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Characterization of the astacin family of metalloproteases in C. elegans Park, Ja-On; Pan, Jie; Möhrlen, Frank; Schupp, Marcus-Oliver; Johnsen, Robert; Baillie, David L; Zapf, Richard; Moerman, Donald G; Hutter, Harald Jan 28, 2010

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RESEARCH ARTICLE Open AccessCharacterization of the astacin family ofmetalloproteases in C. elegansJa-On Park1, Jie Pan1, Frank Möhrlen2, Marcus-Oliver Schupp2, Robert Johnsen3, David L Baillie3, Richard Zapf4,Donald G Moerman4, Harald Hutter1*AbstractBackground: Astacins are a large family of zinc metalloproteases found in bacteria and animals. They have diverseroles ranging from digestion of food to processing of extracellular matrix components. The C. elegans genomecontains an unusually large number of astacins, of which the majority have not been functionally characterized yet.Results: We analyzed the expression pattern of previously uncharacterized members of the astacin family to tryand obtain clues to potential functions. Prominent sites of expression for many members of this family are thehypodermis, the alimentary system and several specialized cells including sensory sheath and sockets cells, whichare located at openings in the body wall. We isolated mutants affecting representative members of the varioussubfamilies. Mutants in nas-5, nas-21 and nas-39 (the BMP-1/Tolloid homologue) are viable and have no apparentphenotypic defects. Mutants in nas-6 and nas-6; nas-7 double mutants are slow growing and have defects in thegrinder of the pharynx, a cuticular structure important for food processing.Conclusions: Expression data and phenotypic characterization of selected family members suggest a diversity offunctions for members of the astacin family in nematodes. In part this might be due to extracellular structuresunique to nematodes.BackgroundAstacins are a family of zinc metalloproteases. There areseveral hundred astacins identified in a variety of differentspecies ranging from bacteria to humans (see [1,2] forreview). The first member of this family, a digestiveenzyme, was identified in the crayfish Astacus astacus [3].A second member of the family, bone morphogenetic pro-tein 1 (BMP-1), was found in vertebrates as a bone-indu-cing factor [4,5], illustrating the range of physiologicalfunctions associated with these proteases. BMP-1 and itsDrosophila homologues, Tolloid and Tolloid-like areamong the best characterized members of the family (see[6] for a recent review). BMP-1/Tolloid is conserved inevolution and found even in cnidarians [7]. In vertebratesit is involved in processing components of the extracellularmatrix, most notably fibrillar collagens, where it acts asprocollagen C-protease [8]. Additional substrates areTGF-b inhibitors like chordin/SOG. Cleavage of chordinby BMP-1 in the embryo leads to activation of the TGF-bsignaling pathway. This has been studied extensively inDrosophila, where activation of the TGF-b decapentaplegic(dpp) on the dorsal side is a key event in patterning thedorso-ventral axis [9]. In vertebrates BMP-1 plays an addi-tional role in the activation of two particular members ofthe TGF-b family. It directly cleaves the prodomain ofmyostatin and GDF11, leading to activation of thesegrowth factors [10,11].A subgroup within the astacin family are meprins, whichare confined to vertebrates and found in the small intes-tine, kidney and skin, where they are thought to cleavebiologically active peptides, cytokines and components ofthe extracellular matrix [12]. The discovery of the closerelationship between meprin and the crayfish astacin ledto the proposal to name this group of zinc metallopro-teases “the astacin family” [13]. The remaining astacinsform a rather diverse group including digestive enzymes,hatching enzymes and also the majority of the astacinsfound in C. elegans [3,14]. C. elegans astacins have beenclustered into six subgroups based on their domain orga-nization [14], specifically on domains found in the C-term-inal extensions adjacent to the catalytic site. Members of* Correspondence: hutter@sfu.ca1Department of Biological Sciences, Simon Fraser University, Burnaby, BC,CanadaPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14© 2010 Park et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.subgroup I (nas-1 to nas-5) have no additional domainsand subgroup II (nas-6 to nas-15) is characterized by thepresence of SXC/ShK toxin domains. Members of sub-group III (nas-16 to nas-30) typically have a single EGFdomain and a single CUB domain. Subgroup IV (nas-31and nas-32) has a single SXC/ShK toxin domain in addi-tion to the EGF and CUB domains, whereas members ofsubgroup V (nas-33 to nas-38) have a TSP1 domaininstead. Subgroup VI (nas-39) consists of the single BMP-1/Tolloid homologue in C. elegans.Only a few C. elegans astacins have been functionallycharacterized so far. hch-1/nas-34 is required for diges-tion of the outer eggshell and migration of a neuroblast[15,16]. nas-36 and nas-37 are required for molting[17,18]. They are expressed and probably secreted fromthe hypodermis and are thought to digest componentsof the cuticle to allow it to be shed. dpy-31/nas-35mutants are embryonic lethal and have characteristiccuticle synthesis defects [19]. DPY-31 is the only C. ele-gans astacin with a likely substrate identified. DPY-31 isthought to be responsible for C-terminal cleavage of thecuticular collagen SQT-3 [19], a function reminiscent ofthe role of BMP-1 in cleaving fibrillar collagens in verte-brates [8]. DPY-31 from two parasitic nematodes,H. contortus and B. malayi, has been shown recently tohave an evolutionary conserved function and a similarrange of protease activity [20].To begin a characterization of the remaining membersof this family we first determined the expression pattern ofpreviously uncharacterized genes. We then tried to isolatemutations in selected members of the different subfamiliesand were able to obtain mutations in nas-5,6,7,21 and 39,representing all but one of the previously uncharacterizedsubgroups. Mutant animals are viable in all cases indicat-ing that none of these genes is essential for survival. nas-6and nas-7 mutants show an incompletely penetrant slowgrowth and partial larval arrest phenotype. A moredetailed examination of these animals revealed structuraldefects in the pharynx, suggesting a role for these genes inpharyngeal development. We were not able to detect anyobvious defects in mutants in nas-39 mutants, the onlyC. elegans BMP-1/Tolloid homologue. The lack of pheno-types related to collagen processing or TGF-b signaling,characteristic phenotypes of its homologues in Drosophilaand vertebrates, suggests that this gene, while structurallyconserved, has functionally evolved independently innematodes.ResultsNematode astacin phylogenyThe C. elegans genome contains 40 astacin genes. In aphylogenetic tree based on alignments of the proteasedomain subgroups I and II cluster together and also sub-groups III-V (Figure 1). A comparison with sequences ofother nematodes like C. remanei, C. briggsae andB. malayi shows that the astacin family has undergone sig-nificant evolution within the nematodes. The phylogeneticanalysis points to a complex evolutionary history withinthe genus Caenorhabditis with multiple gene losses andduplications (Figure 1). The genome of B. malayi, ahuman parasite not closely related to C. elegans, containsonly 13 astacins. These represent five of the six subgroups.Not found in B. malayi are a large number of members ofsubgroup III as well as nas-39/BMP1/Tolloid. Recently thegenome sequences of several other nematodes havebecome available [21-23]. A preliminary analysis revealsthe presence of about 53 astacins in Pristionchus pacificus,about 30 in Meloidogyne hapla and about 37 in Meloido-gyne incognita. This would support the idea of a more gen-eral expansion of this protein family within nematodes.The recently sequenced genomes of the flatworms Schist-soma mansoni [24] and Schistosoma japonicum [25](phylum: Platyhelminthes) each contain only two astacins,orthologs of nas-4 and nas-39 (Additional file 1, Table S1).Astacins are expressed predominantly in tissues exposedto the outside environmentWe used two independent approaches to identify sites ofexpression for previously uncharacterized members ofthe astacin family: ‘green fluorescent protein’ (GFP)reporter constructs (Figure 2; Tables 1, 2) and serial ana-lysis of gene expression (SAGE) of different developmen-tal stages and embryonic tissues (Tables 3, 4). GFPreporter constructs were generated by fusing putativepromoter regions with a cDNA encoding GFP. Trans-genic animals were assayed for GFP expression. GFPreporters for eight of the genes gave no detectableexpression (see Table 2). Some of the remaining astacinsshowed expression in multiple tissues, but the majority ofthe genes were expressed in only a few cells or cell types(Table 1). Prominent sites of expression at the tissue andorgan level are the digestive system (pharynx and intes-tine) and the hypodermis, which express a large numberof astacins (Table 2). Notably underrepresented are‘internal tissues’ like body wall muscle, the nervous sys-tem and reproductive organs (gonad, uterus). These tis-sues only express a few astacins. Expression within thepharynx is essentially confined to two cell types, musclecells and marginal cells. Marginal cells lie between phar-yngeal muscle cells and form an integral part of the phar-yngeal myoepithelium. They are thought to providecontinuity and strength to the epithelium. Several asta-cins are expressed in various interfacial cells, many ofwhich are responsible for generating openings in thebody wall. Examples are the rectal and vulval epithelium,sensory sheath and socket cells, the excretory duct celland the uterine-seam attachment (Table 2). Five astacinsare expressed in gland cells of the alimentary tract, nas-5and nas-12 in pharyngeal glands and nas-2, nas-19 andPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 2 of 13+++Figure 1 Phylogenetic relationships of astacins within Nematodes. The tree was deduced by Bayesian analysis based on the alignment ofthe amino-acid sequences of the catalytic chain, covering the region from Ala-1 to Leu-200 in the prototype, crayfish astacin. The main clustersare shaded: Cluster 1: subgroups I and II; cluster 2: subgroups III-V, The typical domain composition of each cluster is depicted. S: Pre-Pro-Sequences; Astacin: protease domain; EGF: epidermal growth factor like modules; CUB: CUB domain; TSP1: Thrombospondin type 1 domains;SXC: ShK toxin/SXC domain. The scale bar represents a distance of 0.1 accepted point mutations per site (PAM). The number of orthologs inC. briggsae (Cbr), C. remanei (Cre) and B. malayi (Bma) is indicated in brackets. Genes with exactly one ortholog in each of these species are inred, genes missing specifically in B. malayi are indicated in blue, duplicated genes in other species are in bold print.Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 3 of 13nas-25 in rectal glands, suggesting a putative role asdigestive enzymes.Previously characterized astacins of subgroup V (hch-1/nas-34, dpy-31/nas-35, nas-36,37,) are all expressed in thehypodermis [16-19] and probably process cuticle compo-nents like cuticular collagens. We found eight more asta-cins expressed in hypodermis (Table 2). Four of thosebelong to subgroup II and four belong to subgroup IV,suggesting that certain members of these subgroups mightalso be involved in processing cuticle components.16 astacins across almost all subgroups are expressed inthe intestine, suggesting a possible role as food digestiveenzymes. With the exception of nas-16, all of these genesare expressed outside the alimentary tract as well. Amongthese are the characterized astacins dpy-31/nas-35 andnas-37, for which no role in food digestion has yet beenproposed [17-19]. Members of subgroup I have nodomains in addition to the metalloprotease domain. Theymight have broad substrate specificity, a characteristic fea-ture of food digestive enzymes. While two members ofthis subgroup are expressed in gland cells of the alimen-tary system (nas-2 and nas-5), or the gut (nas-2 and nas-3), there is also specific expression of some members inspecialized cells like anterior hypodermal cells, arcade cellsFigure 2 Astacin expression. Representative expression patterns of astacins are shown. Panels H and N show ventral aspects of the midbodyregions, all other panels show side views of head regions. Anterior is to the left. Scale bar 20 μm.Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 4 of 13or even certain neurons, arguing against a simple fooddigestive function.In an independent approach expression data for astacinswas extracted from a number of SAGE experiments wheredifferent larval stages and individual embryonic tissueswere sampled for general gene expression (Tables 3, 4).Overall only a minority of the astacins is represented inthese libraries (14 out of 40). The sequencing depth ofthese SAGE libraries is such that genes expressed at lowlevels or in only a few cells are not necessarily representedin the libraries. Nevertheless we did find representationfor three genes that did not give noticeable expressionwith GFP reporters. nas-8 was found in the L1 stagelibrary, nas-38 in the L2 library and nas-29 in the embryoTable 1 Astacin expression according to GFP reporter constructsgene subgrouppharynx intestine hypodermis muscle neurons othernas-1 I mu, mc arcade cellsnas-2 I all cells rectal glands, 2 cells in the headnas-3 I mu3-5 all cells hyp1, seam (weak) PDE ILso?nas-4 I mc(weak)nas-5 I mc,glandsrectal glands, utsenas-6 II mu, mc2 all cells major hyp body wall musclenas-7 II mu, mc all cells seam (strong), otherhyp (weak)arcade cells, spermatheca, vulva,rectal epithelial cells,nas-9 II major hyp uterus, spermathecanas-11 II anteriormost cellsmajor hyp CEPsh, AMsh, PHshnas-12 II glandsnas-13 II IL2L/R 1 pair ofamphid neuronsnas-14 II mu, mcnas-15 II mc mu3-6nas-16 III anteriormost cellsnas-19 III mu, mc all cells rectal glands, spermathecanas-21 III all cells major hyp (weak) utse, gonadnas-22 III utsenas-23 III mu(weak)weak, allcellsmajor hyp rectumnas-25 III mc rectal gland cells, pha-int valve,arcade cellsnas-26/toh-1III weak, allcellsmajor hyp uterus, vulva epithelium, AMsh,arcade cells, PHsh, rectalepitheliumnas-27 III all cells major hyp vulva epithelium, rectalepitheliumnas-28 III mu all cells coelomocytesnas-30 III all cells rectal epithelial cellsnas-31 IV exc. cell, AMsh, PHsh,nas-32 IV mu anal depressor muscle,intestinal muscle, vulvamuscleunidentifiedhead neuronshead mesodermal cellnas-33 V all cells tail hypdpy-31/nas-35V weak, allcellsmajor hyp vulva epithelium, rectalepithelium, AMsh, IL/OLso, exc.duct cellnas-37 V weak, allcellsmajor hyp incl. seam(weak), rectal epi. cellsvulval epithelium, rectalepitheliumnas-39 VI mu all cells vulva muscle, bwm, many/mostneuronsPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 5 of 13and several larval stages. In all cases absolute expressionlevels were very low with one to four tags per library.A comparison of expression levels across developmentalstages revealed stage-specific changes for three astacins.hch-1/nas-34 is strongly expressed in ooctyes andembryos, as expected from its function as hatchingenzyme [16]. nas-9 and nas-11 are both predominantlyexpressed in the third larval stage. Both genes areexpressed in the hypodermis and could play a role instage-specific processing of cuticle components.Very few astacins are represented in SAGE librariesfrom various embryonic tissues. hch-1/nas-34 is enrichedin embryonic hypodermal cells, again as expected from itsproposed function. The other astacins found in theselibraries show no strong tissue enrichment and aregenerally found in those tissues that show GFP expressionin the corresponding reporter strain - with the exceptionof a low level expression of nas-6, 7 and nas-11 in neu-rons, which was not seen with the GFP reporter expres-sion constructs.Functional analysis of astacin genesnas-5,6 and 7In order to study possible functions of astacins moredirectly we attempted to isolate deletion alleles for sev-eral members of the family across the various sub-groups. We were able to isolate deletions in nas-5,6,7,21and nas-39, members of all but one of the previouslyuncharacterized subgroups. All mutant strains wereviable and only one had obvious defects. nas-6(hd108)mutant animals displayed a slow growth phenotype,Table 2 Astacin expression summarized by tissuetissue No of genes genesno expression 8 nas-8, 10, 17, 18, 20, 24, 29, 38Major tissuesintestine 16 nas-2, 3, 6, 7, 11, 16, 19, 21, 23, 26, 27, 28, 30, 33, 35, 37, 39hypodermis (major hyp) 10 nas-6, 7, 9, 11, 21, 23, 26, 27, 35, 37muscle (pharynx) 10 nas-1, 3, 6, 7, 14, 15, 19, 23, 28, 32, 39muscle (other) 3 nas-6, 32, 39neurons 3 nas-3, 13, 32,39reproductive system (gonad, spermatheca, uterus) 5 nas-7, 9, 19, 21, 26glands (pharyngeal, rectal) 5 nas-3, 5, 12, 19, 25Interfacial epithelial cellsrectal epithelium 6 nas-7, 23, 26, 27, 35, 37vulva epithelium 5 nas-7, 26, 27, 35, 37pharyngeal marginal cells 9 nas-1, 4, 5, 6, 7, 14, 15, 19, 25other interfacial cellsarcade cells 4 nas-1, 3, 7, 26,sensory sheath and socket cells 5 nas-3, 11, 26, 31, 35,uterine-seam attachment 3 nas-5, 21, 22excretory duct cell 1 nas-35Table 3 Astacin expression in stage-specific SAGE librariesgene oocyte embryo L1 L2 L3 L4 adultnas-4 - - - - 1.05 1.05 -nas-7 0.26 1.54 0.65 1.33 - - -nas-8 - - 1.31 - - - -nas-9 0.31 - - - 2.53 0.42 -nas-11 0.07 0.11 - 0.38 4.21 0.9 0.47nas-14 0.17 0.26 - - - 1.05 -nas-28 0.52 0.77 - - - 1.05 -nas-29 - 0.38 - 1.33 1.58 0.53 -nas-31 - - 0.65 0.67 - - -hch-1/nas-34 11.01 5.12 - 1.78 - 2.11 -nas-37 0.1 - - 0.8 0.63 1.26 -nas-38 - - - 1.33 - - -enrichment with respect to a mixed stage library; only genes represented in mixed stage library (contains all genes with tags in any of these libraries)Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 6 of 13with a significant fraction of the animals not reachingthe L4 stage at normal speed (Table 5). About 10% ofthe animals arrested development at various larval stagesand never reached the adult stage. Upon closer exami-nation we detected defects in the pharynx of those moreslowly growing or arrested animals. Minor morphologi-cal abnormalities were apparent in the terminal bulb ofthe pharynx (Figure 3B). More significantly, the grinder,a cuticular tooth-like specialization in the lumen of theterminal bulb, looked highly abnormal in slow-growinganimals (Figure 3C). The role of the grinder is to grindup food (bacteria) before it is passed to the intestine.The abnormalities in the grinder probably do not allowan efficient processing of the food, which would explainthe slow growth or even arrest. Consistent with this ideawe find reduced pharyngeal pumping rates in slowgrowing animals (Table 6). The degree of visibleabnormality in the grinder correlates with the slowgrowth or arrest phenotype (stronger grinder defectscorrelating with stronger growth defects). Grinderdefects are apparent in embryos suggesting that thedefect is developmental in origin.Since the slow growth phenotype in nas-6 mutants isincompletely penetrant and since nas-5 and nas-7 areexpressed in the pharynx as well, we investigatedwhether double or triple mutant combinations enhancethe phenotype. We found that nas-6(hd108); nas-7(hd116) double mutants had a significantly increasednumber of slow growing animals, suggesting that nas-7(hd116) despite having no phenotype on its own, hassome role in grinder development as well. The pheno-type of individual slow-growing animals does not changein nas-6(hd108); nas-7(hd116) double mutants judgingfrom their morphology and their pharyngeal pumpingrate. nas-5(hd96) does not seem to be involved in thisprocess, since nas-5(hd96); nas-6(hd108) double mutantsare not different from nas-6(hd108) single mutants andsince nas-5(hd96); nas-6(hd108); nas-7(hd116) triplemutant animals are not different from nas-6(hd108);nas-7(hd116) double mutants (Tables 5, 6).Table 4 Astacin expression in tissue-specific SAGE librariesgene pharynx intestine hypodermis muscle neuronsnas-6a 0.36 1.99 0.45 - 0.74nas-7 0.18 - 0.23 - 1.11nas-11a - - 0.9 - 1.48nas-12 0.73 - - 0.59 -nas-14 2.18 - 1.81 - -nas-28 - - 0.9 1.19 -hch-1/nas-34 0.51 0.4 3.98 1.13 1.41enrichment with respect to an embryonic library; only genes represented in embryonic library (contains all genes with tags in any of these libraries)Figure 3 Morphological defects in nas-6 mutants. A) Head region of a wild type animal. The arrow points to the grinder in the second bulbof the pharynx. B) Morphological defects in the second bulb of the pharynx in nas-6 mutants (arrow). C) Morphological abnormalities of thegrinder in nas-6 mutants (arrow). Scale bar 10 μm.Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 7 of 13nas-21 and nas-39nas-21 is a member of subgroup III and is expressed inthe major hypodermis and the uterine-seam attachmentcell (Table 1). Because of this expression pattern poten-tial phenotypes to consider would be defects in the cuti-cle or in the attachment of the uterus to the vulva.None of these defects or any other obvious morphologi-cal abnormalities were found in nas-21(hd119) mutantanimals, leaving the cellular function of this geneunclear at this point. It is worth noting that nas-22 isstructurally very similar and has an overlapping expres-sion in both hypodermis and uterine-seam attachment.It is conceivable that there is functional redundancybetween those two genes.nas-39 is the only homolog of BMP-1/Tolloid inC. elegans. It shares the unique domain composition offive CUB domains and 2 EGF modules following themetalloprotease domain. Drosophila and vertebrateshomologs play prominent roles in embryonic develop-ment most notably in TGF-b signaling and processingof ECM components. Mutants for Bmp1 in mouse orTolloid in Drosophila are lethal. In contrast, nas-39(hd104) mutant animals are viable and show no obviousdevelopmental defects. The zinc coordination site in thecatalytic centre of the metalloprotease domain is deletedin nas-39(hd104), so that this mutation should result ina non-functional protease and hence represent a nullallele. A second allele nas-39(gk343) eliminates the firstexon and is also expected to be a null allele. nas-39(gk343) mutant animals also display no obvious defects.TGF-b signaling in C. elegans is involved in severaldevelopmental processes and mutants in the variousTGF-b genes have characteristic defects such as consti-tutive dauer formation [26], a reduced body length [27]or uncoordinated movement due to axon navigationdefects [28]. Neither of the nas-39 alleles shows any ofthese defects, suggesting that TGF-b signaling is notaffected in nas-39 mutants. The overall structure of thenervous system was examined in more detail with apan-neuronal marker in nas-39(hd104) mutant animals.We were not able to detect significant defects in thearrangement of neuronal cell bodies or obvious axonguidance defects, suggesting that these animals do nothave major neuronal defects. The function of this strik-ingly conserved gene in C. elegans currently is unclear.DiscussionNematode astacin phylogenyThe astacin family of metalloproteases with 40 memberspresent in C. elegans has expanded in nematodes morethan in any other phylogenetic group [14]. Orthologs formany of these genes are present in other members ofthe genus Caenorhabditis, like C. remanei or C. briggsae,suggesting that the major expansion of this family didnot occur very recently, i.e. not within the C. eleganslineage itself. This can be contrasted to the currentannotation of the parasitic nematode B. malayi whichcontains only 13 astacin genes. This suggests that eitherthere was a dramatic expansion of astacins within thelineage leading to the genus Caenorhabditis or thatB. malayi has lost members of this family. Gene losswithin nematodes seems to be a frequent phenomenon[29] and the overall number of genes in the B. malayigenome is estimated to be significantly smaller than inTable 5 slow larval growth in astacin mutantsGenotype animals hatching within 24 hours animals reaching L4 stage within 48 hours after hatchingwild type 99% (n = 494) 100% (n = 94)nas-5(hd96) 97% (n = 440) 100% (n = 111)nas-6(hd108) 98% (n = 214) 33%* (n = 205)nas-7(hd116) 100% (n = 208) 98% (n = 129)nas-5; nas-6 98% (n = 257) 31%* (n = 232)nas-5; nas-7 100% (n = 115) 100% (n = 115)nas-6; nas-7 99% (n = 260) 17%* (n = 135)nas-5; nas-6; nas-7 96% (n = 164) 14%* (n = 124)nas-21 (hd119) 98% (n = 508) 100% (n = 628)nas-39(hd104) 96% (n = 307) 99% (n = 650)nas-39(gk343) 100% (n = 233) 100% (n = 634)* significant with p < 0.01 (c2 test)Table 6 Pharyngeal pumping rate (pumps/minute) inslow growing animalsGenotype averageratemaximumrate% animals notpumpingwild type 160 ± 13 188 0%nas-6 39 ± 33 110 17%nas-5; nas-6 47 ± 37 124 17%nas-6; nas-7 37 ± 31 115 17%nas-5; nas-6;nas-740 ± 38 130 27%n = 30; animals not reaching L4 stage after 48 hours were scoredPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 8 of 13C. elegans or C. briggsae [30,31], which supports thesecond hypothesis. A large number of astacins in othernematodes like Pristionchus pacificus, Meloidogynehapla and Meloidogyne incognita and a small number inmembers of other phyla like Schistosoma mansoni alsopoint to a more general expansion of the astacin familywithin nematodes. Details of the evolutionary historycurrently remain unclear because of the limited numberof complete nematode genomes currently available.Astacin expression and function in nematodesOnly four of the astacins in C. elegans have so far beenfunctionally characterized. They are required for eitherdigesting egg-shell [15,16], shedding old cuticle duringmolting [17,18] or processing a cuticle component [19].All four genes belong to subgroup V and are expressedin the hypodermis, the tissue responsible for cuticlesynthesis and turnover. Site of expression and structuralfeatures defining the subgroups apparently are good pre-dictors of potential functions for these genes.With this idea in mind we started to characterize theexpression of the remaining astacins. Expression wasmainly analyzed by using reporter constructs under thecontrol of the putative promoter regions of the gene.While this strategy greatly simplifies expression analysisand allows high-throughput studies [32], one has tokeep in mind that reporter constructs do not alwaysfaithfully recapitulate the expression of the native gene.In cases where there is a discrepancy between reportergene expression and other expression data (e.g. SAGEdata) it is probably wise to consider that the reportergene expression may be problematic. Similarly ourobservation that some reporter constructs did not resultin any visible GFP expression most likely points to lackof essential control elements in the reporter constructrather than a genuine lack of expression of the corre-sponding gene. Keeping these limitations in mind, theobserved gene expression patterns allow us to assesstentative sites of action and provide suggestions forpotential functions.Putative cuticle-components processing enzymesThe comparison of expression pattern within subgroupsdoes not confirm a simple functional subdivision ofthese genes along the structurally defined subgroups.Expression in the major hypodermal cells - typical ofthe characterized members of subgroup V mentionedabove - is found in ten astacins belonging to subgroupsII, III and V. In a previous study one of the uncharacter-ized members of subgroup V (nas-38) was found to beexpressed in hypodermal cells as well [32]. Two of thegenes with hypodermal expression, nas-9 and nas-11,are upregulated in the L3 stage according to the SAGEdata and could therefore be involved in stage-specificprocessing of cuticle components. The other hypoder-mal astacins do not show any stage-specific expressionand might function in several or even all stages. nas-9was found in one RNAi experiment to cause a lowpenetrance of embryonic lethality [33] and in a differentexperiment RNAi against nas-11 resulted in retardedgrowth [34]. The relevance of these results with respectto the function of these genes currently is unclear.Putative digestive enzymesThe digestive tract consisting of pharynx and intestine isanother major hub of astacin expression. As with hypo-dermal expression there is little correlation betweensubgroups and expression and we find members ofalmost all subgroups expressed here. Some of these asta-cins might be food digestive enzymes. Known digestiveenzymes among astacins typically have no additionaldomains besides the protease domain [3]. C. elegansastacins of this type fall into category I (nas-1,2,3,4,5).nas-2 and nas-3 are expressed in the intestine and nas-5is expressed in pharyngeal glands, promoting thosethree astacins as the most promising digestive enzymecandidates. Three additional astacins (nas-12,19,25) arealso expressed in either pharyngeal or rectal glands andmight also function in digestion.Putative basement membrane processing enzymesA surprisingly large number of astacins are expressed inthe marginal cells of the pharynx. These cells are sand-wiched between the pharyngeal muscle cells and providecontinuity across the musculature of the pharynx. Thesecells face the lumen of the pharynx on one side and thebasement membrane surrounding the pharynx on theopposite side. It is unclear whether astacins produced bythese cells are secreted towards the luminal side ortowards the basement membrane. Currently there is noevidence that marginal cells produce digestive enzymes,so it seems more likely that marginal cell astacins aresecreted towards the basement membrane and involvedin processing basement membrane components of thepharynx.Muscle cells in C. elegans produce major basementmembrane components including collagen and laminins,which are known substrates of astacins in other animals.Ten astacins are expressed in pharyngeal muscle cellsand three are expressed in body wall muscle. Theseastacins potentially cleave components of the basementmembrane.Astacins in interfacial cellsInternal organs like the nervous system, body wall mus-cle cells and the reproductive system express only asmall number of astacins. In contrast, a large number ofastacins are expressed in a variety of interfacial cells, inparticular cells associated with openings in the bodywall, like rectal epithelial cells, sensory sheath andsocket cells or the arcade cells of the pharynx. It is con-ceivable that astacins expressed in these cells have anactive role in generating openings in the body wallPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 9 of 13through local breakdown of components of the cuticleand/or basement membrane.nas-6 and nas-7 in pharynx developmentMutations in nas-6 lead to characteristic defects in phar-ynx development, most notably abnormalities in the grin-der, a cuticular structure required for food processing.The simplest explanation for the defects is to assume arole for nas-6 in the processing of cuticular grinder com-ponents. nas-6 is expressed in pharyngeal muscle andmarginal cells, which are close to the grinder, supportingthis idea. Neither the molecular composition of the grin-der nor its development is known in any detail. The nat-ure of putative substrates for NAS-6 therefore iscurrently unclear. nas-7 seems to be required for thisprocess as well, since nas-6; nas-7 double mutants showsignificantly more defects compared to single mutants.Grinder formation might be controlled redundantly byother astacins as well, since even nas-6; nas-7 doublemutants only showed partially penetrant defects. A lackof mutants in most of the pharyngeal astacins currentlyprevents us from exploring this idea further.Redundancy in functionAlmost all astacins have been tested in several genome-wide RNAi screens. In addition to those astacins dis-cussed above, only nas-5, nas-7, nas-18 and nas-38 havebeen identified with phenotypes in RNAi screens: nas-5in a screen for axon navigation defects [35], nas-7 ashaving a reduced brood size or being embryonic lethaldepending on the genotypic context [36], nas-18 as regu-lating fat content [37] and nas-38 as being involved incontrolling life span [38]. These results point to a varietyof different physiological roles for these secreted pro-teases. It should be noted that the axon guidance defectsseen in the earlier RNAi screen with nas-5 could not beconfirmed in the nas-5 mutants and that the slow growthand pharyngeal defects observed in nas-6 mutants herehave not been reported in any of the published RNAiexperiments. The overwhelming majority of astacinshave not produced noticeable phenotypes in a number ofgenome-wide RNAi screens [33,34,39]. This might besimply due to the limited number of phenotypes scoredin these screens, but it could also be a sign of functionalredundancy within the family. In our study we foundenhanced defects in nas-6; nas-7 double mutants com-pared to nas-6 single mutants, but no defects in nas-7mutants alone. This kind of functional overlap with clo-sely related family members might be a common phe-nomenon within the astacins in C. elegans. In particular,the lack of observable phenotypes in nas-21 mutants inparticular at the uterine-seam junction might be due tofunctional overlap with nas-22 and/or nas-5, both ofwhich are also expressed in the uterine-seam attachmentcell. Further exploration of potential redundant functionof astacins would require the isolation of mutants in theremaining family members, since RNAi experiments donot always recapitulate the phenotypes expected fromthe mutants. Furthermore, targeting more than one genesimultaneously in RNAi experiments leads to a signifi-cant drop in the effectiveness of RNAi, which makes itdifficult to address functional redundancies with RNAialone [40].One evolutionarily conserved member of the astacinfamily in C. elegans is a unique member with no closerelative. NAS-39 is the BMP-1/Tolloid homologue,which in Drosophila and vertebrates has important rolesin TGF-b signaling and basement membrane collagenprocessing [6]. Developmental processes in C. elegansthat are known to depend on TGF-b signaling like axonguidance [28] or regulation of dauer formation [26] andbody length [27] are unaffected in nas-39 mutants, sug-gesting that nas-39 is not required to activate TGF-bsignals. In Drosophila and vertebrates nas-39 homolo-gues activate TGF-b signals by cleaving chordin/SOG,an inhibitor keeping the TGF-b in inactive form. Sincethere is no obvious chordin homologue in the C. elegansgenome, the lack of TGF-b related phenotypes is maybenot entirely surprising. Similarly, fibrillar collagens andlysyl oxidases, which are major substrates for BMP-1 invertebrates are also absent in C. elegans. The strongconservation of the unique domain composition of theBMP-1/Tolloid homologue is particularly striking andsomewhat puzzling in this context. There are severalpossible evolutionary scenarios to explain this: firstly,some identified substrates in vertebrates including base-ment membrane components laminin and perlecan arepresent in C. elegans and could be NAS-39 substrates.Secondly, it is possible that the original substrate for theBMP-1/Tolloid protease is still present in C. elegans andhasn’t been identified (neither here nor in other ani-mals). Thirdly, NAS-39 might have acquired additionalnematode-specific substrates before the original sub-strate had been lost from its genome. A good candidatefor the evolutionary oldest substrate in this case is chor-din, since it is found even in Cnidarians [41,42]).ConclusionsExpression data and phenotypic characterization ofselected family members suggest a diversity of functionsfor members of the astacin family in nematodes. Thelarge expansion of the astacin family in nematodes andthe documented functions of those members wheremutants are available suggest that the majority of theseproteins has evolved within the nematode clade to pro-cess components of the extracellular matrix and cuticle.The size of the family and potential redundancy amongclosely related family members complicates the func-tional analysis of astacins, most of which still remainfunctionally uncharacterized.Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 10 of 13MethodsPhylogenetic analysisThe C. elegans genome contains 40 Astacin genes(NAS-1-40, Möhrlen et al. 2003). NAS-40 was pre-viously annotated as pseudogene but the current genemodel (F54B8.15 in Wormbase release WS 198) predictsa complete protein coding sequence and has thereforebeen included in the phylogenetic analysis. The C. ele-gans genome contains a large duplication on chromo-some V, which contains a duplicate of nas-2. This geneis called Y19D10A.6 and is identical to nas-2 at theDNA and protein level. Consequently it has not beenincluded in this analysis. To identify orthologs in thecompletely sequenced Nematode genomes of C. brigg-sae, C. remanei, Brugia malayi and Pristionchus pacifi-cus we used representative C. elegans and vertebrateastacins, or their conserved domains, as queries forBLAST searches of WormBase (WS198 for C. briggsaeand C. remanei, WS207 for B. malayi and P. pacificus)and NCBI (Entrez Gene 10-30-2009 for the Schistosomaand Melodoigyne genomes).For phylogenetic studies the active protease domainsfrom all nematode astacins were aligned using CLUS-TAL and imported into GeneDoc for further manipula-tion. The alignment is available form the authors uponrequest. Bayesian phylogenetic analyses were performedby MrBayes 3.0beta4 [43] with the WAG matrix [44],assuming a gamma distribution of substitution rates.Prior probabilities for all trees and amino acid replace-ment models were equal, the starting trees were ran-dom. Metropolis-coupled Markov chain Monte Carlosampling was performed with one cold and three heatedchains that were run for 80,000 generations. Trees weresampled every 10th generation. Posterior probabilitieswere estimated on 3,000 trees (burnin = 5,000). Thetree presented here was visualised using TreeView.Generation of transgenic strains for expression analysisPutative promoter regions of astacins were amplified byPCR following the strategy described in [32]. 5’-upstreamregions extending either to the next gene or to a maxi-mum of 3 kb were used. Primers used and regions ampli-fied are described in Additional file 1, Table S2.Promoter::GFP fusions were generated by PCR-stitching[45]. Transgenic animals were generated as described[32].Analysis of GFP expression patternsMixed stage transgenic animals were examined for GFPexpression using a Zeiss Axioplan II microscope. Stacksof confocal images with 0.2 to 0.5 μm distance betweenfocal planes were recorded with a Quorum WaveFXspinning disc system. Image acquisition and analysis wasdone with the Volocity software package (Improvision).Cells were identified by location and cell morphology incomparison with reference images from Wormatlashttp://www.wormatlas.org/. Maximum intensity projec-tions of all focal planes were used to generate imagesfor the figures.SAGE analysisSAGE libraries were prepared and processed asdescribed elsewhere [46]. SAGE tags were mapped tothe latest stable release of Wormbase (WS190). Onlytags that could be unambiguously mapped to a singlegene were used. All tags mapping to the same genewere added up. Tags were normalized with respect tolibrary size and enrichment was calculated as ratio ofnormalized tags in a particular library and tags in thereference library. Reference libraries used were a mixedstage library for the stage-specific libraries and a wholeembryo library for the embryonic tissue libraries.Generation of mutantsDeletion alleles were isolated from a library of EMS-mutagenized animals using a poison primer approach toidentify small deletions in certain region of the gene[47]. PCR primer sets were designed using AcePrimer[48]. Details about primers and deletions are given inAdditional file 1, Table S3.Phenotypic characterization of mutantsTen young adult hermaphrodites were placed on anNGM plate with E. coli OP50. After one hour, adultworms were removed and the eggs were incubated at20°C. 24 hours after the eggs were laid, the numbers ofhatched animals and the numbers of embryos that didnot hatch was counted. 48 hours after the eggs werelaid the numbers of total animals on the plate and thenumbers of animals reaching L4 stage were counted.Pharyngeal pumping was scored under stereomicroscopefor 1 minute in 30 worms, which did not reach L4 stage.Additional file 1: lists of orthologs, primer sequences and deletionalleles. Table S1: Orthologs of C. elegans astacins. Table S2: Primers usedto amplify promoter regions. Table S3: Details of deletion alleles used forfunctional analysis.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-213X-10-14-S1.DOC ]AcknowledgementsWe would like to thank members of our labs for critical discussion of theexperiments and for comments on the manuscript, Steven Jones forbioinformatic support and Marco Marra for assistance with sequencing. Thiswork was supported initially by the Max Planck Society and later by a grantfrom the National Sciences and Engineering Research Council of Canada(NSERC) to HH, who is also a MSFHR Senior Scholar. DLB is a CanadaResearch Chair in Genomics and is also supported by an NSERC grant. Thecontributions of DLB and DGM were supported by a grant from GenomeCanada and Genome B.C.Author details1Department of Biological Sciences, Simon Fraser University, Burnaby, BC,Canada. 2Department of Zoology, University of Heidelberg, Heidelberg,Park et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 11 of 13Germany. 3Department of Molecular Biology and Biochemistry, Simon FraserUniversity, Burnaby, BC, Canada. 4Department of Zoology, University ofBritish Columbia, Vancouver, BC, Canada.Authors’ contributionsJ-O P, JP and MS generated GFP reporter strains and isolated and analyzedastacin mutants. FM characterized GFP reporter strains and did thephylogenetic analysis. RJ and DB produced the majority of the GFP reporterstrains. RZ, MM, SJ and DM provided the SAGE data, HH analysed expressionpatterns and wrote the manuscript together with FM, DGM and JP. Allauthors read and approved the final manuscript.Received: 9 June 2009Accepted: 28 January 2010 Published: 28 January 2010References1. Barret AJ, Rawlings ND, Waessner JF: Handbook of Proteolytic EnzymesAcademic Press, London 2004.2. Zwilling R, Stöcker W: The Astacins: Structure and Function of a New ProteinFamily Verlag Dr. Kovac, Hamburg 1997.3. Titani K, Torff HJ, Hormel S, Kumar S, Walsh KA, Rodl J, Neurath H,Zwilling R: Amino acid sequence of a unique protease from the crayfishAstacus fluviatilis. Biochemistry 1987, 26:222-226.4. Wang EA, Rosen V, Cordes P, Hewick RM, Kriz MJ, Luxenberg DP, Sibley BS,Wozney JM: Purification and characterization of other distinct bone-inducing factors. 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McKay SJ, Jones SJ: AcePrimer: automation of PCR primer design basedon gene structure. Bioinformatics 2002, 18:1538-1539.doi:10.1186/1471-213X-10-14Cite this article as: Park et al.: Characterization of the astacin family ofmetalloproteases in C. elegans. BMC Developmental Biology 2010 10:14.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitPark et al. BMC Developmental Biology 2010, 10:14http://www.biomedcentral.com/1471-213X/10/14Page 13 of 13

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