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Originally published In Press as doi:10.1074/jbc.M109973200 on December 20, 2001

J. Biol. Chem., Vol. 277, Issue 8, 6688-6695, February 22, 2002
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Versatile Biosynthetic Engineering of Sialic Acid in Living Cells Using Synthetic Sialic Acid Analogues*

Cornelia OetkeDagger , Reinhard Brossmer§, Lars R. Mantey||, Stephan Hinderlich||, Rainer Isecke§**, Werner Reutter||, Oliver T. KepplerDagger Dagger Dagger , and Michael PawlitaDagger §§

From the Dagger  Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, § Biochemie-Zentrum, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany and || Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.

* This work was supported by the Sonnenfeld-Stiftung, Berlin, Germany, the Fonds der Chemischen Industrie, Frankfurt/Main, Germany, the Wilhelm Sander-Stiftung, München, Germany, and the Human Frontier Science Program, Strasbourg, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Biochemie-Zentrum, Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Tel.: 49-6221-544170; Fax: 49-6221-545586; E-mail: reinhard.brossmer@urz.uni-heidelberg.de.

** Present address: CHESS GmbH, Dr.-Albert-Reimann-Str. 2, D-68526 Ladenburg, Germany.

Dagger Dagger Present address: Gladstone Institute of Virology and Immunology, P. O. Box 419100, San Francisco, CA 94141-9100.

§§ To whom correspondence may be addressed: Deutsches Krebsforschungszentrum, ATV F0200, Im Neuenheimer Feld 280, D-69120 Heidelberg. Tel.: 49-6221-424645; Fax: 49-6221-424932; E-mail: m.pawlita@dkfz.de.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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