Novel Purification and Detailed Characterization of Perlecan Isolated from the Engelbreth-Holm-Swarm Tumor for Use in an Animal Model of Fibrillar A  Amyloid Persistence in Brain

Abstract

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.

Beta-amyloid deposition and Alzheimer's type changes induced by Borrelia spirochetes

“…A␤PP is a proteoglycan core protein [32,35]. It is well established that increased proteoglycan synthesis plays a significant role in amyloidogenesis, including the formation of A␤ in AD [4]. Proteoglycans are present in early stages of amyloid deposition, but their exact role is yet to be determined.…”

Perlecan Binds to the β‐Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability

“…The final purity and quality of the perlecan preparations were assessed further by Alcian blue staining, Coomassie Blue staining, silver staining, and a series of western blots using antibodies against perlecan, laminin, type IV collagen, and fibronectin (Castillo et al, 1996). The perlecan used in the present study was found to contain no other contamination by other basement membrane components or other proteins!…”

“…Perlecan used in the present study was purified from the EHS tumor as recently described (Castillo et a!., 1996). The final purity and quality of the perlecan preparations were assessed further by Alcian blue staining, Coomassie Blue staining, silver staining, and a series of western blots using antibodies against perlecan, laminin, type IV collagen, and fibronectin (Castillo et al, 1996).…”

“…In the present study, we used purified perlecan isolated from the Engelbreth-Holm-Swarm (EHS) tumor (Castillo et al, 1996) to define A/I's binding interactions with substrate-bound perlecan and to determine if perlecan plays roles in A/I fibril formation and maintenance of fibrillar stability, as previously hypothesized . Full-length A/I peptides [A/I(1-42)andA/I(1-40)] wereused, becausethey represent the predominant isoforms of A/I present in amyloid plaques and cerebrovascular amyloid deposits.…”

“…Full-length A/I peptides [A/I(1-42)andA/I(1-40)] wereused, becausethey represent the predominant isoforms of A/I present in amyloid plaques and cerebrovascular amyloid deposits. The perlecan used for these in vitro assays was essentially clean of other potentially contaminating basement membrane components (e.g., laminin, fibronectin, and type IV collagen) and other PGs/proteins known to be produced by the EHS tumor, as recently described (Castillo et al, 1996). By using a newly developed solid-phase binding immunoassay, substrate-bound EHS perlecan interacted with full-length A/I peptides with both high-affinity (low-capacity) and lower-affinity (high-capacity) binding sites.…”