title: Laminin inhibition of beta-amyloid protein (Abeta) fibrillogenesis and identification of an Abeta binding site localized to the globular domain repeats on the laminin a chain.
abstract: beta-Amyloid protein (Abeta) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimer's disease (AD). Inhibitors of Abeta fibrillogenesis are currently sought as potential future therapeutics for AD and related disorders. In the present study, the basement membrane protein laminin was found to bind Abeta 1-40 with a single dissociation constant, K(d) = 2.7 x 10(-9) M, and serve as a potent inhibitor of Abeta fibril formation. 25 microM of Abeta 1-40 was incubated at 37 degrees C for 1 week in the presence of 100 nM of laminin or other basement membrane components, including perlecan, type IV collagen, and fibronectin to determine their effects on Abeta fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Abeta-amyloid fibril formation, causing a ninefold inhibition at 1 and 3 days and a 21-fold inhibition at 1 week. The inhibitory effects of laminin on Abeta fibrillogenesis occurred in a dose-dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Abeta 1-40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Abeta fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Abeta-amyloid fibrils following 4 days of coincubation. Laminin was not effective as an inhibitor of islet amyloid polypeptide fibril formation, suggesting that laminin's amyloid inhibitory effects were Abeta-specific. To identify a potential Abeta-binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Abeta-binding elastase digestion product of approximately 120-130 kDa was found. In addition, a approximately 55 kDa fragment derived from V8 and elastase-digested laminin interacted with biotinylated Abeta 1-40. Amino acid sequencing of the approximately 55 kDa fragment identified a conformationally dependent Abeta-binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Abeta with relatively high affinity but is a potent inhibitor of Abeta-amyloid fibril formation. In addition, further identification of an Abeta-binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Abeta fibrillogenesis.
authors.affiliation: ProteoTech Inc., Kirkland, Washington 98034, USA. castillo@proteotech.com
authors.forename: G M
authors.initials: GM
authors.lastname: Castillo
authors.forename: W
authors.initials: W
authors.lastname: Lukito
authors.forename: E
authors.initials: E
authors.lastname: Peskind
authors.forename: M
authors.initials: M
authors.lastname: Raskind
authors.forename: D A
authors.initials: DA
authors.lastname: Kirschner
authors.forename: A G
authors.initials: AG
authors.lastname: Yee
authors.forename: A D
authors.initials: AD
authors.lastname: Snow
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D001485:Basement Membrane; D052160:Benzothiazoles; D001665:Binding Sites / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry; D007118:Immunoassay; D058228:Islet Amyloid Polypeptide; D007797:Laminin / Q000737:chemistry* / Q000494:pharmacology; D008854:Microscopy, Electron; D016624:Negative Staining; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology*; D013844:Thiazoles
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D058228:Islet Amyloid Polypeptide; D007797:Laminin; D010446:Peptide Fragments; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C084098:laminin A; C009462:thioflavin T