title: Fetuin A stabilizes m-calpain and facilitates plasma membrane repair.
abstract: Yeast two-hybrid experiments identified alpha(2)-Heremans-Schmid glycoprotein (human fetuin A) as a binding partner for calpain domain III (DIII). The tandem DIIIs of calpain-10 interacted under the most selective culture conditions, but DIIIs of m-calpain, calpain-3, and calpain-5 also interacted under less stringent selection. DIIIs of mu-calpain, calpain-6, and the tandem DIII-like domains of the Dictyostelium Cpl protein did not interact with alpha(2)-Heremans-Schmid glycoprotein in the yeast two-hybrid system. Bovine fetuin A stabilized proteolytic activity of purified m-calpain incubated in the presence of mm calcium chloride and prevented calcium-dependent m-calpain aggregation. Consistent with the yeast two-hybrid studies, fetuin A neither stabilized mu-calpain nor prevented its aggregation. Confocal immunofluorescence microscopy of scratch-damaged L6 myotubes demonstrated accumulation of m-calpain at the wound site in association with the membrane repair protein, dysferlin. m-Calpain also co-localized with fluorescein-labeled fetuin A at the wound site. The effect of fetuin A on calpain-mediated plasma membrane resealing was investigated using fibroblasts from Capns1(-/-) and Capns1(+/+) mouse embryos. Capns1 encodes the small noncatalytic subunit that is required for the proteolytic function of m- and mu-calpains. Thus, Capns1(-/-) fibroblasts do not express these calpains in active form. Fetuin A increased resealing of scrape-damaged wild-type fibroblasts but not Capns1(-/-) fibroblasts. These studies identify fetuin A as a potential extracellular regulator of m-calpain at nascent sites of plasma membrane wounding.
authors.affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, Ohio 43614, USA. ronald.mellgren@utoleco.edu
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Mellgren
authors.forename: Xinhua
authors.initials: X
authors.lastname: Huang
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism*; D002122:Calcium Chloride / Q000737:chemistry; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004023:Dictyostelium; D004622:Embryo, Mammalian / Q000378:metabolism* / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D020798:Two-Hybrid System Techniques; D060749:alpha-2-HS-Glycoprotein
chemical_list: C556354:AHSG protein, human; C556357:Ahsg protein, mouse; D001798:Blood Proteins; D060749:alpha-2-HS-Glycoprotein; D002154:Calpain; C449000:m-calpain; D002122:Calcium Chloride