title: Novel UNC-44 AO13 ankyrin is required for axonal guidance in C. elegans, contains six highly repetitive STEP blocks separated by seven potential transmembrane domains, and is localized to neuronal processes and the periphery of neural cell bodies.
abstract: Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin-spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin-binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X(n)VVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin-binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin-binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin-specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development.
authors.affiliation: Department of Biological Sciences, Illinois State University, Campus Box 4120, Normal, Illinois 61790-4120, USA. ajotsuka@ilstu.edu
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Otsuka
authors.forename: Pratumtip
authors.initials: P
authors.lastname: Boontrakulpoontawee
authors.forename: Natalie
authors.initials: N
authors.lastname: Rebeiz
authors.forename: Marc
authors.initials: M
authors.lastname: Domanus
authors.forename: Dawn
authors.initials: D
authors.lastname: Otsuka
authors.forename: Nena
authors.initials: N
authors.lastname: Velamparampil
authors.forename: Sabrina
authors.initials: S
authors.lastname: Chan
authors.forename: Marshall
authors.initials: M
authors.lastname: Vande Wyngaerde
authors.forename: Sarah
authors.initials: S
authors.lastname: Campagna
authors.forename: Andrea
authors.initials: A
authors.lastname: Cox
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000302:isolation & purification*; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology* / Q000254:growth & development; D029742:Caenorhabditis elegans Proteins; D002451:Cell Compartmentation / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis / Q000235:genetics*; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000502:physiology; D009420:Nervous System / Q000166:cytology / Q000196:embryology* / Q000254:growth & development; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics
chemical_list: D017487:Ankyrins; D029742:Caenorhabditis elegans Proteins; D016900:Neurofilament Proteins; D020033:Protein Isoforms; C093514:unc-44 protein, C elegans