Genomes and Genes
Llama-derived phage display antibody arrays for FSHD
Principal Investigator: Silvere M van der Maarel
Abstract: The aim of this project is to gain insight in the cellular and molecular processes leading to dysfunction of the neuromuscular system in FSHD patients by large scale analysis of protein homeostasis in tissues and cell lines of patients and controls. In the past few years, projects have been launched to study deregulation of biological pathways in FSHD on RNA level. These strategies include differential display and RNA profiling experiments on commercial and custom made DNA chips and arrays. Despite their limitations, DNA arrays are now one of the most commonly used and successful methods to determine the molecular and cellular aspects of many acquired and genetic diseases. It is anticipated that also for FSHD, this approach will provide a valuable contribution in understanding its pathology. Nevertheless, protein levels, including the level of modified proteins and the composition of protein complexes are of an order of importance larger to understand FSHD pathophysiology. Consequently there is a need for protein arrays. The llama antibody technology provides a unique opportunity to develop protein arrays. The power of the llama system for this purpose is that this animal makes single (heavy) chain antibodies. Using the genetic information for this single-chain repertoire for the construction of phage-display antibody libraries abolishes the need to combine heavy- and light-chains, one of the major drawbacks of conventional phage-display libraries. Moreover, these single-chain antibodies tend to have a very high affinity and stability. It has already been demonstrated that large naive and directed libraries of antibodies can be generated. Experience in cloning, production and isolation of these llama antibodies is available. We propose to generate muscle-specific antibody arrays derived from Llama single-chain phage-display clones. To this end, a Llama will be immunized with human muscle protein homogenates, and after peak response, a phage display library will be constructed. Antibody clones will be selected with a variety of selection procedures (e.g. with recombinant proteins or with muscle homogenates from different species) and arrayed on glass slides. Well characterized single chain antibody arrays will be used to study FSHD pathophysiology on fluorescently labeled protein homogenates of tissues and cell cultures of patients and controls. Evidently, these antibodies can also be used individually for specific immunohistochemical and immunocytochemical studies.
Funding Period: 2001-09-30 - 2004-08-31
more information: NIH RePORT
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B107 Chemical and Life Sciences Laboratory, Department of Cell and Developmental Biology, University of Illinois at Urbana Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, USA
J Mol Biol 411:397-416. 2011....
- Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage displayYanchao Huang
1Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands
Eur J Hum Genet 13:721-30. 2005..Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin...
- Facioscapulohumeral muscular dystrophySilvere M van der Maarel
Leiden University Medical Center LUMC, Department of Human Genetics, Postal zone S 3 P, PO Box 9600, 2300 RC Leiden, The Netherlands
Biochim Biophys Acta 1772:186-94. 2007..Clearly, better disease models need to be developed to identify and test novel intervention strategies to eventually improve the quality of life for patients with FSHD...
- Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validationPeter Verheesen
University of Utrecht, Department of Molecular and Cellular Biology, Utrecht, The Netherlands
Biochim Biophys Acta 1764:1307-19. 2006....
- FRG1P-mediated aggregation of proteins involved in pre-mRNA processingSilvana van Koningsbruggen
Department of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, Netherlands
Chromosoma 116:53-64. 2007..Intriguingly, among FRG1P-associated proteins are SMN and PABPN1, both being involved in neuromuscular disorders, possibly through RNA biogenesis-related processes...
- AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regenerationYanchao Huang
Center for Human and Clinical Genetics, Leiden Univesity Medical Center, Leiden, The Netherlands
FASEB J 21:732-42. 2007..It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair...
- Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscleYanchao Huang
Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
Hum Mol Genet 17:1855-66. 2008..Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex...