Abstract
Amyloid beta-peptide (Abeta) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with Abeta in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Abeta plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Abeta. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Abeta-(1-40) and Abeta-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Abeta. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Abeta deposits.
Highlights
Alzheimer’s disease (AD)1 is a progressive neurodegenerative disorder characterized by selective neuronal loss associated with intracellular neurofibrillary tangle formation and extracellular amyloid plaques (1)
Purification of Recombinant CLAC—To characterize CLAC and to study its interaction with A, we developed a method for the expression and purification of recombinant CLAC
CLAC fused to a C-terminal myc/His tag was purified from conditioned medium from human embryonic kidney (HEK) 293 cells stably expressing CLAC-P-Myc/His using a cation-exchange column and a nickel-chelating column
Summary
Construction of CLAC-P and Mutant Expression Vectors—A splice variant of CLAC-P/collagen type XXV consisting of 580 residues was cloned from a human brain cDNA library into the pcDNA3.1myc/His expression vector, which contains a C-terminal myc/His tag, as described previously (19). Fractions containing recombinant CLAC were dialyzed overnight against Tris-buffered saline (TBS) at pH 7.4. Heparin Binding Analysis—Conditioned medium from cells stably expressing CLAC-P-Myc/His was loaded onto a heparin column (HiTrap heparin, 1 ml; Amersham Biosciences AB) equilibrated with PBS and washed with PBS, followed by stepwise elution with phosphate buffer containing 0.1, 0.25, 0.5, and 1 M NaCl. Fractions were analyzed for the presence of CLAC by SDS-PAGE and immunoblotting using anti-Myc antibody. The wells were washed three times with TBS-T and incubated with anti-Myc antibody (1:5000 in blocking buffer) for 1 h. Five nM CLAC was mixed with different amounts of peptide (1.0, 10.0, and 25 M) and incubated in wells coated with aggregated A-(1– 42) for 2 h, followed by detection as described above. The aggregated peptides were diluted in PBS to a final peptide concentration of 25
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