Abstract:Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind ␤-amyloid protein (A␤) 1-40 and 1-42, but are also potent enhancers of A␤ fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of A␤ fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on A␤ 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of A␤ fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of A␤ 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of A␤ amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) A␤ amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of A␤ amyloid deposition in Alzheimer's disease brain may also participate in the enhancement of A␤ amyloidosis. Key Words: ␤-Amyloid protein-Alzheimer's disease-Glycosaminoglycans-Sulfate-Fibrillogenesis.

β‐Amyloid protein (Aβ) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimer's disease (AD). Inhibitors of Aβ 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 Aβ 1–40 with a single dissociation constant, Kd = 2.7 × 10–9 M, and serve as a potent inhibitor of Aβ fibril formation. 25 μM of Aβ 1–40 was incubated at 37°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 Aβ fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Aβ‐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 Aβ fibrillogenesis occurred in a dose‐dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Aβ 1–40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Aβ fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Aβ‐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 Aβ‐specific. To identify a potential Aβ‐binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Aβ‐binding elastase digestion product of ∼120–130 kDa was found. In addition, a ∼55 kDa fragment derived from V8 and elastase‐digested laminin interacted with biotinylated Aβ 1–40. Amino acid sequencing of the ∼55 kDa fragment identified a conformationally dependent Aβ‐binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Aβ with relatively high affinity but is a potent inhibitor of Aβ‐amyloid fibril formation. In addition, further identification of an Aβ‐binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Aβ fibrillogenesis. J. Neurosci. Res. 62:451–462, 2000. © 2000 Wiley‐Liss, Inc.