Molecular defects that cause loss of polysialic acid in the complementation group 2A10.
Polysialic acid (PSA) is a dynamically regulated posttranslational modification of the neural cell adhesion molecule (NCAM), which modulates NCAM binding functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unknown. In Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expression. In the complementation group 2A10, the ST8SiaIV gene is disrupted. Investigating the molecular defects in this complementation group, seven clones with missense mutations in ST8SiaIV were found. Mutations cause replacement 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 hamster 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 -M333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be partially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysialylation was achieved in the presence of NCAM. The data presented in this study suggest a functional link between auto- and NCAM polysialylation.
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
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M
Windfuhr
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A
Manegold
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M
Muhlenhoff
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M
Eckhardt
R
R
Gerardy-Schahn
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002645:Chickens; D017124:Conserved Sequence; D006224:Cricetinae; D003545:Cysteine; D005816:Genetic Complementation Test; D014644:Genetic Variation; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000096:biosynthesis* / Q000235:genetics; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D019006:Neural Cell Adhesion Molecules; D011994:Recombinant Proteins; D012794:Sialic Acids; C021319:polysialic acid; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases; C515117:ST8SIA4 protein, human; D003545:Cysteine