title: Preparation, crystallization, and preliminary crystallographic analysis of wild-type and mutant human TREM-2 ectodomains linked to neurodegenerative and inflammatory diseases.
abstract: TREM-2 (triggering receptor expressed on myeloid cells-2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations. In order to facilitate these studies, we undertook the production and crystallization of these proteins. Here we demonstrate that, unlike many single Ig domain proteins, TREM-2 could not be readily refolded from bacterially-expressed inclusion bodies. Instead, we developed a mammalian-cell based expression system for the successful production of wild-type and mutant TREM-2 proteins in milligram quantities and a single-chromatography-step purification scheme that produced diffraction-quality crystals. These crystals diffract to a resolution of 3.3 Å and produce data sufficient for structure determination. We describe herein the procedures to produce wild-type and mutant human TREM-2 Ig domains in sufficient quantities for structural and biophysical studies. Such studies are crucial to understand the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them.
authors: Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine, St. Louis, MO 63110, United States; Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors: Daniel L
authors: DL
authors: Kober
authors: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors: Kelsey M
authors: KM
authors: Wanhainen
authors: Biochemistry Program, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors: Britney M
authors: BM
authors: Johnson
authors: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors: David T
authors: DT
authors: Randolph
authors: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States; Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, United States; Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States.
authors: Michael J
authors: MJ
authors: Holtzman
authors: Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States; Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, United States; Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, United States; Drug Discovery Program in Pulmonary and Critical Care Medicine, Washington University School of Medicine, St. Louis, MO 63110, United States. Electronic address: tbrett@wustl.edu.
authors: Tom J
authors: TJ
authors: Brett
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006801:Humans; D007249:Inflammation / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000648:ultrastructure*; D009154:Mutation; D019636:Neurodegenerative Diseases / Q000473:pathology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics* / Q000648:ultrastructure*
chemical_list: D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; C498715:TREM2 protein, human
keywords: Alzheimer’s disease; Crystallography; Inflammation; Innate immunity; Neurodegenerative disease; Parkinson’s disease; Receptor