Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.     

A heparin-binding protein from neuroblastoma cells: immunological comparison to beta-amyloid precursor protein.

1. beta-Amyloid precursor protein cross-reactive polypeptides were detected in the membrane extracts of a mouse neuroblastoma cell line, NB41A3. Four immunoreactive polypeptide bands were observed on western blots of a cell membrane extract. Their molecular weights as estimated by polyacrylamide gel electrophoresis ranged from 89.1 to 41 kDa. 2. After heparin affinity chromatography, two of these polypeptides strongly cross-reacted with an antibody that recognizes Alzheimer beta-amyloid precursor protein. 3. From the heparin binding fraction, these protein were further separated by reverse-phase high-performance liquid chromatography. A cross-reactive protein was isolated.     

A developmental biological study of aldolase gene expression in Xenopus laevis

We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism. We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that i     

Detection of soluble forms of the beta-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.     

Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation

beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.     

Calnuc binds to Alzheimer's beta-amyloid precursor protein and affects its biogenesis

Calnuc, a Golgi calcium binding protein, plays a key role in the constitution of calcium storage. Abnormal calcium homeostasis has been linked to Alzheimer's disease (AD). Excessive production and/or accumulation of beta-amyloid (Abeta) peptides that are proteolytically derived from the beta-amyloid precursor protein (APP) have been linked to the pathogenesis of AD. APP has also been indicated to play multiple physiological functions. In this study, we demonstrate that calnuc interacts with APP through direct binding to the carboxyl-terminal region of APP, possibly in a calcium-sensitive manner. Immunofluorescence study revealed that the two proteins co-localize in the Golgi in both cultured cells and mouse brains. Over-expression of calnuc in neuroblastoma cells significantly reduces the level of endogenous APP. Conversely, down-regulation of calnuc by siRNA increases cellular levels of APP. Additionally, we show that over-expression of calnuc down-regulates the APP mRNA level and inhibits APP biosynthesis, which in turn results in a parallel reduction of APP proteolytic metabolites, sAPP, CTFs and Abeta. Furthermore, we found that the level of calnuc was significantly decreased in the brain of AD patients as compared with that of age-matched non-AD controls. Our results suggest a novel function of calnuc in modulating the levels of APP and its proteolytic metabolites, which may further affect the patho/physiological functions of APP including AD pathogenesis.