Figure 6

Vitronectin fibrils contain a protease-resistant core. A) In-solution trypsin digestion of vitronectin fibrils reveals a protease-resistant band of ~5 kDa (lane 2). Partial sequencing by N-terminal Edman degradation identified the peptide AMWLSLFSSEESNLGANNYDD, which corresponds to residues 380–400. These amino acids reside in the C-terminus of the protein and likely comprise a portion the amyloidogenic core. This result was confirmed by immunoblotting with a C-terminal-specific antibody (data not shown). Arrowheads indicate molecular weight (kDa). Lane 1 contains undigested vitronectin fibrils, much of which is unresolved (asterisk). B) Upper panel: Several peptides were identified by MALDI-TOF analysis of the protease-resistant band. The mass/charge (m/z) values, amino acid sequence and residue numbers are displayed in the table. Each m/z does not correspond to any other vitronectin peptides and oxidation had to be invoked for peptide A380-R427. Lower panel: Primary sequence analysis of vitronectin using the TANGO algorithm [68] reveals several C-terminal stretches prone to cross-beta aggregation, two of which correlate well with the peptides A380-R402 (gray) and A380-R427 (hatched) identified by mass spectrometry. Y-axis denotes relative cross-beta aggregation propensity.