European Journal of Human Genetics (2005) 13, 721–730 & 2005 Nature Publishing Group All rights reserved 1018-4813/05 $30.00 www.nature.com/ejhg ARTICLE Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display Yanchao Huang1,4, Peter Verheesen2,4, Andreas Roussis1, Wendy Frankhuizen1, Ieke Ginjaar1, Faye Haldane3, Steve Laval3, Louise VB Anderson3, Theo Verrips2, Rune R Frants1, Hans de Haard2, Kate Bushby3, Johan den Dunnen1 and Silve`re M van der Maarel*,1 1Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands; 2Department of Molecular and Cellular Biology, University of Utrecht, Utrecht, The Netherlands; 3Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK 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. European Journal of Human Genetics (2005) 13, 721–730. doi:10.1038/sj.ejhg.5201414 Published online 13 April 2005 Keywords: dysferlin; HCAb; phage display; immunohistochemistry; immunoprecipitation Introduction early age, but show progressive decline in muscle function The limb-girdle muscular dystrophies (LGMD) comprise a and increased muscle weakness from their late teens to large group of clinically and genetically heterogeneous early 20s. Mutations in DYSF not only cause LGMD2B with disorders with autosomal dominant or recessive inheri- prominent involvement of the proximal muscles of the tance. Recessive LGMD2B is typically slowly progressive limb and trunk but also the clinically distinct Myoshi and caused by mutations in the dysferlin gene (DYSF, MIM myopathy (MM),1,2 which mainly affects the posterior 603009).1 Usually, LGMD2B patients appear normal at an distal muscles of the lower limb. Mutations in DYSF were also shown to cause distal anterior compartment myo- pathy (DMAT).3 To date, approximately 100 DYSF muta- *Correspondence: Dr SM van der Maarel, Leiden University Medical Center, Center for Human and Clinical Genetics, Wassenaarseweg 72, tions have been reported (Leiden Muscular Dystrophy 2333 AL Leiden, The Netherlands. pages: http://www.dmd.nl/md.html) and there is no Tel: þ 31 71 527 1915; apparent genotype–phenotype correlation, with consider- Fax: þ 31 71 527 6075; E-mail: [email protected] able intrafamilial clinical heterogeneity.4 4These authors contributed equally to this work Received 13 October 2004; revised 8 February 2005; accepted 22 February Dysferlin is a member of the fer1-like family also 2005 including otoferlin, myoferlin and Fer1L4 and defined by Dysferlin antibody fragments Y Huang et al 722 homology to the Caenorhabditis elegans fer-1 gene.2,5,6 HCAb fragments or single-domain antibody fragments Dysferlin seems to play a crucial role in membrane repair.7 can be produced efficiently by microorganisms such as Dysferlin-null mice develop slowly progressive muscular Escherichia coli15 and Saccharomyces cerevisiae.16 dystrophy with vesicular accumulations and sarcolemmal We have already shown the utility of the selection and disruptions similar to those observed in human dysferlino- use of HCAb fragments from an immune library specific for pathy. Defects in these mice suggest a role for dysferlin in poly(A) binding protein nuclear 1 (PABPN1), which is Ca2 þ -dependent membrane repair. Specifically, these mice mutated in autosomal dominant and recessive oculophar- show an impairment of the process whereby the sarcolem- yngeal muscular dystrophy.17 We describe here the identi- ma of isolated myofibres was resealed following high- fication of HCAb fragments specific for different domains intensity laser irradiation. of dysferlin from a nonimmune library (ie generated from Patients with mutations in dysferlin often show reduc- nonimmunized animals), which eliminates the need for a tion or almost complete absence of dysferlin at the time-consuming immunization protocol. Using these anti- sarcolemmal membrane.8,9 Dysferlin was shown to interact body fragments for protein expression analyses in a panel with caveolin-3,10 which is also a sarcolemmal protein that of proven dysferlinopathy patients, we illustrate the is important for the formation of caveolae and mutated in variable pattern of protein expression in this group and the autosomal dominant LGMD1C.11 Interestingly, abnor- its application in functional analysis of dysferlin. mal intracellular localization of dysferlin can be observed in LGMD1C patients10 as well as other forms of LGMD.9 Only a few antibodies for dysferlin have been reported and Materials and methods the availability of a range of highly specific dysferlin Recombinant antigen production antibodies might facilitate diagnosis and improve the Two recombinant fusion proteins were generated to select understanding of this disease. HCAb fragments. DYSF1 contains amino-acid (aa) residues Typically, monoclonal antibodies are generated in 2–245 and has high homology with myoferlin and hybridoma cell lines, a laborious and expensive process otoferlin, while DYSF2 contains residues 1666–1788 and that has relatively limited application since antibody shows no homology to any known human protein.18 To design is impossible and mouse monoclonal antibodies generate DYSF1, a 740 bp fragment was PCR amplified from can be immunogenic when used in therapies. As an human muscle cDNA with forward primer (50-CGA GAT alternative, phage-display antibody libraries in which CTC TGA GGG TCT TCA TCC TCT ATG-30) and reverse antigen-binding domains are presented by phage particles primer (50-CTC AAG CTT AGC GGT AAC CTT GAC CAC can be screened for the presence of antibody fragments of AGG-30) and cloned into the TOPO 2.1 vector (Invitrogen, interest. As the genetic information is available, phage- Carlsbad, CA, USA). To generate DYSF2, a 375 bp fragment display antibody fragments are amenable to genetic was similarly PCR amplified with forward primer (50-GGA modification including antibody design and humanization TCC CTG GAG AAC AGG CT-30) and reverse primer to overcome immunogenicity.12 (50-GTC GAC CCA CAT CTG CAG CT-30) and also cloned The success of phage-display selections with conven- into TOPO 2.1. tional antibody repertoires strongly depends on the correct To test whether phage-display antibody fragments combination of heavy (VH) and light (VL) chains. How- against DYSF1 crossreact to myoferlin, recombinant myo- ever, construction of phage-display libraries by random ferlin protein (MYOF, aa 2–245) was produced by PCR assembly of VH and VL genes renders most of the amplification of a 740 bp fragment from cDNA clone combinations nonproductive or nonfunctional since the (Clone ID: DKFZp686C16167Q2, RZPD, Berlin, Germany) original VH and VL pairing is lost. Therefore, the with forward primer (50-GGG AAT TCC TGC GAG TGA construction of very large phage libraries is necessary. TTG T-30) and reverse primer (50-GGG AAG CTT AAA CAA An alternative phage-display approach makes use of un- CTC ATC-30) and cloned into the TOPO 2.1 vector. conventional antibody repertoire specific to Camelidae.13 Subsequently, fragments DYSF1, DYSF2 and MYOF were In addition to the conventional IgG repertoire, Camelidae digested with BglII/HindIII, EcoRI/SalI and EcoRI/SalI re- carry a heavy-chain antibody (HCAb) repertoire. HCAbs are striction, respectively, and ligated in the prokaryotic composed of two identical heavy chains and their func- expression vector pET28a (Novagen, Madison, WI, USA). tional antigen-binding domains are solely formed by their All constructs were sequence verified (LGTC, Leiden, The variable domains. This HCAb repertoire is eminently suited Netherlands). for phage-display applications since single-domain anti- Recombinant protein production was carried out in BL21 body fragments derived from the variable region (VHH) of (DE3)-RIL E. coli (Stratagene, La Jolla, CA, USA) according HCAbs represent the smallest naturally occurring intact to the manufacturer’s instructions. After 3.5 h induction, antigen-binding units (17 kDa) reported with antigen- the cells were collected by centrifugation, and lysed with binding affinities fully comparable with those reported BugBuster protein extraction reagent (Novagen) according for intact conventional antibodies.14 Moreover, these to the manufacturer’s instructions. All recombinant pro- European Journal of Human Genetics Dysferlin antibody fragments Y Huang et al 723 tein products were insoluble and purified by virtue of their Subcloning and purification of HCAb fragments hexahistidine tags using