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Amino acid specificity of glycation and protein–AGE crosslinking reactivities determined with a dipeptide SPOT library

Nature Biotechnology volume 17, pages 1006–1010 (1999)Cite this article

Abstract

Advanced glycation end products (AGEs) contribute to changes in protein conformation, loss of function, and irreversible crosslinking. Using a library of dipeptides on cellulose membranes (SPOT library), we have developed an approach to systematically assay the relative reactivities of amino acid side chains and the N-terminal amino group to sugars and protein–AGEs. The sugars react preferentially with cysteine or tryptophan when both the α-amino group and the side chains are free. In peptides with blocked N-terminus and free side chains, cysteine, lysine, and histidine were preferred. Crosslinking of protein–AGEs to dipeptides with free side chains and blocked N termini occurred preferentially to arginine and tryptophan. Dipeptide SPOT libraries are excellent tools for comparing individual reactivities of amino acids for nonenzymatic modifications, and could be extended to other chemically reactive molecules.

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Figure 1: Configuration of dipeptide libraries.
Figure 2: Selective deprotection of the side chains and the N terminus of the dipeptides.
Figure 3: Mapping of the reactivity of fructose with the N-terminal amino function of peptides displayed on a library of 440 immobilized dipeptides.
Figure 4: Mapping of the reactivity of glucose with free side chains of N-terminally acetylated peptides displayed on a library of 440 immobilized dipeptides.
Figure 5: Mapping of the reactivity of glucose with free side chains and the free N-terminal amino function of unprotected peptides displayed on a library of 440 immobilized dipeptides.
Figure 6: Crosslinking of preformed BSA-AGE to peptides displayed on a library of 440 immobilized dipeptides.

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Acknowledgements

We thank Rosemarie Kientsch-Engel, Helmut Lill, Alan Hipkiss, Jürgen Michaelis, Siegfried Hoyer, August Heidland, Geoff Grigg, Ronald Frank, Markus Herderich, Stefanie Diem, and Robin Holliday for inspiring discussions; Bernhard Spengler for performing the matrix-assisted laser desorption/ionization time-of-flight analysis of the spot peptides; and ASTA Medica for continuous moral support. The assistance of Amanda Wong in preparing the manuscript is also acknowledged. This work was supported by the Claussen-Stiftung and by the Alzheimer Forschung International (to G.M).

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Authors and Affiliations

  1. Department of Physiological Chemistry I, University of Würzburg, Würzburg, Germany

    Gerald Münch, Dorothee Schicktanz, Andrea Behme, Dieter Palm & Reinhard Schinzel

  2. Department of Clinical Neurochemistry, Psychiatric Hospital, University of Würzburg, Germany

    Manfred Gerlach & Peter Riederer

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  1. Gerald Münch
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Correspondence to Gerald Münch.

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Münch, G., Schicktanz, D., Behme, A. et al. Amino acid specificity of glycation and protein–AGE crosslinking reactivities determined with a dipeptide SPOT library. Nat Biotechnol 17, 1006–1010 (1999). https://doi.org/10.1038/13704

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