Unique Disulfide Bond Structures Found in ST8Sia IV Polysialyltransferase Are Required for Its Activity

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 sialylt...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2001-05, Vol.276 (18), p.15369-15377
Main Authors: Angata, Kiyohiko, Yen, Ten-Yang, El-Battari, Assou, Macher, Bruce A., Fukuda, Minoru
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Chromatography, High Pressure Liquid
Cysteine - metabolism
Disulfides - chemistry
Disulfides - metabolism
DNA Primers
DNA, Complementary
Insecta
Molecular Sequence Data
Mutagenesis, Site-Directed
neural cell adhesion molecule
polysialyltransferase
Sequence Homology, Amino Acid
Sialyltransferases - chemistry
Sialyltransferases - genetics
Sialyltransferases - metabolism
Spectrometry, Mass, Electrospray Ionization
ST8Sia gene
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M100576200