Alcadein Cleavages by Amyloid β-Precursor Protein (APP) α- and γ-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related γ-Secretase Dysfunction

Saori Hata,Sayaka Fujishige,Yoichi Araki,Naoko Kato,Masahiko Araseki,Masaki Nishimura,Dieter Hartmann,Paul Saftig,Falk Fahrenholz,Miyako Taniguchi,Katsuya Urakami,Hiroyasu Akatsu,Ralph N Martins,Kazuo Yamamoto,Masahiro Maeda,Tohru Yamamoto,Tadashi Nakaya,Sam Gandy,Toshiharu Suzuki

doi:10.1074/jbc.m109.057497

Abstract

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.

Highlights

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc␣, Alc␤, and Alc␥
5 The abbreviations used are: Alc, alcadein; AD, Alzheimer disease; A␤, amyloid ␤-protein; ADAM, a disintegrin and metalloproteinase; amyloid precursor protein (APP), amyloid ␤-precursor protein; sAPP, soluble large extracellular N-terminal domain of APP truncated at the primary cleavage site; sAlc, soluble large extracellular N-terminal domain of Alc truncated at the primary cleavage site; p3-Alc, small peptide generated by serial primary and secondary cleavages of Alc; CSF, cerebrospinal fluid; C-terminal fragments (CTFs), C-terminal fragment of APP or Alc truncated at the primary cleavage site; BACE, ␤-site APP-cleaving enzyme; MEF, mouse embryonic fibroblast; PS, presenilin; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MS/MS, tandem MS
Alc proteins are complexed to X11L molecules, which, in turn, are complexed with the amyloid ␤-precursor protein (APP), a type I transmembrane protein that is believed to play a seminal role in the pathogenesis of familial and sporadic Alzheimer disease

Summary

The abbreviations used are

Alcadein; AD, Alzheimer disease; A␤, amyloid ␤-protein; ADAM, a disintegrin and metalloproteinase; APP, amyloid ␤-precursor protein; sAPP, soluble large extracellular N-terminal domain of APP truncated at the primary cleavage site; sAlc, soluble large extracellular N-terminal domain of Alc truncated at the primary cleavage site; p3-Alc, small peptide generated by serial primary and secondary cleavages of Alc; CSF, cerebrospinal fluid; CTF, C-terminal fragment of APP or Alc truncated at the primary cleavage site; BACE, ␤-site APP-cleaving enzyme; MEF, mouse embryonic fibroblast; PS, presenilin; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MS/MS, tandem MS. Alc proteins are complexed to X11L molecules, which, in turn, are complexed with the amyloid ␤-precursor protein (APP), a type I transmembrane protein that is believed to play a seminal role in the pathogenesis of familial and sporadic Alzheimer disease We report that all three members of the Alc family (Alc␣, Alc␤, and Alc␥) are cleaved by ADAM 10 and ADAM 17, which have been identified as the ␣-secretases for APP [15,16,17]. The detailed biochemical analysis for the cleavages of Alc proteins is significant for understanding the features of p3-Alc peptides in human subjects. We found that various FAD-linked PS1 mutations appeared, at different magnitudes, with the alterations of C termini of p3-Alcs, suggesting that one type of ␥-secretase dysfunction appears in various phenotypes upon cleavage of Alc proteins and APP. We characterized all p3-Alc species generated from Alc␣, Alc␤, and Alc␥ in detail

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION