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Tissue-Specific Co-Expression and in Vitro Heteropolymer Formation of the Two Small Branchiostoma Intermediate Filament Proteins A3 and B2

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Tissue-Specific Co-Expression and in Vitro Heteropolymer Formation of the Two Small Branchiostoma Intermediate Filament Proteins A3 and B2 doi:10.1006/jmbi.2001.5298availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2002) 316, 127±137 Tissue-Specific Co-expression and in vitro Heteropolymer Formation of the Two Small Branchiostoma Intermediate Filament Proteins A3 and B2 AntonKarabinos1,JuÈrgenSchuÈnemann1,DavidA.D.Parry2 andKlausWeber1* 1Max Planck Institute for The two small intermediate ®lament (IF) proteins A3 and B2 of the Biophysical Chemistry cephalochordate Amphioxus were investigated. Blot overlays indicated a Department of Biochemistry heterotypic interaction pattern of the recombinant proteins. While the Am Fassberg 11 individual proteins formed only aggregates, the stoichiometric mixture 37077 Goettingen, Germany formed obligatory heteropolymeric ®laments. Mutant proteins with a single cysteine residue in equivalent positions gave rise to ®laments that 2Institute of Fundamental oxidize to the disul®de-linked heterodimer, which can again form IF. Sciences, Massey University Thus the A3/B2 ®laments, which are expressed in the intestinal Palmerston North, New epithelium, are based on a hetero coiled coil. This keratin-like assembly Zealand process of A3 plus B2 was unexpected, since previous evolutionary tree calculations performed by two laboratories on the various Amphioxus IF proteins identi®ed keratin I and II orthologs but left the A/B group as a separate branch. We discuss obvious evolutionary aspects of the Amphioxus IF multigene family, including the previously made obser- vation that B1, the closest relative of B2, forms homopolymeric IF in vitro and is, like vertebrate type III proteins, expressed in mesodermally derived tissues. # 2002 Elsevier Science Ltd. Keywords: Amphioxus; Branchiostoma; cephalochordate; coiled coil; *Corresponding author developmental expression Introduction IV spans ®ve neuronal IF proteins. Two large mol- ecular mass proteins (synemin and paranemin), and the two IF proteins of the eye lens (phakinin A variety of epidermal fragility syndromes are and ®lensin) fall outside these subfamilies. Finally, caused by mutations in human keratin genes, type V covers the nuclear lamins. suggesting that an important function of cyto- plasmic intermediate ®laments (IF) is to provide Lamins differ from vertebrate cytoplasmic IF resistanceagainstmechanicalstress.1Mostofthe proteins by an extra 42 residues in the coil 1b sub- close to 60 members of the mammalian multigene domain of the central rod domain and by unique family of IF proteins fall into one of the ®ve major carboxy-terminal domains that harbor a nuclear subgroups.2,3TypeIandtypeIIkeratinsarethe localisation signal and end, in most cases, with a largest subfamilies and give rise to the epithelial CaaXbox.4,5Theshortcoil1bdomain,®rstde®ned keratin ®laments that are based on obligatory het- in vertebrate cytoplasmic IF proteins, extends to eromeric double-stranded coiled coils formed by a thecephalochordatesandurochordates,6±9while type I and a type II keratin. Type III covers four cytoplasmic IF proteins from the protostomia show mesenchymally expressed proteins, which, at least the long coil 1b version of the nuclear lamins and, in part, are able to form homopolymeric IF. Type in most cases, display a lamin homology segment intheirtaildomains.10,11 Recent studies on the lancelet, the cephalochor- Abbreviations used: IF, intermediate ®lament; PVDF, 6,12±14 poly(vinylidene ¯uoride). dateBranchiostoma(Amphioxus), andthe E-mail address of the corresponding author: urochordateStyela8,9documentthattheearlychor- [email protected] dates have among their cytoplasmic IF proteins 0022-2836/02/010127±11 $35.00/0 # 2002 Elsevier Science Ltd. 128 Novel Heterotypic IF System in Amphioxus orthologs of the type I to III proteins originally sion numbers of the revised sequences are de®ned in the vertebrates. AJ223575 and AJ223576). The molecular masses We previously cloned 13 cytoplasmic IF proteins calculated from the revised sequences are in good from Amphioxus. Evolutionary tree calculations agreement with those obtained experimentally. indicated three type I and two type II keratins. ThealignmentinFigure1showsthatIFproteins This assignment was con®rmed by the obligatory A3 and B2 essentially lack a C-terminal tail heteropolymeric ®lament formation of the recombi- domain. Only two (B2) and four (A3) residues fol- nant proteins. Any stoichiometric mixture of type I low the well-conserved end of the rod domain. (k1, Y1 and E1) and type II (D1 and E2) proteins Unusually short tail domains are known for the provided IF. In addition, two of the lancelet type I Amphioxus keratin E1 and the human keratin 19 keratins formed chimeric IF when mixed with (6,12andreferencestherein). humankeratin8,atypeIIkeratin.6Threekeratins (k1, Y1 and D1) and protein X1 are the only IF pro- Blot overlays reveal heterotypic interactions teins expressed in the gastrula, which is reminis- between A3 and B2 cent of the expression of two type I keratins (K18 and K19) and one type II keratin (K8) in the mur- The ten currently available recombinant IF pro- inegastrula.15ThenumberoflanceletIFproteins teins of the lancelet were analysed in blot overlays increases at the neurula and early larval stages to using biotinylated A3 and B2 proteins. After exten- seven and 11, respectively. However, 13 different sive washing steps, the biotinylated proteins were proteins are found in the adult. The keratins are detected by the streptavidin-peroxidase system. the major IF proteins in the Amphioxus nerve Figure2(middlepanel)showsthatbiotinylatedA3 cord. Proteins X1 and C2 were found, by immu- binds strongly to B2. Except for a very weak reac- noelectron microscopy, to be integrated into the tion on B1, no other IF protein was detected by the epidermal and nerve cord IF, which are built from A3 probe. The biotinylated B2 protein reacted keratins. The latter two proteins and the C1 protein strongly with the A3 protein and more weakly seemtobelancelet-speci®cIFproteins.13The®ve withtheproteinB1(Figure2,lowerpanel).These remaining IF proteins form the A/B group in the results show a strong heterotypic interaction evolutionarytreepreviouslycalculated(Figure4of between A3 and B2, and indicate a lack of homo- Karabinosetal.6).Sofar,onlytheB1proteinhas typic interactions, i.e. A3 with A3 or B2 with B2, in been characterized. It can form homopolymeric IF the overlay system. These observations raised the in vitro and is expressed in the mesodermally question of whether A3 and B2 could form hetero- derived muscle tails and in coelomic epithelia. polymeric IF. These properties indicate some type III character 12,13 fortheB1protein. Here,weshowthatA3and Equimolar amounts of A3 and B2 form B2, which are co-expressed in the intestinal epi- heteropolymeric IF thelial cells, form obligatory heteropolymeric IF based on a hetero coiled coil consisting of one A3 Puri®ed recombinant proteins A3 and B2 were and one B2 polypeptide chain. dialysed either alone or in equimolar mixture to remove the urea, and the self-assembly products were analysed by electron microscopy after nega- Results tive staining. While the individual proteins yielded Branchiostoma IF proteins A3 and B2 only aggregated material, the mixture of A3 and essentially lack a tail domain B2formedlongIF(Figure3).Thus,A3andB2lack the ability to form homopolymeric IF but partici- Cloning and recombinant expression of A3 and pate in obligatory heteropolymeric IF assembly. B2havebeendescribed.6,7Duringthecurrent study we noted that the apparent molecular The structural unit of A3/B2 filaments is masses of both proteins observed by SDS-PAGE the heterodimer (A3, about 37 kDa; B2, about 39 kDa) were smaller than the masses calculated from the protein To decide whether the A3/B2 ®laments are sequences predicted from the cDNAs (A3, 41,679 based on a A3/B2 heterodimer or the A3 and B2 Da; B2, 49,326 Da). Automated sequencing of the homodimers, we used the approach previously proteins blotted on to a poly(vinylidene ¯uoride) taken to demonstrate the heterodimeric nature of (PVDF) membrane provided the predicted N-term- keratin®laments.9,16Itisbasedontheobservation inal sequences. The experimentally inferred masses that in parallel and unstaggered a helices of a derived by mass spectrometry (A3, 37,278 Da; B2, double-stranded coiled coil, a cysteine residue pre- 39,694 Da) were lower than the values calculated sent in an a or d position of the heptads can form a from the predicted sequences. We therefore sub- disul®de bond crosslink, provided both a helices jected the coding fragments to a detailed sequence have the cysteine residue in equivalent positions. analysisandfoundthattheoriginalanalysis7had Thus we aimed at mutants of A3 and B2 that have overlooked an earlier stop codon. Thus, the open a single cysteine residue, which is located in the reading frames of A3 and B2 were shortened by 39 same position as in the previously studied ker- and 92 codons, respectively (the GenBank acces- atins.9,16Thispositionisexactly40residuesfrom Novel Heterotypic IF System in Amphioxus 129 Figure 1. Alignment of the amino acid sequences of the ®ve related Branchiostoma IF proteins A1, A2, A3, B1 and B2. The typical structural organisation consisting of head, rod and tail domains is indicated. Arrowheads pointing down or up mark the start and end of the four subdomains of the helical rod domain (coils 1a, b, 2a and 2b), which are connected by three non-helical linkers (L1, L12 and L2). Asterisks (*) between the rod domains of A3 and B2 mark the a and d positions of the heptad repeat pattern with the
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