M. Windfuhr et al., Molecular defects that cause loss of polysialic acid in the complementation group 2A10, J BIOL CHEM, 275(42), 2000, pp. 32861-32870
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.