Molecular defects that cause loss of polysialic acid in the complementation group 2A10

Polysialic acid (PSA) is a dynamically regulated posttranslational modifica tion of the neural cell adhesion molecule (NCAM), which modulates NCAM bind ing functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unkno wn. In. Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expres sion. In the complementation group 2A10, the ST8SiaIV gene is disrupted. In vestigating the molecular defects in this complementation group, seven clon es with missense mutations in ST8SiaIV were found. Mutations cause replacem ent of amino acids that are highly conserved in alpha2,8-sialyltransferases . To verify the physiological relevance of identified mutations, identical amino acid substitutions were introduced into epitope-tagged variants of ha mster ST8SiaIV and murine ST8SiaII and recombinant proteins were tested in vivo and in vitro. None of these constructs reconstituted PSA synthesis in 2A10 cells, although the proteins were expressed and with the exception of the cysteine variants ST8SiaIV-C356F and ST8SiaII-C371F correctly targeted to the Golgi apparatus. Interestingly, two mutations (ST8SiaIV-R277G and -M 333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be part ially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysial ylation was achieved in the presence of NCAM. The data presented in this st udy suggest a functional link between auto- and NCAM polysialylation.