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CellAdhesion&Migration7:1,122–134;January/February2013;G2013LandesBioscience Laminin 332 processing impacts cellular behavior Patricia Rousselle1,* and Konrad Beck2 1SFRBioSciencesGerland-LyonSud;InstitutdeBiologieetChimiedesProtéines;UMR5305,CNRS;UniversitéLyon1;Lyon,France;2CardiffUniversitySchoolofDentistry;Cardiff,UK Keywords: laminin 332, LG45 domain, LE domain, syndecan, basement membrane, keratinocyte Abbreviations: ECM, extracellular matrix; LG, laminin globular domain; pre-laminin, precursor-laminin; MMP, matrix metalloproteinase; mTLD, mammalian tolloid; BMP-1, bone morphogenic protein 1; JEB, junctional epidermolysis bullosa; pdb, protein data bank Laminin 332 and Its Maturation Events Laminin 332, composed of the a3, b3 and c2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial- Laminin 332, composed of the a3, β3 and c2 chains, is an mesenchymal cohesion in tissues exposed to external forces, epithelial-basement membrane specific variant. The a3 chain is including skin and stratified squamous mucosa. After being encoded by the LAMA3 gene, which has two transcript variants secretedanddepositedintheextracellularmatrix,laminin332 a3Aanda3B.AsthetruncatedLAMA3Avariantisexpressedand undergoes physiological maturation processes consisting in incorporatedintolaminin332heterotrimers,8wewillrefertothe the proteolytic processing of domains located within the a3 a3Aβ3c2 trimer as representing laminin 332 in this review. Its and the c2 chains. These maturation events are essential for main role in normal tissues is the maintenance of epithelial- laminin332integrationintothebasementmembranewhereit mesenchymal cohesion in tissues exposed to external forces plays an important function in the nucleation and mainten- including skin and stratified squamous mucosa.9,10 To fulfill this ance of anchoring structures. Studies in normal and patho- function, laminin 332 undergoes several physiological post- logical situations have revealed that laminin 332 can trigger translational processing events allowing its supramolecular distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and integration into the basement membrane. In other situations structuralcharacteristicsoflaminin332domainsarepresented such as wound healing or carcinoma, additional cleavages may andwediscuss whetherthey trigger specificfunctions. take place delivering pro-migratory signals to cells therefore compromising the adhesive function of the protein. Laminin 332 processing and integration into the skin basement membrane. In skin, laminin 332 is synthesized by Laminins are large extracellular glycoproteins that are important keratinocytesasahighmolecularweightprecursorproteinof460 components of all basement membranes. They are involved in kDa.AftersecretionanddepositionintotheECM,thea3andc2 severalbiologicalprocesses,includingselfpolymerization,binding chainsundergomaturationeventsconsistinginspecificprocessing to the extracellular matrix (ECM) and cellular interactions.1,2 All to smaller forms.4,11 The 190–200 kDa a3 chain (a3 ) can be 200 lamininsarecomposedofthreedifferentgeneproducts,termeda, successively processed at both C- and N-terminal extremities β and c chains assembled into a cross-shaped heterotrimer aβc. producing 165 (a3 ) and 145 kDa (a3 ) maturation 165 145 The three chains assemble within the endoplasmic reticulum products. The 155 kDa c2 chain (c2 ) is processed in the N- 155 through their C-terminal domains to form a triple stranded terminal region leading to a 105 kDa product (c2 ). 105 a-helical coiled coil rod.3,4 Sixteen laminin isoforms of different Processing of the a3 chain consists of cleavage of the C- subunitcompositionselectedfromfiveindividualachains(a1to terminal globular domains 4 and 5 (LG45) within the spacer a5), three β chains (β1 to β3) and three c chains (c1 to c3), are betweenLG3andLG4(Fig.1AandB).12,13Invitrostudiesusing known with variable cell and tissue specific expression, and they human laminin 332 have revealed that enzymes involved in the are differentially recognized by cellular receptors.5 All laminin a processing include plasmin,12 MMP-2, MT1-MMP and the C- chains possess a large globule at the carboxyl-terminal end that proteinase family of enzymes, especially mammalian tolloid consists of five similar domains LG1 to LG5 each containing (mTLD) and the bone morphogenic protein 1 (BMP-1).14,15 In about 200 residues.6,7 addition, thrombin was shown to have the potency to cleave LG45specifically.16Alternatively,theremaybeothermechanisms that control the rate of laminin a3 LG45 processing, such as the tissue plasminogen proteolytic cascade.12 N-terminal amino acid sequencing of human LG45 purified from the conditioned *Correspondenceto:PatriciaRousselle;Email:[email protected] medium of either primary or immortalized human keratinocytes Submitted:07/16/12;Revised:11/12/12;Accepted:12/05/12 andofthehumangastricadenocarcinomacellsSTKM-1revealed http://dx.doi.org/10.4161/cam.23132 thatthea3chain iscleaved betweenQ and D withinthe 1337 1338 122 CellAdhesion&Migration Volume7Issue1 SPECIALFOCUSREVIEW:LAMININS hingeregionbetweenLG3andLG4suggestingthataproteolytic Laminin 332 was shown to be the major component of cleavagesiteoftheLG45domainmatchestheminimalconsensus anchoring filaments in skin9 where it mediates cell adhesion via sequence LLQD (Fig.1B).17,18 It is not known what endopepti- interaction of the a3 carboxyl-terminal LG1–3 triplet domain dase catalyzes the hydrolysis of this cleavage sequence, and the with both a3β1 and a6β4 integrins,26-29 while the N-terminal existence of additional proteolytic cleavage sites has been shortarmsconnecttobasementmembranecomponents.Laminin suspected both in the hinge and the adjacent regions of the 332canbeincorporatedintothebasementmembranethroughat LG3 and LG4 domains.7,13,14,19 This hypothesis is compatible least two mechanisms. The first involves cross-linking of laminin with the spacer length of the laminin a3 chain, which is longer 332 with laminin 311 (a3β1c1) in the skin basement than those of the a1, a2 and a5 chains.7 Punctual mutations membrane.21 The complex of laminin 311 with laminin 332 is within the spacer region and/or deletion of the sequence LLQD mostlikelytoderivefromaninteractionofdomainLNintheβ3 did not protect the a3 LG3-LG4 linker from cleavage.14,19 As chain of laminin 332 with domain LE of the a3 chain short arm proteolyticprocessingofLGdomainsoccursinallachainsexcept inlaminin311(seebelow).Asseenonrotaryshadowingelectron a1,thesubstitutionofthea3LG3-LG4hingewiththatofthea1 micrographs of the complex, the LN domain of the short arm chain was an elegant way to render the laminin a3 chain interacts with a laminin 311 domain near the intersection of the uncleavable.19 This was also the case when a 46 amino-acid laminin 311 short arms.21 These complexes are most likely portion within the a3 LG3-LG4 hinge was deleted.16 These stabilized by a disulfide bridge between an unpaired cysteine in LG45-uncleavablea3chainconstructswereexpressedinhuman19 domain LN of the β3-chain and domain LE of processed a3 in or mouse16 skin keratinocytes deficient for expression of the a3 laminin311.Asimilarcomplexbetweenlaminin332andlaminin chain, and in both cases the resulting LG45-uncleavable 321 (a3β2c1) is found in the basement membrane of the heterotrimeric laminin 332 was deposited in the ECM. amnions. According to the 3-arm interaction hypothesis of Information gained from the structure of the mouse a2 chain laminin polymerization,30 the dimers could self-associate. The LG45 domain pair revealed an unusual path of the LG3-LG4 secondmechanismreportsadirectinteractionbetweenanchoring linker as it forms an inter-domain disulfide bridge to LG5.20 filaments and anchoring fibrils. Anchoring fibrils are disulfide BeingintegratedintotheLG45tandemthelinkeristhereforean bondstabilizeddimersoftypeVIIcollagen.31Monomericlaminin integral part of the structure, a feature that is conserved in all 332 as well as the laminin 332/311 dimer directly bind the lamininachains.Itistemptingtospeculatethatproteinasesmay N-terminal globular domain NC1 of type VII collagen.10,32 The have easy access to the LG3-LG4 cleavage sites located within a interactionislikelytooccurwithintheshortarmoftheβ3and/or well-exposed linker.7 c2 subunit.32 Further cleavage occurs in theN-terminal LEregion of the a3 Other processing events in laminin 332. In addition to the chain releasing the full short arm which might be important for laminin 332 maturation aimed at regulating the skin basement laminin 332 function (Fig.1A).14,21 A mutation causing an N- membrane structural integrity, other studies have reported two terminal deletion of 226 amino acids in the human a3A isoform specific migration-inducing MMP-2 cleavages of the laminin c2 was identified in the laryngo-oncho-cutaneous syndrome, a rare N-termini producing c2 chain fragments of 100 and 80 kDa.33 autosomal recessive disorder characterized by chronic production Thesec2processingcouldalsobecarriedbyMT1-MMPincolon of vascularized granulation tissue.22 Cultured keratinocytes andbreastcarcinomacells.34Bothenzymes,thatwerefirstshown obtained from patients revealed that heterotrimeric laminin 332 tocleaveratlaminin332,wereproposedtoalsocutdownhuman carrying the mutant a3 chain is assembled and secreted. The laminin 332 in both physiological and pathological situations mutated laminin was regularly expressed in the skin basement such as tumorigenesis.35,36 MMP-3, -12, -13, -19 and -20 were membrane and only very subtle ultrastructural changes were seen also shown to process the c2 chain inducing epithelial cell such as focal widening of the lamina lucida in places where migration.37,38 Migration events might therefore result from the hemidesmosome plaques were smaller. These studies suggest that interaction of newly exposed laminin domains with cell surface thelaminina3N-terminaldomainmaybeakeyregulatorofthe signaling receptors. Rat recombinant c2LEb1-LEb4’ was shown granulation tissue response during wound healing. to bind and activate the EGF receptor to stimulate cell In human skin laminin 332, the processing of the c2 chain migration.39 Studies with cancer cells have reported that MMP- 155 leadstothec2 subunitandwasshowntobeachievedbyBMP- 7,MT1-MMPandhepsincleave lamininβ3leading toincreased 105 1 and mTLD metalloproteinases.11,14,15,23,24 The N-terminal migration.40-42 The molecular mechanisms of laminin 332 in processing is complex as it occurs within a disulfide-linked loop squamous carcinoma have been reviewed in detail by of the L4 domain followed by reshuffling of disulfide bonds for Marinkovich.43 release of the cleaved fragments.24 The immunohistochemical analysis of laminin 332 in mTLD/BMP-1-deficient mouse skin Laminin 332 Sequence-Structure Relationship revealedastrongexpressionoftheprecursorc2chainconfirming that these enzymes are involved in the c2 chain maturation in The structure of the heterotrimeric laminin 332 molecule vivo.25 Incontrast,theprocessingoftheLG45domaininthe a3 consisting of disulfide-linked a3, β3 and c2 chains was first chain seemed, at least partially, unaffected reinforcing the revealedbyrotaryshadowingelectronmicroscopy,whichshowed hypothesis that several enzymes are involved in this maturation a rod 107 nm in length terminated by one large and two small process. globules at opposite ends9 which correspond to the C-terminal www.landesbioscience.com CellAdhesion&Migration 123 Figure1.Structureofhumanlaminin332anditsphysiologicalmaturationprocess.(A)Laminin332iscomposedofthreesubunitsa3A,b3andc2.Each chainiscomposedofdifferentdomainsthatareindicated.DomainL4inthec2chaincorrespondstoanLEdomainwitha~180residuesinsertbetween thethirdandfourthcysteineofthecanonical8-Cyspattern.LE4’andLEb4’ofthea3Aandc2chainaretruncatedLErepeatscontainingonlythefirstfour andsixcysteines,respectively,ofthepattern.Basedontheoddnumberofcysteines,b3LN,a3ALE4’,b3LE6andc2LEb1(aftermaturation),andthecoiled coilofa3havefreeSHgroupsasindicated.ThecoiledcoilisstabilizedbydisulfidebondsattheN-terminus(dottedlines)thoughtheconnectivityisyet unknown.ThelargeLGstructurelocatedattheC-terminalendofthea3chaincontainsfiverepeatingLGdomains.Thefirstthreerepeats(LG1–3) interactwitha3b1,a6b1anda6b4integrinswhilethelasttwo(LG45)containbindingsitesforsyndecan-1and-4.Laminin332issynthesizedasa precursormoleculethatundergoesmaturationbyproteolyticprocessingsatthea3AchainN-andC-terminusaswellasatthec2chainN-terminal extremity.Thecleavagesitesareindicatedbyarrowsaswellasenzymesinvolvedidentifiedsofar.(B)SchematicstructuresofLG.ThefiveLGdomains containnumerouscysteinesthatarenumbered.Disulfidebridgedandfreecysteines(SH)withinthehumansequenceareindicated.Atthebottom,an alignmentofthehumanandmouseLG3-LG4linkerisshown.Eachsequencedisplaysthesamecleavagesite(underlined)andacysteine(printedin inversefont)thatformsaninter-domaindisulfidebondtoLG5.20ThesequenceP PFLMLLKGSTRsupposedtobecrucialforintegrina3b1binding58is 1312 doubleunderlined. 124 CellAdhesion&Migration Volume7Issue1 LG and N-terminal LN and LE/L4 domains, respectively for protease susceptibility. Indeed, major processing occurs by (Fig.1A). This length is consistent with the later determined cleavage of the G -D site (Fig.1A).23 434 435 sequences which indicate that ca. 570 residues per chain TheL4domainseparatingLEa3andLEb1canberegardedasa contribute to the three stranded a-helical coiled coil structure specialized LE domain with a large, ~180 residues long insert giving a length of ~80 nm, and ~7 tandem LE domains betweenthethird(C )andfourth(C )cysteineoftheregular 196 382 accounting for a further 20 nm. Circular dichroism spectroscopy pattern.Incaseofthec2chains,theL4insertishighlyconserved revealed an a-helical content of ~30%44 similar to that from human down to Xenopus and zebrafish, and contains one determined for laminin 11145 suggesting that most of this unique cysteine absent in all other L4 domains. For L4-LEb1 it secondary structure contribution relates to the coiled coil. wasshownthatthisC iscovalentlylinkedwiththelastcysteine 309 Assembly of the laminin 332 trimer occurs in the endoplasmic C of LEb1, which contains an unusual S-S bridge between 459 reticulum where first a disulfide linked β3-c2 dimer is formed to C -C corresponding to C6-C7. After cleavage by BMP-1 442 445 whichthea3chainalignsviathecoiledcoil.Trimerformationis between G and D , protein disulfideisomerase was required 434 435 aprerequisitefortransporttotheGolgicomplexandsecretionbut to release the fragments.24 It remains unclear whether after does not require N-linked glycosylation.4 processing C stays in a reduced state, or whether disulfide 459 Lamininβ3LNdomain.Sofarnohighresolutionstructuresof reshuffling might form the usual C7-C8 connectivity and leaves laminin 332 domains have been solved. However, based on C6, i.e., C , with a free SH group.24 The mouse L4-LEb1 442 sequence similarity, some insight into the molecular organization domain pair was modified probably via one or both of the two of various domains can be anticipated from homology modeling. putativeNxTacceptorsitesforN-linkedglycosylation,whichare Recently, the crystal structure of the N-terminal LN domain alsofoundinthehumansequence(N andN ).TheN xT 342 362 342 togetherwithfouradjacentLEmodulesofthemouselamininβ1 site is conserved down to zebrafish. Mouse L4-LEb1 binds chain has been solved (pdb code: 4aqs).46 The corresponding heparin, nidogen-1 (but not -2), and fibulin-1 and -2; it remains regions of human laminin β3 can be fitted to this structure unclearwhether theseinteractions occur between L4 or LEb1,or (Fig.2) suggesting that LN (residues 18–248; accession number whether epitopes from both are required.24 NP_000219) forms a β-sandwich consisting of eight β-strands In conclusion, besides the β3 LN domain, each of the three foldedintoajellyrollmotif.ThefirstLEdomain(res.249–314) irregular LE modules, a3A-LE4’, β3-LE6 and c2-LEb1 after istightlyintegratedintoLN,whichstartswithadisulfide-bonded processing,haveonefreecysteinewhichcouldparticipateeitherin reverse turn connected by C –C , and the N-terminal region interchaindisulfidebondingorcovalentbindingtootherproteins 21 26 (res. 18–28) associates to LE1. Pro , which is conserved in all like laminins 311 and/or 321. Based on the canonical cysteine 28 lamininLNsequences,fitsintoapocketofLE1.Furtherdisulfide pattern, the most likely residues are a3A-C , β3-C and 199 572 bondscanbeexpectedbetweenC -C ,C -C andC -C c2-C or c2-C . 47 69 56 66 153 173 442 459 (Fig.2A). This leaves C , which is unique for β3 LN domains Laminin332coiledcoilandLβdomain.Carboxylterminalto 67 and surface accessible (Fig.2B), in a reduced state making it the LE modules, the three laminin chains have a C-x-x-C motif availableforcovalentlybindingtootherproteinslikelaminin331. (a3A:C -C ;β3:C -C ;c2:C -C )whichcysteines 202 205 581 582 609 612 TheN-linkedglycosylationsitesfoundwithinβ1LNatN and are conserved in all laminins and most likely serve for interchain 120 in c1 LN at N /N are absent in the β3 chain. The critical covalent linkage of the heterotrimer.The actual disulfide pattern 58 132 residues D and T of c1 LN involved in calcium binding,46 has not yet been established. The following ~570 residues 106 114 which might be important for laminin polymerization, are associate via formation of an a-helical coiled coil represented on replaced by S and R in the β3 chain indicating that this site the sequence level by a 3–4 heptad pattern of hydrophobic 70 76 will not be functional. residues.3 As it is the case for all β chains, the β3 coiled coil is Laminin 332 LE and L4 domains. A common feature of LE interrupted by domain Lβ ~200 residues away from the C-x-x-C modulesistheirconnectivityoftheeightcysteineresiduesasC1- motif containing six cysteines (human β3: C -C ) probably 788 818 C3, C2-C4, C5-C6 and C7-C8. The loop formed between C5- forming intrachain disulfide bonds. The a3 chain coiled coil C6usuallyconsistsofeightresidueswithaglycineinpositionsix. contains one cysteine (human a3: C ) about 50 nm from the 551 In human laminin 332, the following LE domains deviate from C-x-x-C motif, which has no adjacent counterpart in the other the 8-Cys pattern (Fig.1A): β3-LE6 preceding the coiled coil twochains.Thecoiledcoilregionisterminatedbycysteinesclose contains one extra cysteine (C ) between C7 and C8 whereas totheC-terminus(humanβ3:C ;c2:C ),whichbasedon 572 1171 1184 the corresponding region within a3A-LE4’ is truncated after the similarity to the β1 and c1 chains most probably form an fourthcysteine(C ; accessionnumberAAA59483)followedby interchain disulfide bond.48 185 an irregular cysteine (C ) just before C /C , which are Besides its structural importance for assembly, it has recently 199 202 205 presumablyformingintrachaindisulfidesattheN-terminusofthe been found that in the absence of the LG domains the laminin coiled coil. The a3A chain is processed just after LE4’ between 111 coiled coil has anti-adhesive properties. When cultured on K and D .14 LEb1 following domain L4 of the human c2 such truncated laminin 111, cell adhesion and spreading were 191 192 chain is missing the fifth cysteine of the 8-Cys pattern and does inhibited;genescompatiblewithapro-migratoryandpro-invasive not contain the glycine residue N-terminal to the sixth cysteine function like MMPs and various matricellular proteins were (C ; accession number NP_005553). This probably results in upregulated.47 No comparable studies have yet been performed 442 anincreasedmobilityoftheloopbetweenC andC allowing forlaminin332,butanantagonisticinterplaybetweencoiled-coil 431 442 www.landesbioscience.com CellAdhesion&Migration 125 Figure2.Structuralmodelofthehumanlamininb3LN-LE1–4domains.(A)ThestructureshowsresiduesQ toQ asacartoonwithredcylindersand 18 464 bluearrowsindicatinga-helicesandb-strands,respectively.DisulfidebondedcysteineswithintheLEdomainsaredepictedinball-and-stickpresentation usingdifferentcolors.Domainboundariesareindicatedbydashedlines.P fitsintoapocketsharedbytheLNandLE1domain,andtheN-terminal 28 residuesareinteractingwithLE1.DisulfidesandthefreecysteineresidueoftheLNdomainareindicated.(B)Theelectrostaticsurfacepotentialis representedinthesameorientationandonthesamescaleasin(A).Negativeandpositivepotentialsareshowninredandblue,respectively.Homology basedmodelingwasperformedusingSwiss-Model106inmanualalignmentmodewiththemouseb1structure(pdbcode4aqs46)asatemplate. and LG domains (see below) could be of importance in cancer be used to model the corresponding a3 LG1-LG5 domains metastasis. (Fig.3). LG domains fold to a β-sandwich consisting of 14 Laminin a3 LG domains. The large globule seen in electron strands, where the two sheets form a concave and convex micrographsatthetipofthelongarmconsistsoffiveLGdomains interface. Close to the C-terminus, each LG sequence contains formed by the a chains. Domains LG4-LG5 are separated from two cysteines ~30 residues apart, which form intradomain LG1 to LG3 by a flexible linker (Fig.1B). As these domains are disulfide bonds (Fig.3A and C). In contrast to electron involved in many interactions (see below), particular interest has micrographs suggesting close proximity of LG1 to LG3,53,54 the beenfocusedontheirstructuralcharacterization(forareview,see a2 LG1–3 structure shows LG1 dissociated from the LG2-LG3 ref.7). X-ray structureshave beensolved forthemousea5 LG1- pair.49Previousworksuggestedthattrimerizationoftheawithβ LG3 triplet (pdb code: 2wjs),49 mouse a1 and a2 LG45 pair and c chains is required for the LG domain to exert its integrin (2jd4,50 1dyk,20 1okq51), and mouse a2 LG5 (1qu052)which can bindingactivity,andthatspecificallyaglutamicacidtworesidues 126 CellAdhesion&Migration Volume7Issue1 Figure3.Forfigurelenged,seenextpage. www.landesbioscience.com CellAdhesion&Migration 127 Figure3(Seepreviouspage).Structuralmodelsofhumanlaminina3LGdomains.(A)ThestructureofLG1toLG3encompassingresiduesM toS 771 1308 isshownasacartoonasdescribedforFigure2.Allcysteineresiduesaredepictedinspacefilling(CPK)style.ThedisulfidebridgebetweenC andC 941 971 (reddottedline)couldnotbecorrectlymodeledasthetemplatesequencehasashorterloopbetweenthecorrespondingresidues.Allothercysteines formdisulfidebridgesexceptforC inLG1whichsidechainpointsintotheinnerspaceoftheb-sandwich.(B)Theelectrostaticsurfacepotentialis 840 representedinthesameorientationandonthesamescaleasin(A).Negativeandpositivepotentialsareshowninredandblue,respectively.(C)A modelofLG45correspondingtoresiduesC toQ isshownwithallcysteinesinCPKstyle.C fromtheloopregionconnectingLG3andLG4 1354 1713 1354 formsaninterdomaindisulfidebondwithC .C inLG4andC inLG5arepointedtothespacebetweenthebsheetsofthesandwichstructures 1617 1443 1587 andarenotsurfaceaccessible,whereasC -C andC -C formdisulfidebonds.(D)AmodelofLG4interactingwithaheparin-likecarbohydrate 1507 1530 1682 1710 chainconsistingoffourGlcNS(6S)-IdoA(2S)disaccharides(ball-and-stickrepresentationwithasurfaceenvelope)isshown.ThesidechainsofK ,R , 1434 1436 andK ,whichwereshowntobecriticallyinvolvedinsyndecan-1interactionaredepictedinred,whereasthoseofK andK involvedin 1438 1440 1442 syndecan-4interactionareshowningreen.59Theε-aminoandguanidiniumgroupsofthelysineandarginineresidues,respectively,pointtothesulfate groupsofthecarbohydratechains.(E)Thesurfaceviewofthemodelshownin(D)indicatesthetightpackingoftheheparinfragmentintothegroove formedbytheconcavesurfaceoftheLG4b-sandwichwithpositivesurfacepotentialspacedtofitthedistanceofsulfategroups.Thetemplatestructures usedformodelingweremouselaminina2LG1-LG3(pdbcode:2wjs)49andmouselaminina1LG4-LG5(2jd4,chainB).50Dockingofaheparinfragment (1hpn)60toLG4wasperformedwithPatchDock.107 apart from the C-terminus of the c chain is essential.55 This Due to their physiological relevance, many studies have been residue is indeed highly conserved in the c1 and c2, but not c3 performed to elucidate the interaction of laminins with subunits. A lack of integrin a3β1 binding activity in the absence proteoglycans. As a model for the complex glycosaminoglycan ofatleastshortβ3andc2segmentshasbeenshownfora3LG1- chains, most experiments are performed with heparin. We have LG3.54,56 The absence of such crucial parts of the β and c chains recently reported on the specific interaction of human a3 LG45 couldresultintheopenLG1-LG2conformation(Fig.3AandB) withsyndecansandfoundthatK ,R andK arecrucial 1434 1436 1438 and might not reflect the conformation of the native molecule.49 for syndecan-1 binding, whereas K and K show a 1440 1442 TheLG3andLG4domainsareconnectedbyaflexiblelinker, preference for syndecan-4 (see below).59 Using the modeled a3 which was realized very early by the proteolytic susceptibility of LG4 domain and a truncated heparin-like structure (pdb code mouse laminin 111.45 Based on the C- and N-terminal residues, 1hpn),60 docking simulations predict that the carbohydrate chain which can be resolved in the mouse a5 LG1–3 (2wjs49) and a1 aligns to the concave surface of LG4 with the positively charged LG4–5(2jd450)structures,respectively,forhumana3thetermini residues filling the grooves formed by the repeating sulfates of the corresponding loop can be defined as N and D (Fig.3D and E). 1310 1352 (Fig.1B).Closetothemiddle,a3LGisprocessedbetweenQ 1337 and D .17,57 This leaves a segment P PFLMLLKGSTR Functions of LG45 in Precursor a3 1338 1312 attached to LG3 which, based on a recombinantly overexpressed LG and synthetic peptide approach, was reported as crucial for A potential function for the tandem LG45 domains was initially integrin a3β1 binding.58 C-terminally truncated peptides, suspected based on the ability of laminin 332 to trigger distinct however, abolished this activity. The difference of this part in cellular events depending on the level of processing of its a3 mousea3(PPFLMLFKSPKG, Fig.1B),however,would suggest chain. A form of laminin 332 that lacked LG45 was found in that this interaction is rather species specific. mature basement membranes, where it was shown to play an Based on the structuresof the mousea1 and a2 LG4–5 pairs, important function in the nucleation and maintenance of modeling predicts that human a3 C within the loop region anchoring structures through a3β1 and a6β4 integrin interac- 1354 forms a disulfide bond with C in LG5 (Fig.3C). tions.61-63In contrast, laminin 332with intact LG45(a3 ) was 1617 200 Corresponding cysteines within the loop and LG5 are found in found in migratory/remodelling situations such as epidermal all laminin a chains down to Drosophila and C. elegans. repair.64-66Indeed,laminin332withana3 chainwasfoundin 200 Interestingly, human a3 LG4 (C ) and a1 LG3 contain the ECM of keratinocytes migrating on collagen I or after 1443 furthercysteinesinasimilarposition,whicharenotconservedin stimulationbyTGF-β1.67,68Invivo,epidermalinjuryactivatesthe mouseandrat.Withinthemodel(Fig.3C),theC andC transcription and deposition of laminin 332 into the provisional 1443 1587 SH groups point into the hydrophobic core of the β sandwich. matrix by the leading keratinocytes in the process of epidermal The crystal structures of mouse a5 LG1, LG2, and a1, a2 outgrowth and migration at the wound edge.8,61,69 Noteworthy, LG4, LG5 contain tightly bound calcium or magnesium ions a3 laminin332isfoundinthisprovisionalmatrixbutisabsent 200 interacting with pairs of aspartate residues.20,49-51 Our human a3 from mature basement membranes.16,61,62 Recently, laminin 332 LGmodels(Fig.3)suggestthatthesecationbindingsitesarenot comprising an a3 chain was proposed to be involved in the 200 conserved. Although within LG1 and LG2 D and D , invasion of squamous cell carcinomas in vivo.70 Polyclonal 827 1090 respectively, are present, their partner residues are changed to antibodies targeting the LG45 domains induced squamous cell N and R . Within LG4 and LG5, the corresponding carcinoma apoptosis in vivo and thus inhibited tumor 910 1023 residues are replaced by S /S and K /S . This does proliferation.70 1413 1481 1584 1654 not exclude that a3 LG domains might contain other divalent Function in laminin 332/311 deposition into the ECM. A cation binding sites, but so far no conclusive experimental data functionforLG45inthedepositionoflaminin 332intheECM exist. has been first hypothesized by Carter and coworkers,16 who 128 CellAdhesion&Migration Volume7Issue1 showedthatexogenoushumana3 laminin332,butnota3 laminin 332 accumulation in the basement membrane is related 200 165 laminin 332, was trapped by cultured LAMA3 deficient mouse to this processing defect. Only one study reported a missense keratinocytes and deposited within their ECM. Moreover, when mutation in the LAMA3 gene affecting LG4 in a patient with a these keratinocytes were transfected with constructs encoding mild non-Herlitz JEB phenotype.71 This mutation resulting in either an LG45-uncleavable or a precleaved a3 chain, they G1506E triggers an imperfect local protein folding that, without deposited a larger amount of the LG45-uncleavable laminin 332 impairing trimerization of the coiled coil, causes laminin 332 into their ECM as compared with the LG45 pre-cleaved laminin intracellular accumulation within the endoplasmic reticulum. 332thatwaspreferentiallysecretedintotheculturemedium.The Only a small amount of the laminin 332 harboring the mutated importanceofLG45inlaminin332depositionwasconfirmedby a3 chain is secreted and physiologically processed thus providing a study reporting that a mutant laminin 332 lacking the LG45 partial adhesion functions and explaining the mild phenotype. domain, stably expressed in laminin 332 null keratinocytes Therefore structural changes caused by mutations of this highly derivedfromapatientwithjunctionalepidermolysisbullosa(JEB) conserved residue throughout laminin LG4 domains highlights withunderlyingLAMA3 genemutations,waslessretainedinthe potential important functions of LG45 in laminin secretion. ECM but rather found in the culture medium as compared with Whenalaminina3cDNAwastransfectedintoHT1080cells, the WT.70 Co-expression of LG45 in these a3 laminin the exogenous a3 chain was assembled with the endogenous 165 expressing keratinocytes enhanced laminin 332 deposition into β3/c2 and β1/c1 to produce laminin 332 and laminin 311 the ECM, reinforced keratinocyte adhesion to the ECM and heterotrimers.72 Out of the two laminin isoforms found in the decreasedmigration.Inthesecells,LG45co-localizedwiththea6 culture medium, laminin 311 was found primarily with a integrin subunit in stable adhesion contacts in a manner precursor a3 chain while the laminin 332 was found with an 200 comparable to the LG45 domain of WT laminin 332 a3 chain.72 Sigle et al.16 showed that exogenous human a3 165 200 keratinocytes.70 laminin311wasnottrappedbyculturedLAMA3deficientmouse Another study, however, found no difference in laminin 332 keratinocytesnordepositedwithintheirECMlikea3 laminin 200 deposited bykeratinocytes expressing either LG45uncleavable or 332 (see above). When these keratinocytes were transfected with pre-cleaved laminin 332.19 Skin equivalents epithelialized with constructs encoding an LG45-uncleavable a3 chain, they keratinocytes expressing the a3 laminin 332 displayed a depositedlarge amountof LG45-uncleavable laminin 332 within 165 dermal-epidermal junction identical to that obtained with wild their ECM, and the LG45-uncleavable laminin 311 was found typekeratinocytessuggestingthata3 laminin332wasproperly exclusivelyintheculturemedium.16Theseresultssuggestthatthe 165 deposited into the basement membrane or at least in an amount two laminin isoforms integrate within the basement membrane sufficient to allow dermal-epidermal cohesion.19 Besides, skin through different mechanisms. That a3 laminin 311 was not 200 equivalents designed with keratinocytes expressing the LG45 depositedwithintheECMfirstshowsthatLG45couldnotfulfil uncleavable laminin 332 revealed absence of hemidesmosomes this event on its own. It also reinforces the hypothesis that a accompanied by an appreciable thickening of the lamina lucida proteindomainpresentinlaminin332butabsentinlaminin311, likely secondary to the reduced cohesion of the tissue.19 These possibly in the c2 chain,73 may contribute to the laminin experimentsshowthatLG45cleavageappearstobeaprerequisite deposition. for hemidesmosome formation in skin equivalent models. Sincelaminin311formsacomplexwithlaminin332invivo,it These studies show that LG45 has the ability to integrate into is tempting to speculate that laminin 311 deposition within the the keratinocyte ECM either on its own or when present in the ECMmightmaydependonitscovalentassociationwithlaminin a3 chain. It targets a6β4 integrin containing stable adhesion 332. Most interestingly, co-polymers of perlecan with mature 200 contacts suggesting that it may play a role in a6β4 integrin laminin311wereidentifiedinECMformedbyalveolarepithelial clustering in vitro. Although LG45 removal decreased laminin cells.74,75 Through nidogen linkage to the laminin 311 c1 chain, 332depositionintheECM,itdidnotfullypreventit,suggesting perlecan was proposed to nucleate formation of laminin 311 thatothermechanismsmaycontributeincontrollinglaminin332 fibrils. deposition. Functionincelladhesionandmigration.Ithasbeenproposed Defective a3 and c2 processing was seen in cylindromatosis, a that precursor laminin 332, together with integrin a3β1, plays a rare genetic human disorder characterized by the occurrence of central role in the polarization and migration of cells.12,66 multipleirregular benignepithelialtumors intheupperdermis.63 Processing of the laminin 332 a chain was suggested to alter its These nodules display a dramatically enlarged basement mem- binding ability to various integrins by differential exposure of brane (up to 4.3 mm thickness) as well as ultrastructural binding sites/sequences to integrin receptors. Cell adhesion abnormalities as no clear lamina densa could be detected. The studies have shown that immobilized purified a3 laminin 200 different integrin receptors are found in improper ratios as β1 332 promotes adhesion of keratinocytes, but to a lesser extent integrins are upregulated while a6β4 integrin expression and than that obtained with mature laminin 332.76 Further analysis hemidesmosome numbers are decreased.63 Despite a massive revealedthatthea3β1integrinwasthemajorreceptortointeract accumulation of laminin 332 throughout the entire basal lamina, with a3 laminin 332, while both a3β1 and a6β4 integrins 200 a thin labeling of the LG45 domain detected at the interface were involved in cell adhesion to a3 laminin 332.76 These 165 between cylindroma cells and the basal lamina suggests that the findings correlate with previous results showing that cells plated LG45 processing may have been delayed. It is not clear whether on an a3 laminin 332 rich matrix failed to form a6β4 200 www.landesbioscience.com CellAdhesion&Migration 129 Table1.HeparinbindingsitesidentifiedwithinhumanandmouseLG45domains Laminina3LG45heparinbindingsites Methodusedforidentification Bindingpartner References Human N SFMALYLSKGR Peptide Syndecan-2 82 1412 Syndecan-4 L GTDGKKLRIKSKEKCNDG LG45/heparincross-link Heparin 85 1428 K KLRIK Directedmutagenesis Syndecan-1 59 1433 R IKSKEK Directedmutagenesis Syndecan-4 59 1436 L GSPPSGKPKSL LG45/heparincross-link Heparin 85 1488 V TPKQSLCD LG45/heparincross-link Heparin 85 1610 Mouse K ARSFNVNQLLQD Peptide Unknownreceptor 81 1336 K PRLQFSLDIQT Peptide Unknownreceptor 81 1398 D GQWHSVTVSIK Peptide Unknownreceptor 81 1629 integrin-dependent hemidesmosomes, whereas plasmin cleavage the a3LG45 sequence (Table 1). Some of these conferred of the LG45 domain inhibited migration and promoted heparin-dependent cell adhesion properties, which suggested that hemidesmosome formation.12 These data were corroborated by this region in laminin 332 could interact with a heparan sulfate studiesshowingthata3 laminin332isapreferentialligandfor proteoglycan receptor.29,59 An early report described the char- 200 integrin a3β1 and/or a6β1, whereas processed a3 LG domains acterization of aheparin-binding synthetic peptide corresponding are the preferred ligand for integrin a6β4.65 Besides, the analysis to residues K PRLQFSLDIQT derived from the murine a1 1398 ofthebindingcapacityofsolublerecombinanta3β1integrintoa LG4sequence.80Thispeptideinducesadhesion ofthemelanoma mini-laminin 332 comprising the five LG domains has revealed cells B16F10 through an heparan sulfate proteoglycan type anincreasedaffinityforintegrinbindingwhenLG45wascleaved receptorthatremainstobeidentified.Apeptidicscreeningofthe off.56 Information gained from the structural model of the LG murinea3LG45domainsallowedidentificationofthreeheparin predicts that LG1–3 have the shape of a cloverleaf from which bindingsiteswithcelladhesionproperties.81Peptidicscreeningof LG45extends.7ThespecificlengthoftheLG3-LG4linkerresults the human sequence lead to the identification of a motif that inLG5beingclosertotheLG1–3cloverleafthanLG4suggesting included residues N SFMALYLSKGR, which was shown to 1412 that the integrin-binding domain may be differently exposed in induce syndecan-2 and -4 mediated cell adhesion, neurite a3 and a3 laminin 332. Other studies have contradicted outgrowth and MMP-1 and -9 secretion.78,79,82,83 Further work 200 165 this hypothesis as both a6β4 and a3β1 integrins were found to suggested that this motif also induced keratinocyte migration by interact with an LG45-uncleavable laminin 33216 and were triggering syndecan-4 clustering and subsequent β1 integrin colocalized in precursor laminin 332 trails left behind migrating activation.84 Later, three novel heparin-binding sites were cells.57 Moreover, a6β4 integrin containing stable anchoring identified based on cross-linking the native protein to heparin complexes77 were detected in cultured keratinocytes expressing beads.85Recently,throughasite-directedmutagenesisapproachto precursor- or LG45-uncleavable laminin 33216,19 in which LG45 alter the most critical basic residues in a recombinant LG45 and a6β4 integrins colocalized.70 Although the β4 integrin was protein, we identified a unique heparin-binding site surrounded correctlyexpressedandlocalizedattheapicalsideoftheepidermal by a track of converging low affinity, positively charged basal layer, LG45-uncleavable laminin 332 expressing keratino- residues (Fig.3D and E).49 We further showed that this cytes failed toform hemidesmosomes when seeded over adermal K KLRIKSKEK sequence region, which matches one of the 1433 equivalent model.19 sites identified by Vives et al.85 harbours distinctive syndecan-1 Other studies have reported that soluble LG45 may have a and-4interactionsites(Table1).Besides,ourgrouphasreported function when released from laminin 332. Experiments have thatsyndecan-1isthecellularreceptorinvolvedincelladhesionto shown that adding either recombinant a3LG45 or LG4 to the the a3 LG45 domain57,86 and this interaction may participate in culture medium could induce migration of keratinocytes in a keratinocytemigrationbysupportingtheformationofactin-based MMP-9-dependent manner.78 In similar experiments, LG4 cellular protrusions (Fig.4A and B).76,87 The development of induced activation of pro-migratory MMP-1.79 Moreover, Ras/ these membrane protrusions remarkably requires dephosphoryla- IkBa transformed keratinocytes that lacked the LG45 domain tion of tyrosine residues in the cytoplasmic tail of syndecan-1, a showed a deficiency in MMP-9 and MMP-1 expression. This condition essential for syntenin-1 recruitment.87,88 In the deficiency was reversed by replacing LG45 through retroviral epidermis, syndecan-1 is located in the pericellular region of transduction.70 keratinocytes and displays a modest expression in the basal cell Bindingsiteswithina3LG45.Themechanismunderlyingthe layer,whichbecomesincreasinglyintenseinthesuprabasallayers. function of the LG45 domain in laminin 332 remains poorly Remarkably, syndecan-1 is strongly induced in wound edge understood. Several heparin-binding sites have been identified in keratinocytes during wound healing.89-91 This elevated expression 130 CellAdhesion&Migration Volume7Issue1 inkeratinocytesappearstobespecifictosyndecan-1asnochanges defective c2 processing was found in the rare inherited have been detected for other syndecans.92 Moreover, syndecan-1 cylindromatosis disease (see above), in which abnormal basement deficient mice display a defect in keratinocyte proliferation and membranes and excessive ectopic expression of collagen VII were migration during wound healing.93 In light of these data, found.63,96 The defective processing of the c2 chain may have syndecan family members stimulate interest as potential laminin resulted in a deficient interaction with its basement membrane 332 co-receptors. Recent data has provided evidence that when partners collagen VII causing defaults in anchoring fibrils human squamous carcinoma A431 cells are plated on laminin linkage.32,95 332, syndecan-1 forms a complex with the a6β4 integrin. This Numerousstudiesconductedwithnormalbreastepithelialand triggers Fyn-mediated phosphorylation of the β4 integrin carcinomacellshaveshownthatcellmigration-inducingfunctions cytoplasmic tail activating PI3K- and Akt-mediated signaling, of the c2 chain rely on its proteolysis.35 Recently, it has been protecting the cells against apoptosis.94 This is of particular reported that a syndecan-1 interaction with the N-terminal interest as LG45 in a3 laminin 332 was proposed as an domain in precursor c2 could negatively regulate β4 integrin 200 essential PI3K pathway activation promoter.70 phosphorylationleadingtoenhancedcarcinomacelladhesionand decreased motility97 reinforcing the idea that the precursor c2 Functions of Precursor c2 N-Terminal Domain chain does not support cell migration. In contrast, other reports have suggested that precursor c2 may play a role in keratinocyte It has been shown that in human skin basement membrane, the migrationduringthewoundhealingprocess.Aninteractionofits N-terminal domain of the c2 chain is absent suggesting that the N-terminal portion with a2β1 integrin was shown to occur c2 processing could have an important physiological role.11,63 In during TGF-β1 induced keratinocyte migration in vitro and was contrast to these findings, antibodies specific for the released L4 proposed to take place at the wound margin of the skin healing domain were shown to label the basal epidermal cells in mouse process in vivo.68 Keratinocytes at the edge of wounds made in skin.24 A positive signal was also found in the basement early passage cultures shown to co-express the laminin 332 c2 membranes of early stages of skin development suggesting that chain and the cell cycle inhibitor p16INK4a, displayed increased the precursor c2 chain or its released N-terminal domain directional motility.98 Additional studies revealed that this expression might persist in vivo.24 The analysis of mTLD/BMP- keratinocyte hypermotility/growth arrest response relied on both 1 deficient mice revealed that although defaults in skin cohesion the precursor state of the laminin 332 c2 chain and the were not clinically apparent, ultrastructural examination of the participation of a serum co-factor through a TGFβ receptor I basement membrane showed places where anchoring structures dependent mechanism.99 This apparent contradiction in experi- were disconnected at the level of the lamina densa with presence mentalfindingsislikelyduetotissuespecificdifferences.Astudy of rudimentary hemidesmosomes.15,25 A strong expression of the indicatedthattheL4modulemediatestheintegrationoflaminin precursor c2 chain suggested that c2 chain maturation takes part 332 into the extracellular matrix through intermolecular in the basement membrane cohesion and stability. Besides, interactions.73 The question arises whether the heparin-binding domains located within the a3 LG45 domains and the N-terminal portion of the c2 chain are involved in laminin 332 deposition. Heparinwasshowntoinhibitlaminin332depositionwithinthe ECM suggesting that a heparan sulfate proteoglycan might be involved in binding to both precursor a3 and c2.13 Null mutation in syndecan-1100 and -4101 has not reported defects in laminin deposition, which suggests that any of these two receptors is solely responsible for this mechanism. Generation of double syndecan-1 and -4 knockout mice would reveal whether both these receptors are implicated. Future studies should examine whether a heparan sulfate proteoglycan cell surface receptor or a basement membrane components are is implicated inprecursorlaminin332deposition.Interestingly,arecentstudy Figure4.Syndecan-1mediatedcelladhesiontolaminin332LG45 domain.(A)FibrosarcomaHT1080cellswereplatedonsurfacescoated hasreportedthattheperlecancolocalizeswithlaminin332atthe withpurifiedLG45domainfor1h,fixedandstainedwithphalloidin- wound margin of healing full thickness wounds generated in FITC.Theanalysisoftheactinstainingrevealedanorganizationofthe mice suggesting proximity of these two components during cytoskeletoninradialarraysofmicrospikes,protrusiveadhesion basement membrane remodelling.102 However, as perlecan was structuresknowntobeinvolvedintransientcell-substratuminteractions. shown not to be essential for matrix assembly in perlecan-null (B)ConfocalimageofHT1080cellsexpressingGFP-syndecan-1platedon theLG45domain.Thephasecontrastimageshowsthetypicalinduced mouse embryos,103 its presence in the ECM might not be an cellshapecharacterizedbytheformationofspike-likeactin-based absolute requirement for laminin 332 deposition. Considering protrusivestructures.Thereceptorsyndecan-1(green)promotescell the important function of laminin 332 in skin homeostasis, adhesiontothelamininfragmentandalignsalongtheactinfilaments, severalextracellularligandsmightbeinvolvedinitsdepositionin whichareorganizedintoparallelbundles.Bars,10mm. the basement membrane. www.landesbioscience.com CellAdhesion&Migration 131

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CellAdhesion&Migration7:1,122–134;January/February2013;G2013LandesBioscience Laminin 332 processing impacts cellular behavior Patricia Rousselle1,* and Konrad Beck2 1SFRBioSciencesGerland-LyonSud;InstitutdeBiologieetChimiedesProtéines;UMR5305,CNRS;UniversitéLyon1;Lyon,France;2CardiffUniversitySchoolofDentistry;Cardiff,UK Keywords: laminin 332, LG45 domain, LE domain, syndecan, basement membrane, keratinocyte Abbreviations: ECM, extracellular matrix; LG, laminin globular domain; pre-laminin, precursor-laminin; MMP, matrix metalloproteinase; mTLD, mammalian tolloid; BMP-1, bone morphogenic protein 1; JEB, junctional epidermolysis bullosa; pdb, protein data bank Laminin 332 and Its Maturation Events Laminin 332, composed of the a3, b3 and c2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial- Laminin 332, composed of the a3, β3 and c2 chains, is an mesenchymal cohesion in tissues exposed to external forces, epithelial-basement membrane specific variant. The a3 chain is including skin and stratified squamous mucosa. After being encoded by the LAMA3 gene, which has two transcript variants secretedanddepositedintheextracellularmatrix,laminin332 a3Aanda3B.AsthetruncatedLAMA3Avariantisexpressedand undergoes physiological maturation processes consisting in incorporatedintolaminin332heterotrimers,8wewillrefertothe the proteolytic processing of domains located within the a3 a3Aβ3c2 trimer as representing laminin 332 in this review. Its and the c2 chains. These maturation events are essential for main role in normal tissues is the maintenance of epithelial- laminin332integrationintothebasementmembranewhereit mesenchymal cohesion in tissues exposed to external forces plays an important function in the nucleation and mainten- including skin and stratified squamous mucosa.9,10 To fulfill this ance of anchoring structures. Studies in normal and patho- function, laminin 332 undergoes several physiological post- logical situations have revealed that laminin 332 can trigger translational processing events allowing its supramolecular distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and integration into the basement membrane. In other situations structuralcharacteristicsoflaminin332domainsarepresented such as wound healing or carcinoma, additional cleavages may andwediscuss whetherthey trigger specificfunctions. take place delivering pro-migratory signals to cells therefore compromising the adhesive function of the protein. Laminin 332 processing and integration into the skin basement membrane. In skin, laminin 332 is synthesized by Laminins are large extracellular glycoproteins that are important keratinocytesasahighmolecularweightprecursorproteinof460 components of all basement membranes. They are involved in kDa.AftersecretionanddepositionintotheECM,thea3andc2 severalbiologicalprocesses,includingselfpolymerization,binding chainsundergomaturationeventsconsistinginspecificprocessing to the extracellular matrix (ECM) and cellular interactions.1,2 All to smaller forms.4,11 The 190–200 kDa a3 chain (a3 ) can be 200 lamininsarecomposedofthreedifferentgeneproducts,termeda, successively processed at both C- and N-terminal extremities β and c chains assembled into a cross-shaped heterotrimer aβc. producing 165 (a3 ) and 145 kDa (a3 ) maturation 165 145 The three chains assemble within the endoplasmic reticulum products. The 155 kDa c2 chain (c2 ) is processed in the N- 155 through their C-terminal domains to form a triple stranded terminal region leading to a 105 kDa product (c2 ). 105 a-helical coiled coil rod.3,4 Sixteen laminin isoforms of different Processing of the a3 chain consists of cleavage of the C- subunitcompositionselectedfromfiveindividualachains(a1to terminal globular domains 4 and 5 (LG45) within the spacer a5), three β chains (β1 to β3) and three c chains (c1 to c3), are betweenLG3andLG4(Fig.1AandB).12,13Invitrostudiesusing known with variable cell and tissue specific expression, and they human laminin 332 have revealed that enzymes involved in the are differentially recognized by cellular receptors.5 All laminin a processing include plasmin,12 MMP-2, MT1-MMP and the C- chains possess a large globule at the carboxyl-terminal end that proteinase family of enzymes, especially mammalian tolloid consists of five similar domains LG1 to LG5 each containing (mTLD) and the bone morphogenic protein 1 (BMP-1).14,15 In about 200 residues.6,7 addition, thrombin was shown to have the potency to cleave LG45specifically.16Alternatively,theremaybeothermechanisms that control the rate of laminin a3 LG45 processing, such as the tissue plasminogen proteolytic cascade.12 N-terminal amino acid sequencing of human LG45 purified from the conditioned *Correspondenceto:PatriciaRousselle;Email:[email protected] medium of either primary or immortalized human keratinocytes Submitted:07/16/12;Revised:11/12/12;Accepted:12/05/12 andofthehumangastricadenocarcinomacellsSTKM-1revealed http://dx.doi.org/10.4161/cam.23132 thatthea3chain iscleaved betweenQ and D withinthe 1337 1338 122 CellAdhesion&Migration Volume7Issue1 SPECIALFOCUSREVIEW:LAMININS hingeregionbetweenLG3andLG4suggestingthataproteolytic Laminin 332 was shown to be the major component of cleavagesiteoftheLG45domainmatchestheminimalconsensus anchoring filaments in skin9 where it mediates cell adhesion via sequence LLQD (Fig.1B).17,18 It is not known what endopepti- interaction of the a3 carboxyl-terminal LG1–3 triplet domain dase catalyzes the hydrolysis of this cleavage sequence, and the with both a3β1 and a6β4 integrins,26-29 while the N-terminal existence of additional proteolytic cleavage sites has been shortarmsconnecttobasementmembranecomponents.Laminin suspected both in the hinge and the adjacent regions of the 332canbeincorporatedintothebasementmembranethroughat LG3 and LG4 domains.7,13,14,19 This hypothesis is compatible least two mechanisms. The first involves cross-linking of laminin with the spacer length of the laminin a3 chain, which is longer 332 with laminin 311 (a3β1c1) in the skin basement than those of the a1, a2 and a5 chains.7 Punctual mutations membrane.21 The complex of laminin 311 with laminin 332 is within the spacer region and/or deletion of the sequence LLQD mostlikelytoderivefromaninteractionofdomainLNintheβ3 did not protect the a3 LG3-LG4 linker from cleavage.14,19 As chain of laminin 332 with domain LE of the a3 chain short arm proteolyticprocessingofLGdomainsoccursinallachainsexcept inlaminin311(seebelow).Asseenonrotaryshadowingelectron a1,thesubstitutionofthea3LG3-LG4hingewiththatofthea1 micrographs of the complex, the LN domain of the short arm chain was an elegant way to render the laminin a3 chain interacts with a laminin 311 domain near the intersection of the uncleavable.19 This was also the case when a 46 amino-acid laminin 311 short arms.21 These complexes are most likely portion within the a3 LG3-LG4 hinge was deleted.16 These stabilized by a disulfide bridge between an unpaired cysteine in LG45-uncleavablea3chainconstructswereexpressedinhuman19 domain LN of the β3-chain and domain LE of processed a3 in or mouse16 skin keratinocytes deficient for expression of the a3 laminin311.Asimilarcomplexbetweenlaminin332andlaminin chain, and in both cases the resulting LG45-uncleavable 321 (a3β2c1) is found in the basement membrane of the heterotrimeric laminin 332 was deposited in the ECM. amnions. According to the 3-arm interaction hypothesis of Information gained from the structure of the mouse a2 chain laminin polymerization,30 the dimers could self-associate. The LG45 domain pair revealed an unusual path of the LG3-LG4 secondmechanismreportsadirectinteractionbetweenanchoring linker as it forms an inter-domain disulfide bridge to LG5.20 filaments and anchoring fibrils. Anchoring fibrils are disulfide BeingintegratedintotheLG45tandemthelinkeristhereforean bondstabilizeddimersoftypeVIIcollagen.31Monomericlaminin integral part of the structure, a feature that is conserved in all 332 as well as the laminin 332/311 dimer directly bind the lamininachains.Itistemptingtospeculatethatproteinasesmay N-terminal globular domain NC1 of type VII collagen.10,32 The have easy access to the LG3-LG4 cleavage sites located within a interactionislikelytooccurwithintheshortarmoftheβ3and/or well-exposed linker.7 c2 subunit.32 Further cleavage occurs in theN-terminal LEregion of the a3 Other processing events in laminin 332. In addition to the chain releasing the full short arm which might be important for laminin 332 maturation aimed at regulating the skin basement laminin 332 function (Fig.1A).14,21 A mutation causing an N- membrane structural integrity, other studies have reported two terminal deletion of 226 amino acids in the human a3A isoform specific migration-inducing MMP-2 cleavages of the laminin c2 was identified in the laryngo-oncho-cutaneous syndrome, a rare N-termini producing c2 chain fragments of 100 and 80 kDa.33 autosomal recessive disorder characterized by chronic production Thesec2processingcouldalsobecarriedbyMT1-MMPincolon of vascularized granulation tissue.22 Cultured keratinocytes andbreastcarcinomacells.34Bothenzymes,thatwerefirstshown obtained from patients revealed that heterotrimeric laminin 332 tocleaveratlaminin332,wereproposedtoalsocutdownhuman carrying the mutant a3 chain is assembled and secreted. The laminin 332 in both physiological and pathological situations mutated laminin was regularly expressed in the skin basement such as tumorigenesis.35,36 MMP-3, -12, -13, -19 and -20 were membrane and only very subtle ultrastructural changes were seen also shown to process the c2 chain inducing epithelial cell such as focal widening of the lamina lucida in places where migration.37,38 Migration events might therefore result from the hemidesmosome plaques were smaller. These studies suggest that interaction of newly exposed laminin domains with cell surface thelaminina3N-terminaldomainmaybeakeyregulatorofthe signaling receptors. Rat recombinant c2LEb1-LEb4’ was shown granulation tissue response during wound healing. to bind and activate the EGF receptor to stimulate cell In human skin laminin 332, the processing of the c2 chain migration.39 Studies with cancer cells have reported that MMP- 155 leadstothec2 subunitandwasshowntobeachievedbyBMP- 7,MT1-MMPandhepsincleave lamininβ3leading toincreased 105 1 and mTLD metalloproteinases.11,14,15,23,24 The N-terminal migration.40-42 The molecular mechanisms of laminin 332 in processing is complex as it occurs within a disulfide-linked loop squamous carcinoma have been reviewed in detail by of the L4 domain followed by reshuffling of disulfide bonds for Marinkovich.43 release of the cleaved fragments.24 The immunohistochemical analysis of laminin 332 in mTLD/BMP-1-deficient mouse skin Laminin 332 Sequence-Structure Relationship revealedastrongexpressionoftheprecursorc2chainconfirming that these enzymes are involved in the c2 chain maturation in The structure of the heterotrimeric laminin 332 molecule vivo.25 Incontrast,theprocessingoftheLG45domaininthe a3 consisting of disulfide-linked a3, β3 and c2 chains was first chain seemed, at least partially, unaffected reinforcing the revealedbyrotaryshadowingelectronmicroscopy,whichshowed hypothesis that several enzymes are involved in this maturation a rod 107 nm in length terminated by one large and two small process. globules at opposite ends9 which correspond to the C-terminal www.landesbioscience.com CellAdhesion&Migration 123 Figure1.Structureofhumanlaminin332anditsphysiologicalmaturationprocess.(A)Laminin332iscomposedofthreesubunitsa3A,b3andc2.Each chainiscomposedofdifferentdomainsthatareindicated.DomainL4inthec2chaincorrespondstoanLEdomainwitha~180residuesinsertbetween thethirdandfourthcysteineofthecanonical8-Cyspattern.LE4’andLEb4’ofthea3Aandc2chainaretruncatedLErepeatscontainingonlythefirstfour andsixcysteines,respectively,ofthepattern.Basedontheoddnumberofcysteines,b3LN,a3ALE4’,b3LE6andc2LEb1(aftermaturation),andthecoiled coilofa3havefreeSHgroupsasindicated.ThecoiledcoilisstabilizedbydisulfidebondsattheN-terminus(dottedlines)thoughtheconnectivityisyet unknown.ThelargeLGstructurelocatedattheC-terminalendofthea3chaincontainsfiverepeatingLGdomains.Thefirstthreerepeats(LG1–3) interactwitha3b1,a6b1anda6b4integrinswhilethelasttwo(LG45)containbindingsitesforsyndecan-1and-4.Laminin332issynthesizedasa precursormoleculethatundergoesmaturationbyproteolyticprocessingsatthea3AchainN-andC-terminusaswellasatthec2chainN-terminal extremity.Thecleavagesitesareindicatedbyarrowsaswellasenzymesinvolvedidentifiedsofar.(B)SchematicstructuresofLG.ThefiveLGdomains containnumerouscysteinesthatarenumbered.Disulfidebridgedandfreecysteines(SH)withinthehumansequenceareindicated.Atthebottom,an alignmentofthehumanandmouseLG3-LG4linkerisshown.Eachsequencedisplaysthesamecleavagesite(underlined)andacysteine(printedin inversefont)thatformsaninter-domaindisulfidebondtoLG5.20ThesequenceP PFLMLLKGSTRsupposedtobecrucialforintegrina3b1binding58is 1312 doubleunderlined. 124 CellAdhesion&Migration Volume7Issue1 LG and N-terminal LN and LE/L4 domains, respectively for protease susceptibility. Indeed, major processing occurs by (Fig.1A). This length is consistent with the later determined cleavage of the G -D site (Fig.1A).23 434 435 sequences which indicate that ca. 570 residues per chain TheL4domainseparatingLEa3andLEb1canberegardedasa contribute to the three stranded a-helical coiled coil structure specialized LE domain with a large, ~180 residues long insert giving a length of ~80 nm, and ~7 tandem LE domains betweenthethird(C )andfourth(C )cysteineoftheregular 196 382 accounting for a further 20 nm. Circular dichroism spectroscopy pattern.Incaseofthec2chains,theL4insertishighlyconserved revealed an a-helical content of ~30%44 similar to that from human down to Xenopus and zebrafish, and contains one determined for laminin 11145 suggesting that most of this unique cysteine absent in all other L4 domains. For L4-LEb1 it secondary structure contribution relates to the coiled coil. wasshownthatthisC iscovalentlylinkedwiththelastcysteine 309 Assembly of the laminin 332 trimer occurs in the endoplasmic C of LEb1, which contains an unusual S-S bridge between 459 reticulum where first a disulfide linked β3-c2 dimer is formed to C -C corresponding to C6-C7. After cleavage by BMP-1 442 445 whichthea3chainalignsviathecoiledcoil.Trimerformationis between G and D , protein disulfideisomerase was required 434 435 aprerequisitefortransporttotheGolgicomplexandsecretionbut to release the fragments.24 It remains unclear whether after does not require N-linked glycosylation.4 processing C stays in a reduced state, or whether disulfide 459 Lamininβ3LNdomain.Sofarnohighresolutionstructuresof reshuffling might form the usual C7-C8 connectivity and leaves laminin 332 domains have been solved. However, based on C6, i.e., C , with a free SH group.24 The mouse L4-LEb1 442 sequence similarity, some insight into the molecular organization domain pair was modified probably via one or both of the two of various domains can be anticipated from homology modeling. putativeNxTacceptorsitesforN-linkedglycosylation,whichare Recently, the crystal structure of the N-terminal LN domain alsofoundinthehumansequence(N andN ).TheN xT 342 362 342 togetherwithfouradjacentLEmodulesofthemouselamininβ1 site is conserved down to zebrafish. Mouse L4-LEb1 binds chain has been solved (pdb code: 4aqs).46 The corresponding heparin, nidogen-1 (but not -2), and fibulin-1 and -2; it remains regions of human laminin β3 can be fitted to this structure unclearwhether theseinteractions occur between L4 or LEb1,or (Fig.2) suggesting that LN (residues 18–248; accession number whether epitopes from both are required.24 NP_000219) forms a β-sandwich consisting of eight β-strands In conclusion, besides the β3 LN domain, each of the three foldedintoajellyrollmotif.ThefirstLEdomain(res.249–314) irregular LE modules, a3A-LE4’, β3-LE6 and c2-LEb1 after istightlyintegratedintoLN,whichstartswithadisulfide-bonded processing,haveonefreecysteinewhichcouldparticipateeitherin reverse turn connected by C –C , and the N-terminal region interchaindisulfidebondingorcovalentbindingtootherproteins 21 26 (res. 18–28) associates to LE1. Pro , which is conserved in all like laminins 311 and/or 321. Based on the canonical cysteine 28 lamininLNsequences,fitsintoapocketofLE1.Furtherdisulfide pattern, the most likely residues are a3A-C , β3-C and 199 572 bondscanbeexpectedbetweenC -C ,C -C andC -C c2-C or c2-C . 47 69 56 66 153 173 442 459 (Fig.2A). This leaves C , which is unique for β3 LN domains Laminin332coiledcoilandLβdomain.Carboxylterminalto 67 and surface accessible (Fig.2B), in a reduced state making it the LE modules, the three laminin chains have a C-x-x-C motif availableforcovalentlybindingtootherproteinslikelaminin331. (a3A:C -C ;β3:C -C ;c2:C -C )whichcysteines 202 205 581 582 609 612 TheN-linkedglycosylationsitesfoundwithinβ1LNatN and are conserved in all laminins and most likely serve for interchain 120 in c1 LN at N /N are absent in the β3 chain. The critical covalent linkage of the heterotrimer.The actual disulfide pattern 58 132 residues D and T of c1 LN involved in calcium binding,46 has not yet been established. The following ~570 residues 106 114 which might be important for laminin polymerization, are associate via formation of an a-helical coiled coil represented on replaced by S and R in the β3 chain indicating that this site the sequence level by a 3–4 heptad pattern of hydrophobic 70 76 will not be functional. residues.3 As it is the case for all β chains, the β3 coiled coil is Laminin 332 LE and L4 domains. A common feature of LE interrupted by domain Lβ ~200 residues away from the C-x-x-C modulesistheirconnectivityoftheeightcysteineresiduesasC1- motif containing six cysteines (human β3: C -C ) probably 788 818 C3, C2-C4, C5-C6 and C7-C8. The loop formed between C5- forming intrachain disulfide bonds. The a3 chain coiled coil C6usuallyconsistsofeightresidueswithaglycineinpositionsix. contains one cysteine (human a3: C ) about 50 nm from the 551 In human laminin 332, the following LE domains deviate from C-x-x-C motif, which has no adjacent counterpart in the other the 8-Cys pattern (Fig.1A): β3-LE6 preceding the coiled coil twochains.Thecoiledcoilregionisterminatedbycysteinesclose contains one extra cysteine (C ) between C7 and C8 whereas totheC-terminus(humanβ3:C ;c2:C ),whichbasedon 572 1171 1184 the corresponding region within a3A-LE4’ is truncated after the similarity to the β1 and c1 chains most probably form an fourthcysteine(C ; accessionnumberAAA59483)followedby interchain disulfide bond.48 185 an irregular cysteine (C ) just before C /C , which are Besides its structural importance for assembly, it has recently 199 202 205 presumablyformingintrachaindisulfidesattheN-terminusofthe been found that in the absence of the LG domains the laminin coiled coil. The a3A chain is processed just after LE4’ between 111 coiled coil has anti-adhesive properties. When cultured on K and D .14 LEb1 following domain L4 of the human c2 such truncated laminin 111, cell adhesion and spreading were 191 192 chain is missing the fifth cysteine of the 8-Cys pattern and does inhibited;genescompatiblewithapro-migratoryandpro-invasive not contain the glycine residue N-terminal to the sixth cysteine function like MMPs and various matricellular proteins were (C ; accession number NP_005553). This probably results in upregulated.47 No comparable studies have yet been performed 442 anincreasedmobilityoftheloopbetweenC andC allowing forlaminin332,butanantagonisticinterplaybetweencoiled-coil 431 442 www.landesbioscience.com CellAdhesion&Migration 125 Figure2.Structuralmodelofthehumanlamininb3LN-LE1–4domains.(A)ThestructureshowsresiduesQ toQ asacartoonwithredcylindersand 18 464 bluearrowsindicatinga-helicesandb-strands,respectively.DisulfidebondedcysteineswithintheLEdomainsaredepictedinball-and-stickpresentation usingdifferentcolors.Domainboundariesareindicatedbydashedlines.P fitsintoapocketsharedbytheLNandLE1domain,andtheN-terminal 28 residuesareinteractingwithLE1.DisulfidesandthefreecysteineresidueoftheLNdomainareindicated.(B)Theelectrostaticsurfacepotentialis representedinthesameorientationandonthesamescaleasin(A).Negativeandpositivepotentialsareshowninredandblue,respectively.Homology basedmodelingwasperformedusingSwiss-Model106inmanualalignmentmodewiththemouseb1structure(pdbcode4aqs46)asatemplate. and LG domains (see below) could be of importance in cancer be used to model the corresponding a3 LG1-LG5 domains metastasis. (Fig.3). LG domains fold to a β-sandwich consisting of 14 Laminin a3 LG domains. The large globule seen in electron strands, where the two sheets form a concave and convex micrographsatthetipofthelongarmconsistsoffiveLGdomains interface. Close to the C-terminus, each LG sequence contains formed by the a chains. Domains LG4-LG5 are separated from two cysteines ~30 residues apart, which form intradomain LG1 to LG3 by a flexible linker (Fig.1B). As these domains are disulfide bonds (Fig.3A and C). In contrast to electron involved in many interactions (see below), particular interest has micrographs suggesting close proximity of LG1 to LG3,53,54 the beenfocusedontheirstructuralcharacterization(forareview,see a2 LG1–3 structure shows LG1 dissociated from the LG2-LG3 ref.7). X-ray structureshave beensolved forthemousea5 LG1- pair.49Previousworksuggestedthattrimerizationoftheawithβ LG3 triplet (pdb code: 2wjs),49 mouse a1 and a2 LG45 pair and c chains is required for the LG domain to exert its integrin (2jd4,50 1dyk,20 1okq51), and mouse a2 LG5 (1qu052)which can bindingactivity,andthatspecificallyaglutamicacidtworesidues 126 CellAdhesion&Migration Volume7Issue1 Figure3.Forfigurelenged,seenextpage. www.landesbioscience.com CellAdhesion&Migration 127 Figure3(Seepreviouspage).Structuralmodelsofhumanlaminina3LGdomains.(A)ThestructureofLG1toLG3encompassingresiduesM toS 771 1308 isshownasacartoonasdescribedforFigure2.Allcysteineresiduesaredepictedinspacefilling(CPK)style.ThedisulfidebridgebetweenC andC 941 971 (reddottedline)couldnotbecorrectlymodeledasthetemplatesequencehasashorterloopbetweenthecorrespondingresidues.Allothercysteines formdisulfidebridgesexceptforC inLG1whichsidechainpointsintotheinnerspaceoftheb-sandwich.(B)Theelectrostaticsurfacepotentialis 840 representedinthesameorientationandonthesamescaleasin(A).Negativeandpositivepotentialsareshowninredandblue,respectively.(C)A modelofLG45correspondingtoresiduesC toQ isshownwithallcysteinesinCPKstyle.C fromtheloopregionconnectingLG3andLG4 1354 1713 1354 formsaninterdomaindisulfidebondwithC .C inLG4andC inLG5arepointedtothespacebetweenthebsheetsofthesandwichstructures 1617 1443 1587 andarenotsurfaceaccessible,whereasC -C andC -C formdisulfidebonds.(D)AmodelofLG4interactingwithaheparin-likecarbohydrate 1507 1530 1682 1710 chainconsistingoffourGlcNS(6S)-IdoA(2S)disaccharides(ball-and-stickrepresentationwithasurfaceenvelope)isshown.ThesidechainsofK ,R , 1434 1436 andK ,whichwereshowntobecriticallyinvolvedinsyndecan-1interactionaredepictedinred,whereasthoseofK andK involvedin 1438 1440 1442 syndecan-4interactionareshowningreen.59Theε-aminoandguanidiniumgroupsofthelysineandarginineresidues,respectively,pointtothesulfate groupsofthecarbohydratechains.(E)Thesurfaceviewofthemodelshownin(D)indicatesthetightpackingoftheheparinfragmentintothegroove formedbytheconcavesurfaceoftheLG4b-sandwichwithpositivesurfacepotentialspacedtofitthedistanceofsulfategroups.Thetemplatestructures usedformodelingweremouselaminina2LG1-LG3(pdbcode:2wjs)49andmouselaminina1LG4-LG5(2jd4,chainB).50Dockingofaheparinfragment (1hpn)60toLG4wasperformedwithPatchDock.107 apart from the C-terminus of the c chain is essential.55 This Due to their physiological relevance, many studies have been residue is indeed highly conserved in the c1 and c2, but not c3 performed to elucidate the interaction of laminins with subunits. A lack of integrin a3β1 binding activity in the absence proteoglycans. As a model for the complex glycosaminoglycan ofatleastshortβ3andc2segmentshasbeenshownfora3LG1- chains, most experiments are performed with heparin. We have LG3.54,56 The absence of such crucial parts of the β and c chains recently reported on the specific interaction of human a3 LG45 couldresultintheopenLG1-LG2conformation(Fig.3AandB) withsyndecansandfoundthatK ,R andK arecrucial 1434 1436 1438 and might not reflect the conformation of the native molecule.49 for syndecan-1 binding, whereas K and K show a 1440 1442 TheLG3andLG4domainsareconnectedbyaflexiblelinker, preference for syndecan-4 (see below).59 Using the modeled a3 which was realized very early by the proteolytic susceptibility of LG4 domain and a truncated heparin-like structure (pdb code mouse laminin 111.45 Based on the C- and N-terminal residues, 1hpn),60 docking simulations predict that the carbohydrate chain which can be resolved in the mouse a5 LG1–3 (2wjs49) and a1 aligns to the concave surface of LG4 with the positively charged LG4–5(2jd450)structures,respectively,forhumana3thetermini residues filling the grooves formed by the repeating sulfates of the corresponding loop can be defined as N and D (Fig.3D and E). 1310 1352 (Fig.1B).Closetothemiddle,a3LGisprocessedbetweenQ 1337 and D .17,57 This leaves a segment P PFLMLLKGSTR Functions of LG45 in Precursor a3 1338 1312 attached to LG3 which, based on a recombinantly overexpressed LG and synthetic peptide approach, was reported as crucial for A potential function for the tandem LG45 domains was initially integrin a3β1 binding.58 C-terminally truncated peptides, suspected based on the ability of laminin 332 to trigger distinct however, abolished this activity. The difference of this part in cellular events depending on the level of processing of its a3 mousea3(PPFLMLFKSPKG, Fig.1B),however,would suggest chain. A form of laminin 332 that lacked LG45 was found in that this interaction is rather species specific. mature basement membranes, where it was shown to play an Based on the structuresof the mousea1 and a2 LG4–5 pairs, important function in the nucleation and maintenance of modeling predicts that human a3 C within the loop region anchoring structures through a3β1 and a6β4 integrin interac- 1354 forms a disulfide bond with C in LG5 (Fig.3C). tions.61-63In contrast, laminin 332with intact LG45(a3 ) was 1617 200 Corresponding cysteines within the loop and LG5 are found in found in migratory/remodelling situations such as epidermal all laminin a chains down to Drosophila and C. elegans. repair.64-66Indeed,laminin332withana3 chainwasfoundin 200 Interestingly, human a3 LG4 (C ) and a1 LG3 contain the ECM of keratinocytes migrating on collagen I or after 1443 furthercysteinesinasimilarposition,whicharenotconservedin stimulationbyTGF-β1.67,68Invivo,epidermalinjuryactivatesthe mouseandrat.Withinthemodel(Fig.3C),theC andC transcription and deposition of laminin 332 into the provisional 1443 1587 SH groups point into the hydrophobic core of the β sandwich. matrix by the leading keratinocytes in the process of epidermal The crystal structures of mouse a5 LG1, LG2, and a1, a2 outgrowth and migration at the wound edge.8,61,69 Noteworthy, LG4, LG5 contain tightly bound calcium or magnesium ions a3 laminin332isfoundinthisprovisionalmatrixbutisabsent 200 interacting with pairs of aspartate residues.20,49-51 Our human a3 from mature basement membranes.16,61,62 Recently, laminin 332 LGmodels(Fig.3)suggestthatthesecationbindingsitesarenot comprising an a3 chain was proposed to be involved in the 200 conserved. Although within LG1 and LG2 D and D , invasion of squamous cell carcinomas in vivo.70 Polyclonal 827 1090 respectively, are present, their partner residues are changed to antibodies targeting the LG45 domains induced squamous cell N and R . Within LG4 and LG5, the corresponding carcinoma apoptosis in vivo and thus inhibited tumor 910 1023 residues are replaced by S /S and K /S . This does proliferation.70 1413 1481 1584 1654 not exclude that a3 LG domains might contain other divalent Function in laminin 332/311 deposition into the ECM. A cation binding sites, but so far no conclusive experimental data functionforLG45inthedepositionoflaminin 332intheECM exist. has been first hypothesized by Carter and coworkers,16 who 128 CellAdhesion&Migration Volume7Issue1 showedthatexogenoushumana3 laminin332,butnota3 laminin 332 accumulation in the basement membrane is related 200 165 laminin 332, was trapped by cultured LAMA3 deficient mouse to this processing defect. Only one study reported a missense keratinocytes and deposited within their ECM. Moreover, when mutation in the LAMA3 gene affecting LG4 in a patient with a these keratinocytes were transfected with constructs encoding mild non-Herlitz JEB phenotype.71 This mutation resulting in either an LG45-uncleavable or a precleaved a3 chain, they G1506E triggers an imperfect local protein folding that, without deposited a larger amount of the LG45-uncleavable laminin 332 impairing trimerization of the coiled coil, causes laminin 332 into their ECM as compared with the LG45 pre-cleaved laminin intracellular accumulation within the endoplasmic reticulum. 332thatwaspreferentiallysecretedintotheculturemedium.The Only a small amount of the laminin 332 harboring the mutated importanceofLG45inlaminin332depositionwasconfirmedby a3 chain is secreted and physiologically processed thus providing a study reporting that a mutant laminin 332 lacking the LG45 partial adhesion functions and explaining the mild phenotype. domain, stably expressed in laminin 332 null keratinocytes Therefore structural changes caused by mutations of this highly derivedfromapatientwithjunctionalepidermolysisbullosa(JEB) conserved residue throughout laminin LG4 domains highlights withunderlyingLAMA3 genemutations,waslessretainedinthe potential important functions of LG45 in laminin secretion. ECM but rather found in the culture medium as compared with Whenalaminina3cDNAwastransfectedintoHT1080cells, the WT.70 Co-expression of LG45 in these a3 laminin the exogenous a3 chain was assembled with the endogenous 165 expressing keratinocytes enhanced laminin 332 deposition into β3/c2 and β1/c1 to produce laminin 332 and laminin 311 the ECM, reinforced keratinocyte adhesion to the ECM and heterotrimers.72 Out of the two laminin isoforms found in the decreasedmigration.Inthesecells,LG45co-localizedwiththea6 culture medium, laminin 311 was found primarily with a integrin subunit in stable adhesion contacts in a manner precursor a3 chain while the laminin 332 was found with an 200 comparable to the LG45 domain of WT laminin 332 a3 chain.72 Sigle et al.16 showed that exogenous human a3 165 200 keratinocytes.70 laminin311wasnottrappedbyculturedLAMA3deficientmouse Another study, however, found no difference in laminin 332 keratinocytesnordepositedwithintheirECMlikea3 laminin 200 deposited bykeratinocytes expressing either LG45uncleavable or 332 (see above). When these keratinocytes were transfected with pre-cleaved laminin 332.19 Skin equivalents epithelialized with constructs encoding an LG45-uncleavable a3 chain, they keratinocytes expressing the a3 laminin 332 displayed a depositedlarge amountof LG45-uncleavable laminin 332 within 165 dermal-epidermal junction identical to that obtained with wild their ECM, and the LG45-uncleavable laminin 311 was found typekeratinocytessuggestingthata3 laminin332wasproperly exclusivelyintheculturemedium.16Theseresultssuggestthatthe 165 deposited into the basement membrane or at least in an amount two laminin isoforms integrate within the basement membrane sufficient to allow dermal-epidermal cohesion.19 Besides, skin through different mechanisms. That a3 laminin 311 was not 200 equivalents designed with keratinocytes expressing the LG45 depositedwithintheECMfirstshowsthatLG45couldnotfulfil uncleavable laminin 332 revealed absence of hemidesmosomes this event on its own. It also reinforces the hypothesis that a accompanied by an appreciable thickening of the lamina lucida proteindomainpresentinlaminin332butabsentinlaminin311, likely secondary to the reduced cohesion of the tissue.19 These possibly in the c2 chain,73 may contribute to the laminin experimentsshowthatLG45cleavageappearstobeaprerequisite deposition. for hemidesmosome formation in skin equivalent models. Sincelaminin311formsacomplexwithlaminin332invivo,it These studies show that LG45 has the ability to integrate into is tempting to speculate that laminin 311 deposition within the the keratinocyte ECM either on its own or when present in the ECMmightmaydependonitscovalentassociationwithlaminin a3 chain. It targets a6β4 integrin containing stable adhesion 332. Most interestingly, co-polymers of perlecan with mature 200 contacts suggesting that it may play a role in a6β4 integrin laminin311wereidentifiedinECMformedbyalveolarepithelial clustering in vitro. Although LG45 removal decreased laminin cells.74,75 Through nidogen linkage to the laminin 311 c1 chain, 332depositionintheECM,itdidnotfullypreventit,suggesting perlecan was proposed to nucleate formation of laminin 311 thatothermechanismsmaycontributeincontrollinglaminin332 fibrils. deposition. Functionincelladhesionandmigration.Ithasbeenproposed Defective a3 and c2 processing was seen in cylindromatosis, a that precursor laminin 332, together with integrin a3β1, plays a rare genetic human disorder characterized by the occurrence of central role in the polarization and migration of cells.12,66 multipleirregular benignepithelialtumors intheupperdermis.63 Processing of the laminin 332 a chain was suggested to alter its These nodules display a dramatically enlarged basement mem- binding ability to various integrins by differential exposure of brane (up to 4.3 mm thickness) as well as ultrastructural binding sites/sequences to integrin receptors. Cell adhesion abnormalities as no clear lamina densa could be detected. The studies have shown that immobilized purified a3 laminin 200 different integrin receptors are found in improper ratios as β1 332 promotes adhesion of keratinocytes, but to a lesser extent integrins are upregulated while a6β4 integrin expression and than that obtained with mature laminin 332.76 Further analysis hemidesmosome numbers are decreased.63 Despite a massive revealedthatthea3β1integrinwasthemajorreceptortointeract accumulation of laminin 332 throughout the entire basal lamina, with a3 laminin 332, while both a3β1 and a6β4 integrins 200 a thin labeling of the LG45 domain detected at the interface were involved in cell adhesion to a3 laminin 332.76 These 165 between cylindroma cells and the basal lamina suggests that the findings correlate with previous results showing that cells plated LG45 processing may have been delayed. It is not clear whether on an a3 laminin 332 rich matrix failed to form a6β4 200 www.landesbioscience.com CellAdhesion&Migration 129 Table1.HeparinbindingsitesidentifiedwithinhumanandmouseLG45domains Laminina3LG45heparinbindingsites Methodusedforidentification Bindingpartner References Human N SFMALYLSKGR Peptide Syndecan-2 82 1412 Syndecan-4 L GTDGKKLRIKSKEKCNDG LG45/heparincross-link Heparin 85 1428 K KLRIK Directedmutagenesis Syndecan-1 59 1433 R IKSKEK Directedmutagenesis Syndecan-4 59 1436 L GSPPSGKPKSL LG45/heparincross-link Heparin 85 1488 V TPKQSLCD LG45/heparincross-link Heparin 85 1610 Mouse K ARSFNVNQLLQD Peptide Unknownreceptor 81 1336 K PRLQFSLDIQT Peptide Unknownreceptor 81 1398 D GQWHSVTVSIK Peptide Unknownreceptor 81 1629 integrin-dependent hemidesmosomes, whereas plasmin cleavage the a3LG45 sequence (Table 1). Some of these conferred of the LG45 domain inhibited migration and promoted heparin-dependent cell adhesion properties, which suggested that hemidesmosome formation.12 These data were corroborated by this region in laminin 332 could interact with a heparan sulfate studiesshowingthata3 laminin332isapreferentialligandfor proteoglycan receptor.29,59 An early report described the char- 200 integrin a3β1 and/or a6β1, whereas processed a3 LG domains acterization of aheparin-binding synthetic peptide corresponding are the preferred ligand for integrin a6β4.65 Besides, the analysis to residues K PRLQFSLDIQT derived from the murine a1 1398 ofthebindingcapacityofsolublerecombinanta3β1integrintoa LG4sequence.80Thispeptideinducesadhesion ofthemelanoma mini-laminin 332 comprising the five LG domains has revealed cells B16F10 through an heparan sulfate proteoglycan type anincreasedaffinityforintegrinbindingwhenLG45wascleaved receptorthatremainstobeidentified.Apeptidicscreeningofthe off.56 Information gained from the structural model of the LG murinea3LG45domainsallowedidentificationofthreeheparin predicts that LG1–3 have the shape of a cloverleaf from which bindingsiteswithcelladhesionproperties.81Peptidicscreeningof LG45extends.7ThespecificlengthoftheLG3-LG4linkerresults the human sequence lead to the identification of a motif that inLG5beingclosertotheLG1–3cloverleafthanLG4suggesting included residues N SFMALYLSKGR, which was shown to 1412 that the integrin-binding domain may be differently exposed in induce syndecan-2 and -4 mediated cell adhesion, neurite a3 and a3 laminin 332. Other studies have contradicted outgrowth and MMP-1 and -9 secretion.78,79,82,83 Further work 200 165 this hypothesis as both a6β4 and a3β1 integrins were found to suggested that this motif also induced keratinocyte migration by interact with an LG45-uncleavable laminin 33216 and were triggering syndecan-4 clustering and subsequent β1 integrin colocalized in precursor laminin 332 trails left behind migrating activation.84 Later, three novel heparin-binding sites were cells.57 Moreover, a6β4 integrin containing stable anchoring identified based on cross-linking the native protein to heparin complexes77 were detected in cultured keratinocytes expressing beads.85Recently,throughasite-directedmutagenesisapproachto precursor- or LG45-uncleavable laminin 33216,19 in which LG45 alter the most critical basic residues in a recombinant LG45 and a6β4 integrins colocalized.70 Although the β4 integrin was protein, we identified a unique heparin-binding site surrounded correctlyexpressedandlocalizedattheapicalsideoftheepidermal by a track of converging low affinity, positively charged basal layer, LG45-uncleavable laminin 332 expressing keratino- residues (Fig.3D and E).49 We further showed that this cytes failed toform hemidesmosomes when seeded over adermal K KLRIKSKEK sequence region, which matches one of the 1433 equivalent model.19 sites identified by Vives et al.85 harbours distinctive syndecan-1 Other studies have reported that soluble LG45 may have a and-4interactionsites(Table1).Besides,ourgrouphasreported function when released from laminin 332. Experiments have thatsyndecan-1isthecellularreceptorinvolvedincelladhesionto shown that adding either recombinant a3LG45 or LG4 to the the a3 LG45 domain57,86 and this interaction may participate in culture medium could induce migration of keratinocytes in a keratinocytemigrationbysupportingtheformationofactin-based MMP-9-dependent manner.78 In similar experiments, LG4 cellular protrusions (Fig.4A and B).76,87 The development of induced activation of pro-migratory MMP-1.79 Moreover, Ras/ these membrane protrusions remarkably requires dephosphoryla- IkBa transformed keratinocytes that lacked the LG45 domain tion of tyrosine residues in the cytoplasmic tail of syndecan-1, a showed a deficiency in MMP-9 and MMP-1 expression. This condition essential for syntenin-1 recruitment.87,88 In the deficiency was reversed by replacing LG45 through retroviral epidermis, syndecan-1 is located in the pericellular region of transduction.70 keratinocytes and displays a modest expression in the basal cell Bindingsiteswithina3LG45.Themechanismunderlyingthe layer,whichbecomesincreasinglyintenseinthesuprabasallayers. function of the LG45 domain in laminin 332 remains poorly Remarkably, syndecan-1 is strongly induced in wound edge understood. Several heparin-binding sites have been identified in keratinocytes during wound healing.89-91 This elevated expression 130 CellAdhesion&Migration Volume7Issue1 inkeratinocytesappearstobespecifictosyndecan-1asnochanges defective c2 processing was found in the rare inherited have been detected for other syndecans.92 Moreover, syndecan-1 cylindromatosis disease (see above), in which abnormal basement deficient mice display a defect in keratinocyte proliferation and membranes and excessive ectopic expression of collagen VII were migration during wound healing.93 In light of these data, found.63,96 The defective processing of the c2 chain may have syndecan family members stimulate interest as potential laminin resulted in a deficient interaction with its basement membrane 332 co-receptors. Recent data has provided evidence that when partners collagen VII causing defaults in anchoring fibrils human squamous carcinoma A431 cells are plated on laminin linkage.32,95 332, syndecan-1 forms a complex with the a6β4 integrin. This Numerousstudiesconductedwithnormalbreastepithelialand triggers Fyn-mediated phosphorylation of the β4 integrin carcinomacellshaveshownthatcellmigration-inducingfunctions cytoplasmic tail activating PI3K- and Akt-mediated signaling, of the c2 chain rely on its proteolysis.35 Recently, it has been protecting the cells against apoptosis.94 This is of particular reported that a syndecan-1 interaction with the N-terminal interest as LG45 in a3 laminin 332 was proposed as an domain in precursor c2 could negatively regulate β4 integrin 200 essential PI3K pathway activation promoter.70 phosphorylationleadingtoenhancedcarcinomacelladhesionand decreased motility97 reinforcing the idea that the precursor c2 Functions of Precursor c2 N-Terminal Domain chain does not support cell migration. In contrast, other reports have suggested that precursor c2 may play a role in keratinocyte It has been shown that in human skin basement membrane, the migrationduringthewoundhealingprocess.Aninteractionofits N-terminal domain of the c2 chain is absent suggesting that the N-terminal portion with a2β1 integrin was shown to occur c2 processing could have an important physiological role.11,63 In during TGF-β1 induced keratinocyte migration in vitro and was contrast to these findings, antibodies specific for the released L4 proposed to take place at the wound margin of the skin healing domain were shown to label the basal epidermal cells in mouse process in vivo.68 Keratinocytes at the edge of wounds made in skin.24 A positive signal was also found in the basement early passage cultures shown to co-express the laminin 332 c2 membranes of early stages of skin development suggesting that chain and the cell cycle inhibitor p16INK4a, displayed increased the precursor c2 chain or its released N-terminal domain directional motility.98 Additional studies revealed that this expression might persist in vivo.24 The analysis of mTLD/BMP- keratinocyte hypermotility/growth arrest response relied on both 1 deficient mice revealed that although defaults in skin cohesion the precursor state of the laminin 332 c2 chain and the were not clinically apparent, ultrastructural examination of the participation of a serum co-factor through a TGFβ receptor I basement membrane showed places where anchoring structures dependent mechanism.99 This apparent contradiction in experi- were disconnected at the level of the lamina densa with presence mentalfindingsislikelyduetotissuespecificdifferences.Astudy of rudimentary hemidesmosomes.15,25 A strong expression of the indicatedthattheL4modulemediatestheintegrationoflaminin precursor c2 chain suggested that c2 chain maturation takes part 332 into the extracellular matrix through intermolecular in the basement membrane cohesion and stability. Besides, interactions.73 The question arises whether the heparin-binding domains located within the a3 LG45 domains and the N-terminal portion of the c2 chain are involved in laminin 332 deposition. Heparinwasshowntoinhibitlaminin332depositionwithinthe ECM suggesting that a heparan sulfate proteoglycan might be involved in binding to both precursor a3 and c2.13 Null mutation in syndecan-1100 and -4101 has not reported defects in laminin deposition, which suggests that any of these two receptors is solely responsible for this mechanism. Generation of double syndecan-1 and -4 knockout mice would reveal whether both these receptors are implicated. Future studies should examine whether a heparan sulfate proteoglycan cell surface receptor or a basement membrane components are is implicated inprecursorlaminin332deposition.Interestingly,arecentstudy Figure4.Syndecan-1mediatedcelladhesiontolaminin332LG45 domain.(A)FibrosarcomaHT1080cellswereplatedonsurfacescoated hasreportedthattheperlecancolocalizeswithlaminin332atthe withpurifiedLG45domainfor1h,fixedandstainedwithphalloidin- wound margin of healing full thickness wounds generated in FITC.Theanalysisoftheactinstainingrevealedanorganizationofthe mice suggesting proximity of these two components during cytoskeletoninradialarraysofmicrospikes,protrusiveadhesion basement membrane remodelling.102 However, as perlecan was structuresknowntobeinvolvedintransientcell-substratuminteractions. shown not to be essential for matrix assembly in perlecan-null (B)ConfocalimageofHT1080cellsexpressingGFP-syndecan-1platedon theLG45domain.Thephasecontrastimageshowsthetypicalinduced mouse embryos,103 its presence in the ECM might not be an cellshapecharacterizedbytheformationofspike-likeactin-based absolute requirement for laminin 332 deposition. Considering protrusivestructures.Thereceptorsyndecan-1(green)promotescell the important function of laminin 332 in skin homeostasis, adhesiontothelamininfragmentandalignsalongtheactinfilaments, severalextracellularligandsmightbeinvolvedinitsdepositionin whichareorganizedintoparallelbundles.Bars,10mm. the basement membrane. www.landesbioscience.com CellAdhesion&Migration 131

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