title: Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.
abstract: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue. Four antibodies were raised in rabbits against two non-overlapping N-terminal NOTCH3 sequences. These reagents were used in immunohistochemical experiments to detect epitopes in post-mortem CADASIL brains (n=8), control brains, and cells overexpressing NOTCH3. To determine the biochemical nature of NOTCH3 epitopes, we used these antibodies to probe pure NOTCH3-Fc fusion proteins treated with acid, urea, guanidinium, ionic detergents, acrylamide, and thiol- and phosphorus-based reductants. All antibodies avidly stained arteries in 8 of 8 CADASIL brain samples. The most prominent staining was in degenerating media of leptomeningeal arteries and sclerotic penetrating vessels. Normal appearing vessels from control brains were not reactive. Antibodies did not react with cultured cells overexpressing NOTCH3 or with purified NOTCH3-Fc protein. Furthermore, treatment of pure protein with acid, chaotropic denaturants, alkylators, and detergents failed to unmask N-terminal NOTCH3 epitopes. Antibodies, however, recognized novel N-terminal epitopes in purified NOTCH3-Fc protein treated with three different reductants (DTT, beta-mercaptoethanol, and TCEP). We conclude that CADASIL arteries feature latent N-terminal NOTCH3 epitopes, suggesting the first evidence in vivo of NOTCH3 structural alterations.
authors.affiliation: Departments of Neurology, University of Michigan, Ann Arbor, MI 48109-5622, USA.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Departments of Neurology, University of Michigan, Ann Arbor, MI 48109-5622, USA.
authors.forename: Soo Jung
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Departments of Neurology, University of Michigan, Ann Arbor, MI 48109-5622, USA.
authors.forename: Kelly Z
authors.initials: KZ
authors.lastname: Young
authors.affiliation: Josephson-Wallack-Munshower Neurology, Indianapolis, IN 46260, USA; Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46260, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Josephson
authors.affiliation: Department of Neurology, Memory and Aging Center, University of California, San Francisco, CA 94143, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Geschwind
authors.affiliation: Departments of Neurology, University of Michigan, Ann Arbor, MI 48109-5622, USA; Molecular and Integrative Physiology, University of Michigan, 7725 Med Sci II 5622, 1137 Catherine Street, Ann Arbor, MI 48109-5622, USA; VA Ann Arbor Healthcare System, Ann Arbor, MI 48105, USA. Electronic address: micwang@umich.edu.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Wang
mesh_terms: D000818:Animals; D001158:Arteries / Q000378:metabolism / Q000473:pathology; D001921:Brain / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D046589:CADASIL / Q000378:metabolism* / Q000473:pathology; D000939:Epitopes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D010084:Oxidation-Reduction; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D000939:Epitopes; C499374:NOTCH3 protein, human; D000071656:Receptor, Notch3; D051880:Receptors, Notch
keywords: CADASIL; Leptomeningeal; NOTCH3; Redox state; Small vessel disease