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Originally published In Press as doi:10.1074/jbc.M100576200 on February 7, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15369-15377, May 4, 2001
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Unique Disulfide Bond Structures Found in ST8Sia IV Polysialyltransferase Are Required for Its Activity*

Kiyohiko AngataDagger , Ten-Yang Yen§, Assou El-Battari, Bruce A. Macher§, and Minoru FukudaDagger ||

From the Dagger  Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037, the § Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132, and  INSERM Unité 260 Faculté de Médecine, 27 Blvd. J. Moulin, 13385 Marseille Cedex 5, France

NCAM polysialylation plays a critical role in neuronal development and regeneration. Polysialylation of the neural cell adhesion molecule (NCAM) is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which contain sialylmotifs L and S conserved in all members of the sialyltransferases. The members of the ST8Sia gene family, including ST8Sia II and ST8Sia IV are unique in having three cysteines in sialylmotif L, one cysteine in sialylmotif S, and one cysteine at the COOH terminus. However, structural information, including how disulfide bonds are formed, has not been determined for any of the sialyltransferases. To obtain insight into the structure/function of ST8Sia IV, we expressed human ST8Sia IV in insect cells, Trichoplusia ni, and found that the enzyme produced in the insect cells catalyzes NCAM polysialylation, although it cannot polysialylate itself ("autopolysialylation"). We also found that ST8Sia IV does not form a dimer through disulfide bonds. By using the same enzyme preparation and performing mass spectrometric analysis, we found that the first cysteine in sialylmotif L and the cysteine in sialylmotif S form a disulfide bridge, whereas the second cysteine in sialylmotif L and the cysteine at the COOH terminus form a second disulfide bridge. Site-directed mutagenesis demonstrated that mutation at cysteine residues involved in the disulfide bridges completely inactivated the enzyme. Moreover, changes in the position of the COOH-terminal cysteine abolished its activity. By contrast, the addition of green fluorescence protein at the COOH terminus of ST8Sia IV did not render the enzyme inactive. These results combined indicate that the sterical structure formed by intramolecular disulfide bonds, which bring the sialylmotifs and the COOH terminus within close proximity, is critical for the catalytic activity of ST8Sia IV.

* This work was supported by National Institutes of Health Research Grants R01 CA33895 (to M. F.) and P20 RR11805 (RIMI) and by National Science Foundation Grant MCB-9816780 (to B. A. M.).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 should be addressed: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax: 858-646-3193; E-mail: minoru@burnham.org.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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