title: Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.
abstract: Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.
authors: Department of Gene Therapy, Ulm University, Ulm, Germany.
authors: Bin
authors: B
authors: Huang
authors: Department of Gene Therapy, Ulm University, Ulm, Germany.
authors: Tanja
authors: T
authors: Lucas
authors: Department of Gene Therapy, Ulm University, Ulm, Germany.
authors: Claudia
authors: C
authors: Kueppers
authors: Department of Gene Therapy, Ulm University, Ulm, Germany.
authors: Xiaomin
authors: X
authors: Dong
authors: Center for Integrated Protein Science Munich, Department of Biotechnology, Technische Universität München, Garching, Germany.
authors: Maike
authors: M
authors: Krause
authors: Center for Integrated Protein Science Munich, Department of Biotechnology, Technische Universität München, Garching, Germany.
authors: Alexander
authors: A
authors: Bepperling
authors: Center for Integrated Protein Science Munich, Department of Biotechnology, Technische Universität München, Garching, Germany.
authors: Johannes
authors: J
authors: Buchner
authors: Novartis Institute for Biomedical Research, Basel, Switzerland.
authors: Hans
authors: H
authors: Voshol
authors: Novartis Institute for Biomedical Research, Basel, Switzerland.
authors: Andreas
authors: A
authors: Weiss
authors: Novartis Institute for Biomedical Research, Basel, Switzerland.
authors: Bertran
authors: B
authors: Gerrits
authors: Department of Gene Therapy, Ulm University, Ulm, Germany.
authors: Stefan
authors: S
authors: Kochanek
mesh_terms: D002460:Cell Line; D002942:Circular Dichroism; D004318:Doxycycline / Q000494:pharmacology; D006801:Humans; D000071058:Huntingtin Protein; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D004318:Doxycycline