Abstract
To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid beta (Abeta), we labeled human Abeta40 (monomeric, with predominant unordered structure) and Abeta42 (mixture of monomers and oligomers in approximately 50:50 ratio, rich in beta-sheet conformation) with either Na(125)I or (125)I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Abeta40 and Abeta42 showed a short half-life of 2.5-3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Abeta species became obviously relevant at doses above 10 microg. The use of biotin-labeled Abeta allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Abeta demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Abeta, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Abeta but the little fluctuation observed with age and disease stages.
Highlights
Alzheimer’s disease (AD)1 is the most frequent type of amyloidosis in humans and the commonest form of clinical dementia
We investigated the excretion/catabolism of human A40 and A42 species in mice using both iodinated peptides and peptides labeled with an intracellularly trapped ligand procedure based on the use of 125I-tyramine-cellobiose (TC), a sugar adduct that is not degraded by mammalian cells and accumulates in the organs involved in the uptake [18]
A42 had a predominant -structure, as indicated by the classical CD profile showing a minimum at 218 nm (Fig. 1B), and interacted with apolipoprotein J (apoJ) with high affinity (Kd ϭ 2.44 nM), as reported previously [23]
Summary
A-(1– 40) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and A-(1– 42) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), homologous to residues 672–711 and 672–713, respectively, of human A precursor protein APP770 as well as a derivative of A-(1– 40) bearing a single biotin molecule at the N-terminal aspartate residue, were all synthesized at the W. Peptides were analyzed by amino acid sequence and MALDI-TOF mass spectrometry and structurally characterized via size exclusion chromatography and CD spectroscopy The integrity of their biological activity was assessed through their binding interaction with apolipoprotein J (apoJ). Two proteins of different molecular masses and well known half-lives in rodents, human IgG (EMD Biosciences, San Diego, CA) and SAP (Sigma), were radioiodinated as above and used as controls for the pharmacokinetic parameters described below Their specific activities were in the range of 50 –70 Ci/g with Ͼ95% trichloroacetic acid precipitability in both cases. Identification of Targeted Amino Acid Residues in the Iodination Procedure—To identify the iodinated amino acid residues, A-(1– 40) and A-(1– 42) were labeled with Na[127I], the nonoxidized species separated by RP-HPLC identically as above, the resulting peaks subjected to separate proteolytic degradations with three different enzymes (trypsin, endoproteinase Asp-N, and endoproteinase Glu-C), and the subsequent proteolytic fragments analyzed by MALDI-TOF mass spectrometry to assess iodine incorporation. Specific activities of the labeled TC peptides after purification (typically 20 Ci/g) were calculated from the integration of the peak areas obtained at 220 nm, and the radioactivity counts were obtained from aliquots of the respective pools
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