title: Ahnak1 abnormally localizes in muscular dystrophies and contributes to muscle vesicle release.
abstract: Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150 nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.
authors: Muscle Research Unit, Experimental and Clinical Research Center, a joint cooperation between  the Charité Medical Faculty and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany.
authors: Ute
authors: U
authors: Zacharias
authors: Bettina
authors: B
authors: Purfürst
authors: Verena
authors: V
authors: Schöwel
authors: Ingo
authors: I
authors: Morano
authors: Simone
authors: S
authors: Spuler
authors: Hannelore
authors: H
authors: Haase
mesh_terms: D002154:Calpain / Q000235:genetics / Q000378:metabolism; D016022:Case-Control Studies; D003238:Connective Tissue / Q000648:ultrastructure*; D000073939:Dysferlin; D006720:Homozygote; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000648:ultrastructure*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D049288:Muscular Dystrophies, Limb-Girdle / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D012508:Sarcolemma / Q000648:ultrastructure*; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: C075176:AHNAK protein, human; C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; C105884:CAPN3 protein, human; D002154:Calpain