Molecular characterization of the genetic lesion in Dystonia musculorum (dt-Alb) mice.

Dystonia musculorum (dt) is an inherited autosomal recessive neuropathy in mice. Homozygous animals display primarily sensory neurodegeneration resulting in a severe loss of coordination. Several dt strains exist, including spontaneous mutants dt-Alb (Albany), dt-J (Jackson Labs), and dt-Frk (Frankel), and a transgene insertion mutant, Tg4. They contain mutations in the gene encoding Bullous Pemphigoid Antigen 1 (BPAG1), or dystonin. BPAG1 is a member of the plakin family of cytolinker proteins. BPAG1 is alternatively spliced to produce several isoforms, including the major brain-specific isoform, BPAG1a. The neurological phenotype observed in dt-Alb mice is thought to result from the absence of BPAG1a protein in the developing nervous system. The goal of this study was to determine the precise molecular nature of the dt-Alb mutation and examine residual BPAG1 expression in homozygous dt-Alb mice. A combination of molecular biological strategies revealed that the dt-Alb lesion is a deletion-insertion eliminating a large part of the coding region of BPAG1a. The molecular lesion in the dt-Alb BPAG1 allele is expected to render it completely non-functional. Although transcripts corresponding to BPAG1 segments still remaining in homozygous dt-Alb mice could be detected by RT-PCR, there was no positive signal for BPAG1 in the brain of dt-Alb mice by Northern blotting. Western blotting with polyclonal anti-BPAG1 antibodies confirmed the absence of functional BPAG1 protein (full-length or truncated) in the dt-Alb brain. Our identification of the 5' junction of the dt-Alb insertion makes it possible to genotype dt-Alb animals by standard PCR.

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

Dissecting the sequence specific functions of alternative N-terminal isoforms of mouse bullous pemphigoid antigen 1.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins that is involved in cross-linking the cytoskeletal elements and attaching them to cell junctions. BPAG1 null mice develop severe degeneration of sensory neurons that was attributed in part due to the absence of a splice variant called BPAG1a that harbors an actin-binding domain at the N-terminus. Additional alternative splicing also results in BPAG1a isoforms with different first exons, leading to three additional types of BPAG1a called isoforms 1, 2 and 3 (or BPAG1a1, BPAG1a2, and BPAG1a3). These unique N-terminal extensions of the BPAG1a isoforms are of variable length. In this study, we characterized these N-terminal isoforms and evaluated the influence of these unique N-terminal sequences to the actin-binding properties. The unique N-terminal region of isoform 1 is very short and was not expected to affect the property of the ABD that followed it. In contrast, transfection studies and mutagenesis analyses signified that the N-terminal sequences of isoform 2 had the ability to bundle actin filaments and the N-terminal region that contained isoform 3 showed cortical localization. Isoforms 1, 2 and 3 also displayed differential tissue expression profiles. Taken together, these data suggested that the unique N-terminal regions of these isoforms have different roles that may be tailored to meet tissue specific functions.