Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia

Abstract

Cerebrovascular pathology at the biochemical level has been informed by the study of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a vascular disorder caused by NOTCH3 mutations. Previous work in CADASIL described N-terminal proteolysis of NOTCH3 generated by specific non-enzymatic cleavage of the first Asp-Pro sequence of the protein. Here, we investigated whether the second Asp-Pro peptide bond (residues 121–122) of NOTCH3 is cleaved in CADASIL. Monospecific antibodies were generated that recognize the neo-epitope predicted to be generated by cleavage after Asp121. These antibodies were used to localize cleavage events at Asp121 in post-mortem CADASIL and control brain tissue and to investigate factors that regulate cleavage at Asp121. We report that cleavage at Asp121 occurs at a high level in the arterial media of CADASIL cerebral arteries. Leptomeningeal arteries demonstrated substantially more cleavage product than penetrating arteries in the white matter, and control vessels harbored only a small amount of cleaved NOTCH3. Proteolysis at Asp121 occurred in purified preparations of NOTCH3 ectodomain, was increased by acidic pH and reductive conditions, and required native protein conformation for cleavage. Increasing the concentration of NOTCH3 EGF-like domain protein elevated the level of proteolysis. On the other hand, several polyanionic chemicals potently blocked cleavage at Asp121. These studies demonstrate that the NOTCH3 protein in CADASIL is cleaved in multiple locations at labile Asp-Pro peptide bonds. As such, chronic brain vascular disease, like other neurodegenerative conditions, features proteolysis of pathological proteins at multiple sites which may generate small pathological peptides.

Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences

“…Cleavage at NOTCH3 residue Asp121, which is required for the generation of NTF2, is enriched in media of CADASIL arteries, the site of mutant NOTCH3 antigen accumulation [ 15 , 22 , 30 ]. To test if NTF2 may preferentially trans-reduce CADASIL mutant NOTCH3, we compared thiol availability of wild type and mutant recombinant NOTCH3 fragments after incubation with NTF2 peptide.…”

“…PLA assays, which localize molecules in extremely close proximity within tissues, were performed on frontal lobe sections of four genetically characterized CADASIL samples and four control samples. Antibodies specific for the Asp121 cleavage product (145H; [ 22 ]) and for the EGF-like domain 17–21 (1E4; full length) of NOTCH3 were used together and individually (as negative controls) in standardized PLA assays. Because 145H recognizes the C-terminus of the cleavage between EGF-like domains 2 and 3, it binds to NTF2 but not the intact large NOTCH3 ectodomain and not to the C-terminal cleavage fragment resulting from Asp121 cutting.…”

“…Both NTF and NTF2 are predicted to result from cleavage of NOTCH3, a process which occurs spontaneously in CADASIL vessels [ 15 , 22 ]. It remains to be determined what the role of cleavage plays in trans-reduction.…”

“…De-identified formalin fixed brain sections from autopsies were procured from the Brain Bank of the National Institute for Developmental and Childhood Disorders at the University of Maryland and from the Alzheimer’s Disease Center at the University of Michigan. CADASIL samples obtained at autopsy from genetically characterized individuals have been described [ 22 ]. All samples were frontal lobe, five-micron sections prepared for proximity ligation assay (PLA) using standard immunohistochemical conditions with antibodies specific against a neo-epitope revealed by cleavage at NOTCH3 Asp121 [ 22 ] (145H; rabbit mAb) and full length NOTCH3 [ 27 ] (1E4; Millipore, Burlington, MA, USA).…”

“…The EGF-like repeats of NOTCH3 and other extracellular domains are homologous, raising the possibility that additional EGF-like sequences, such as NTF, may possess trans-reduction potential. Recently, we identified a second cleavage site in NOTCH3 that occurs in CADASIL vessels; this cleavage is predicted to separate EGF-like domain 2 from domain 3 [ 22 ]. We designated the peptide resulting from sequential cleavages at Asp80 and Asp121 as NTF2, a sequence that is composed of a 41 amino acid peptide (residues 81–121) that is similar to NTF.…”