Association and release of the amyloid protein precursor of Alzheimer's disease from chick brain extracellular matrix

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDSlabile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited crosslinking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of ␣-secretory APP increased, soluble ␤-cleaved APP levels decreased. Because A␤40 and A␤42 decreased to similar levels as soluble ␤-cleaved APP, we conclude either that ␤-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.The amyloid precursor protein (APP) 3 of Alzheimer disease is one of the best studied type I cell surface glycoproteins with a proposed regulatory role in cell adhesion in the peripheral and central nervous system (1). APP is physiologically processed by either ␣-or ␤-secretases, which release the soluble ectodomain (sAPP␣ or sAPP␤) from the cell surface (2-5). Alternatively, the membrane-bound fragment APP-C-terminal fragment, which is derived from ␤-secretase cleavage, is further processed by the ␥-secretase into the cytoplasmic domain of APP and amyloid ␤-peptides (A␤) of varying lengths (6 -9). Recently, elucidation of the ␥-secretase cleavage mechanism has revealed that a conserved GXXXG motif in the transmembrane sequence of APP mediates homophilic helix-helix interactions and has an important role in the processing of A␤40/42 into shorter A␤ species, i.e. A␤38, A␤37, A␤35, and A␤34 (7).Unraveling structural features of APP by NMR spectroscopy and x-ray crystallography has led to the identification of specific regions that function as subdomains (see Fig. 1A). The extracellular N-terminal domain of APP has been dissected into the E1 region consisting of the N-terminal growth factor-like domain (GFLD) followed by the copper-binding domain (CuBD) (10). An acidic region (residues 190 -264) precedes the carbohydrate domain, which contains...

Section: Discussionmentioning

confidence: 99%

Homophilic Interactions of the Amyloid Precursor Protein (APP) Ectodomain Are Regulated by the Loop Region and Affect β-Secretase Cleavage of APP

Kaden

Munter

Joshi

et al. 2008

“…Because most earlyonset familial AD patients have gene mutations that influence the processing or aggregation of A␤, and the neurites associated with A␤ plaques are often damaged (2), the process of A␤ deposition in the brain seems to be strongly associated with AD pathogenesis. Even though APP has been proposed to have a receptor-like function and binds to multiple extracellular matrix proteins such as heparin and collagen (8,9), understanding the biological functions of APP remains an important scientific and intellectual challenge (10). Two paralogs of APP are known in mammals and are designated APPlike proteins 1 and 2.…”

mentioning

confidence: 99%

Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form

Kitazume

Tachida

Kato

et al. 2010

101

Deposition of amyloid ␤ (A␤) in the brain is closely associated with Alzheimer disease (AD). A␤ is generated from amyloid precursor protein (APP) by the actions of ␤-and ␥-secretases. In addition to A␤ deposition in the brain parenchyma, deposition of A␤ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how A␤ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced A␤40 and A␤42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, ⌷-glycosylated APP770 was preferentially processed by both ␣-and ␤-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770␤ in human cerebrospinal fluid, this unique soluble APP770␤ has the potential to serve as a marker for cortical dementias such as AD and vascular dementia. Alzheimer disease (AD)3 is characterized by intracellular accumulation of neurofibrillary tangles and extracellular deposits of amyloid ␤ (A␤) peptides in the brain (1, 2). A␤ is generated from amyloid precursor protein (APP) by the sequential actions of ␤-secretase, BACE1 (␤-site APP-cleaving enzyme 1) (3-6), and ␥-secretase (7). Because most earlyonset familial AD patients have gene mutations that influence the processing or aggregation of A␤, and the neurites associated with A␤ plaques are often damaged (2), the process of A␤ deposition in the brain seems to be strongly associated with AD pathogenesis. Even though APP has been proposed to have a receptor-like function and binds to multiple extracellular matrix proteins such as heparin and collagen (8, 9), understanding the biological functions of APP remains an important scientific and intellectual challenge (10). Two paralogs of APP are known in mammals and are designated APPlike proteins 1 and 2. Interestingly, triple knock-out mice lacking all three APP family members die shortly after birth (11), suggesting redundant functions of the APP family proteins.APP has three alternatively spliced isoforms: APP695, APP751, and APP770 (12, 13). Compared with APP695, APP751 contains an additional Kunitz-type protease inhibitor (KPI) domain, whereas APP770 contains a KPI domain plus an OX2 domain. APP695 is most abundantly expressed in neurons, whereas APP751 and APP770 show more ubiquitous expression patterns (14). The secreted form of APP containing a KPI domain, also known as the protease nexin 2, potentially inhibits certain serine proteases, most notably several prothrombotic enzymes (15); very limited information is available concerning the functions of the OX2 domain. In the present study, we show that brain microvascular endothelial cells (BME...

“…As presynaptic turnover would be suggestive of proteolysis, we also examined presynaptically targeted APSF proteins for evidence of proteolytic processing. Finally, the extracellular domains of APP and APLP2 may carry glycosylations (8) that could potentially interact with extracellular materials (20,21) or cell membranes (22) at the synapse. Thus, we examined neuronally expressed, presynaptically targeted APSF proteins for various types of glycosylation.…”

mentioning

confidence: 99%

Post-translational Processing and Turnover Kinetics of Presynaptically Targeted Amyloid Precursor Superfamily Proteins in the Central Nervous System

Lyckman

Confaloni

Wang

et al. 1998

The amyloid precursor superfamily is composed of three highly conserved transmembrane glycoproteins, the amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1, APLP2), whose functions are unknown. Proteolytic cleavage of APP yields the ␤A4 peptide, the major component of cerebral amyloid in Alzheimer's disease. Here we show that five post-translationally modified, full-length species of APP and APLP2 (but not APLP1) arrive at the mature presynaptic terminal in the fastest wave of axonal transport and are subsequently rapidly cleared (mean halflife of 3.5 h). Rapid turnover of presynaptic APP and APLP2 occurs independently of visual activity. Turnover of the most rapidly arriving APP species was accompanied by a delayed accumulation of a 120-kDa, APP fragment lacking the C terminus, consistent with presynaptic APP turnover via constitutive proteolysis. Turnover of APLP2 was not accompanied by detectable APLP2 fragment peptides, suggesting either that APLP2 either is more rapidly degraded than is APP or is retrogradely transported shortly after reaching the terminus. A single 150-kDa APLP2 species containing the Kunitz protease inhibitor domain is the major amyloid precursor superfamily protein transported to the presynapse. Presynaptic APP and APLP2 are sialylated and N-and O-glycosylated, and some also carry chondroitin sulfate glycosaminoglycan and/or dermatan sulfate glycosaminoglycan. The rapid kinetics for turnover of APP and APLP2 predict a sensitive balance of synthesis, transport, and elimination rates that may be critical to normal neuronal functions and metabolic fates of these proteins.Alzheimer's disease is an age-related human dementia whose principle neuropathological signs are forebrain cholinergic neuronal death, neurofibrilliary tangles, and amyloidosis of cerebral vessels and senile plaques (1). Interest in the regulation of APP 1 expression arose from it being recognized as the parent protein of ␤A4, the major peptide constituent of cerebrovascular and senile plaque amyloid (2). APP is one of the three proteins encoded by genes in the mammalian amyloid precursor superfamily (APSF; see Fig. 1A), which also has two genes encoding APLP1 and APLP2 (3). Although the APP gene is the only APSF gene that encodes ␤A4, the conservation of gene structure (4) and domain homology (5) among the APSF proteins suggests that they may share significant overlap in function and expression, and thus, that amyloidogenic processing of APP may be influenced in parallel with processing of APLP1 and/or APLP2. For instance, APSF proteins from various phyla share highly conserved extracellular zinc and heparin binding domains (3), single membrane-spanning domains (4, 6), and intracellular phosphorylation sites (7). Additionally, APP and APLP2 genes both contain N-linked glycosylation sites (8), splice-specific chondroitin sulfate glycosaminoglycan (CSGAG) attachment sites (9 -11), and, alternatively, spliced exons that encode the Kunitz protease inhibitor (KPI) domain (4, 12). Various combinat...