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Potential βPP-processing proteinase activities from alzheimer's and control brain tissues

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Abstract

Fluorogenic peptide substrates designed to encompass the reportedα-secretory and amyloidogenic cleavage sites of the amyloid-β precursor protein (βPP) were used to analyze proteinase activities in brain extracts from control patients and those with Alzheimer's disease (AD). Activity against the secretory substrate atpH 7.5 in control and AD brains produced a major endopeptidase cleavage at the Lys687-Leu688 bond (βPP770 numbering), consistent with theβPP secretase cleavage. Activity in control brains against the amyloidogenic substrate atpH 7.5 produced one cleavage at the Ala673-Glu674 bond, two residues C-terminal to the amyloidogenic Met-Asp site. However, in three of four AD brains, the major cleavage was at the Asp-Ala bond, one residue from the amyloidogenic site. Both endopeptidase and carboxypeptidase activities in AD brains were lower than in control brains. Proteinase activities against the secretory substrate had a major optimum atpH 3.0–4.0 and another atpH 6.0–7.5. Proteinase activities against the amyloidogenic substrate had a major optimum at or belowpH 3.0 and another atpH 6.0. Using both substrates, activities at lowpH were higher in AD brains than in controls, while atpH above 6.5, activities in control brains were higher than in AD. These results indicate that the levels of proteolytic enzymes in AD brains are altered relative to controls.

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Abbreviations

Aβ:

Amyloid-β

ACN:

acetonitrile

AD:

Alzheimer's disease

βPP:

amyloid-β precursor protein

DABCYL:

4-(4-dimethylaminophenylazo)-benzoic acid

EDANS:

5-{(2-aminoethyl)amino}napthalene-1-sulfonic acid

MES:

morpholinoethane sulfonic acid

MOPS:

morpholino-propane sulfonic acid

RP-HPLC:

reverse-phase high-performance liquid chromatography

SDS-PAGE:

sodium do-decyl sulfate-polyacrylamide gel electrophoresis

TFA:

tri-fluoroacetic acid

Tris:

tris(hydroxyethyl)aminomethane

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Authors and Affiliations

  1. Drug Design and Delivery, Abbott Laboratories, 60064, Abbott Park, Illinois

    Uri S. Ladror, Gary T. Wang, Thomas F. Holzman & Grant A. Krafft

  2. Department of Neurobiology, Northwestern University, 60208, Evanston, Illinois

    William L. Klein

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  1. Uri S. Ladror
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  2. Gary T. Wang
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  3. William L. Klein
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  5. Grant A. Krafft
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Ladror, U.S., Wang, G.T., Klein, W.L. et al. Potential βPP-processing proteinase activities from alzheimer's and control brain tissues. J Protein Chem 13, 357–366 (1994). https://doi.org/10.1007/BF01901691

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