCAfurinB F CCAAGGCATCTACATCAACAC

R ACACCTCTGTGCTGGAAAPCSK5a F AAGCCATGGTACCTGGAAGA

R GGTCAGAGCTGGATTTGCTTPCSK5b F TGTTCCTCGACCCTTACCAC

R ATCTCGCCATGTCAGGAAAGPCSK7 F AGAGTGTTGGACGGGC

R TGCCTAATGGATGCGGTMBTPS1 F GATGTTATAGGTGTTGGAGGG

R TCACGATGTCAGGCTTCAPCSK9 F AGGGCAAGGGTACTGTG

R TGTTTAGGGTGCGACTGAEF1A F CTGGAGGCCAGCTCAAACAT

R ATCAAGAAGAGTAGTACCGCTAGCATTACTGF 1a F CAGGATGAGGATGAGGACTA

R CAGCCGGTAGTCTGGAATA

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

A) noninjected B) RC MO

C) PCSK7 e3 MO low D) PCSK7 e8 MO low

E) PCSK7 e3 MO high F) PCSK7 e8 MO high

G) PCSK7 e3 MO + p53 MO H) PCSK7 e3 MO + e8 MO

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A) non-injected n=216B) RC n=191

E) PCSK7e3 n=175

I)otic vesiclebrain

eye heart

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Figure 5.

0 1 2 3 4 50

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1. PCSK7 e3 MO 0.5pmoln=2632. PCSK7 e3 MO 0.5pmol+ RNA 100pg n=2553. non-injected n=410

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Figure 6.

A)

B)

Primer Sequence

e3 F ATGACATGAAGCGAGGGATG

e3 R CGATGGCCTCCATACTGTCT

e8 F AACCTATGCGGAGGAGTGTG

e8 R TGATGGAAACCAGCTCCATT

1 2 3 4 5 6

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Figure 7.

B) negative control

A) PCSK7 probe C) RC MO

D) PCSK7 e3 MO

photoreceptor layer

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Figure 8.

A) RC MO B) PCSK7 e3 + p53 MO

D) PCSK7 e8 MOC) PCSK7 e3 MO

at 2dpf

MO conc. pmol

1 otolith

per ear (%)

2 otoliths

per ear (%)

3 otoliths

per ear (%) p*

RC 0.5 0 149 (100%) 0

PCSK7 e8 0.25 0 109 (97.3%) 3 (2.7%) NS

PCSK7 e8 0.5 0 57 (73.1) 21 (26.9%) 2.4e-11

PCSK7 e3 0.25 88 (86.3%) 12 (11.7%) 2 (2.0%) 1.9e-55

PCSK7 e3 0.5 94 (92.2%) 6 (5.9%) 2 (2.0%) 7.9e-63

PCSK7 e3+p53 0.5+0.75 93 (86.9%) 14 (13.1%) 0 3.3e-55

* 2-sided 2x3 Fisher exact test PCSK7 MO vs RC

E)

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28

Figure 9.

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29

Figure 10.

TGF 1 - + + + + + + +PCSK - - furinA furinB PCSK7 - furin PCSK7

zebrafish

proTGF 1WB: -myc

Celllysates

A)

C) D)

Super-natants

mature TGF 1WB: -myc

kDa55

17

B)

1.0 6.9 1.5 2.3 1.0 6.4 3.4Mature/proTGF 1

TGF 1a expression at 48hpf

RC PCSK7e3+p530.0

0.5

1.0

1.5 **

Rel

ativ

e m

RN

A e

xpre

ssio

n

human