440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons.

Abstract

Abstract. 440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrins, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. . J. Cell Biol. 236:1372-1379. This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrine, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and prolinedirected protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible a-helix or/3 sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrine with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrins is selectively targeted to premyelinated axons, and that 440-kD ankyrine disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrins. 440-kD ankyrins thus is a specific component of unmyelinated axons and expression of 440-kD ankyrins may be downregulated as a consequence of myelination.KYRINS are a family of spectrin-binding proteins that link the spectrin/actin network to cytoplasmic domains of integral proteins that include ion channels and cell adhesion molecules (Bennett, 1992; Bennett and Giliigan, 1993;Davis et al., 1993). Ankyrins contain three structural domains: (a) an NH2-terminal 89-95-kD membrane-binding domain (Davis and Bennett, 1990a); (b) a 62-kD domain that binds to spectrin (Bennett, 1978); and (c) a COOH-terminai domain that is the target of alternative splicing and represents the most variable domain among different ankyrins. A striking feature of the membranebinding domain is the presence of 24 tandem repeats of 33 amino acids. The 33-residue repeats are necessary and sufliciem for association of ankyrin with the anion exchanger (Davis and Bennett, 1990b;Davis et al., 1991), the voltagedependent sodium channel (Srinivasan et al., 1992), and nervous system cell adhesion molecules related to L1 and neurofascin (Davis et al., 1993).

Giant ankyrin-G: A critical innovation in vertebrate evolution of fast and integrated neuronal signaling

“…Although giant exons of ankyrin-B and AnkG were discovered over 20 y ago (16,34,35), relatively little is known about their function. The 270-kDa AnkG, resulting from an in-frame splicing event that eliminates 1,900 amino acids from the giant exon, retains the ability to target to the AIS of WT neurons (36)(37)(38)(39) as well as AnkG-deficient neurons (40).…”

An ankyrin-related gene (unc-44) is necessary for proper axonal guidance in Caenorhabditis elegans.

“…Paralleling the human ANK2 gene, unc-44 encodes "conventional" ankyrin isoforms (AO49 and AO66 ankyrins) with gross similarities to erythrocyte ankyrin and brain ankyrin 2, as well as a much larger form of ankyrin (AO13 ankyrin). Although AO13 ankyrin is predicted to be similar in size to vertebrate brain ankyrin 1, its carboxyl-terminal domain sequence is highly acidic and distinct from that reported from brain ankyrin 1 (Chan et al, 1993).…”

“…Brain ankyrins 1 and 2 contain different carboxyl termini produced by alternative splicing at aa position 1444 . In brain ankyrin 1, alternative splicing results in insertion of 2085 aa, including 15 repeats of a 12-aa sequence, into the carboxyl-terminal domain to produce a 440-kD product , Chan et al, 1993. For unc-44, the amino acids are numbered from the start of the partial cDNAs, while those expected in the fuU-length protein are noted in parentheses.…”

“…Molecular analysis of human brain ankyrin cDNA clones reveals several alternatively spliced RNAs . The predicted products of two of the RNAs are brain ankyrin 2 (202 kD) with a gross structural similarity to erythrocyte ankyrin, and brain ankyrin 1 ("~440 kD) which contains the typical ankyrin repeat, linker, and spectrin-binding domains, but has an extensive carboxyl-terminal domain Chan et al, 1993). Brain and erythrocyte ankyrins bind to distinct sites on kidney membranes, suggesting different transmembrane protein targets for the different ankyrins (Davis et al, 1989).…”

Rearrangement in the PITX2 and MIPOL1 genes in a patient with a t(4;14) chromosome

“…Studies in rat and Drosophila previously showed that the ANK2 ortholog had predominant expression in embryonic, neonatal and adult brain, particularly in areas rich in axons and dendrites. 23,24 The Ank2 protein is selectively targeted to premyelinated axons, and is possibly involved in neuronal migration. Using a panel of amplimers designed within the PRO0971, T2BP and ANK2 genes, no deletions or rearrangements among these genes were detected (Figures 1 and 3).…”