Autocatalytic polysialylation of polysialyltransferase-1.
Polysialic acid (PSA) is a specific and highly regulated post-translational modification of the neural cell adhesion molecule NCAM. Synthesis of PSA depends on the activity of a single enzyme, the polysialyltransferase-1 (PST-1), recently cloned from three mammalian species. The present study was carried out to investigate the catalytic mechanism of PST-1. Using a newly developed in vitro assay system, we demonstrate autopolysialylation for PST-1. The synthesis of PSA chains, which involved N-glycosylation sites, occurred immediately after contact with the activated sugar donor CMP-Neu5Ac. In contrast to the polysialylation of NCAM, where terminal sialylation in either the alpha2,3 or alpha2,6 position is required, the autopolysialylation could be started in the asialo-PST-1 isolated from CHO cells of the Lec2 complementation group. Pre-formed PSA chains were not transferred to NCAM. Nevertheless, the autocatalytic step is likely to be a prerequisite for enzymatic activity, since agalacto-PST-1 isolated from Lec8 cells was functionally inactive. Our data describe a novel route of autocatalytic maturation of a glycosyltransferase and thereby provide a new basis for studies aimed at elucidating and influencing the catalytic functions of PST-1.
Mühlenhoff
M
M
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Eckhardt
M
M
Bethe
A
A
Frosch
M
M
Gerardy-Schahn
R
R
D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D006031:Glycosylation; D007700:Kinetics; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism
D019006:Neural Cell Adhesion Molecules; D011994:Recombinant Proteins; D012794:Sialic Acids; D013205:Staphylococcal Protein A; C021319:polysialic acid; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases