Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease

Abstract

The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite‐promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin‐binding domain of APP. A fraction 17‐fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity‐purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high‐ and low‐affinity sites with equilibrium dissociation constants of 1.4 × 10−11 and 6.5 × 10−10M, respectively. APP, in conjunction with the affinity‐purified proteoglycan fraction, promoted neurite outgrowth. The affinity‐purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity‐purified fraction with heparitinase I revealed a core protein of 63–69‐kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP‐binding proteoglycans may be an important step in the regulation of the neurite outgrowth‐promoting activity of APP.

Chondroitin sulfates and their binding molecules in the central nervous system

“…The direct interaction of CSPGs and APP has been demonstrated using solid phase binding assay. ChABC digestion of CS reduces the binding by 79%, this confirms the binding is through CS GAG chains [182]. …”

“…Recent study has reported that an overexpression of heparanase decreases the amyloid burden in vivo [179], and that PNN neurons resist neurotoxicity from Aβ and oxidative stress suggesting the neuroprotective role of PNNs [180, 181]. Otherwise, an interaction of APP and PG enhances the neurite outgrowth [182]. The direct interaction of CSPGs and APP has been demonstrated using solid phase binding assay.…”

Determination of dissociation constants for a heparin‐binding domain of amyloid precursor protein and heparins or heparan sulfate by affinity capillary electrophoresis

“…A study of the interaction of heparin binding domains of APP with proteoglycan reported nanomolar dissociation constants between HBD1 of APP from which the (96-110) APP peptide is derived and both heparan sulfate-and chondroitin sulfate-proteoglycans captured by synthetic HBD1 [4]. The proteoglycans were captured from P3 mouse brain cells, indicating there is specificity to this binding interaction.…”

“…It was found that (96-110) APP peptides inhibit the binding of APP and heparin with IC 50 values on the order of 10 -7 M [2]. Additionally, dissociation constants for the binding of proteoglycans purified from P3 mouse brain cells and APP have been investigated using a solid-phase binding assay [4]. However, these studies lack the ability to directly determine dissociation constants between various GAGs and the APP peptide in solution.…”

The Amyloid Precursor Protein (APP) of Alzheimer Disease and Its Paralog, APLP2, Modulate the Cu/Zn-Nitric Oxide-catalyzed Degradation of Glypican-1 Heparan Sulfate in Vivo

“…Results of in vitro studies strongly suggest that HSPGs reg-ulate the effects of APP on neurite outgrowth (17,20,21). When PG from neonatal mouse brain cells was subjected to affinity chromatography on an APP695-substituted matrix, the HSPG glypican-1 (Gpc-1) was the most strongly bound with an affinity in the nanomolar range (22).…”