title: Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display.
abstract: Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.
authors.affiliation: 1Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Huang
authors.forename: Peter
authors.initials: P
authors.lastname: Verheesen
authors.forename: Andreas
authors.initials: A
authors.lastname: Roussis
authors.forename: Wendy
authors.initials: W
authors.lastname: Frankhuizen
authors.forename: Ieke
authors.initials: I
authors.lastname: Ginjaar
authors.forename: Faye
authors.initials: F
authors.lastname: Haldane
authors.forename: Steve
authors.initials: S
authors.lastname: Laval
authors.forename: Louise V B
authors.initials: LV
authors.lastname: Anderson
authors.forename: Theo
authors.initials: T
authors.lastname: Verrips
authors.forename: Rune R
authors.initials: RR
authors.lastname: Frants
authors.forename: Hans
authors.initials: H
authors.lastname: de Haard
authors.forename: Kate
authors.initials: K
authors.lastname: Bushby
authors.forename: Johan
authors.initials: J
authors.lastname: den Dunnen
authors.forename: Silvère M
authors.initials: SM
authors.lastname: van der Maarel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000032:analysis; D002161:Camelids, New World / Q000276:immunology; D004252:DNA Mutational Analysis; D000073939:Dysferlin; D006801:Humans; D007118:Immunoassay; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology*; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000032:analysis* / Q000276:immunology*; D009136:Muscular Dystrophies / Q000378:metabolism*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000276:immunology
chemical_list: C114839:DYSF protein, human; D000073939:Dysferlin; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007527:Isoenzymes; D008565:Membrane Proteins; D009124:Muscle Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C105884:CAPN3 protein, human; D002154:Calpain