Preservation of enzyme activity and antigenicity after mutagenesis of the membrane anchoring domain of GAD65

Autoimmunity
A PlesnerAke Lernmark

Abstract

The smaller isoform of glutamic acid decarboxylase, GAD65, is an important autoantigen implicated in the pathogenesis of type 1 diabetes whereas the larger isoform, GAD67 appears to play no major role. The primary difference between the two isoforms resides in the N-terminal part of the molecule including the GAD65 membrane-anchoring domain. The aim of this study was to generate mutants of the membrane targeting domain spanning amino acids 24 to 31 of GAD65 to determine effects on enzyme activity and antibody recognition. Three GAD65 mutants were generated by substituting two, nine or eleven nucleotides coding for the membrane targeting with the corresponding bases of GAD67. SDS-PAGE and Western blotting wildtype (wt) and mutated GAD65 ascertained that they were of similar size and recognized GAD65-specific antibodies. No difference in enzymatic activity was found between the mutants and wt GAD65. GAD65 antibody positive sera from type 1 diabetes patients immunoprecipitated mutated GAD65 whether two, nine or eleven nucleotides were replaced. Mono-or polyclonal antibodies to the N-terminal region demonstrated that the mutated GAD65 with two or nine nucleotides replaced was immunoprecipitated markedly better than wt whereas no di...Continue Reading

References

Nov 1, 1978·Journal of Neurochemistry·M Covarrubias, R Tapia
Jan 1, 1992·The Journal of Clinical Investigation·D L KaufmanA J Tobin
Mar 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·D F BuA J Tobin
Oct 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·A E KarlsenD Foster
Jul 1, 1991·Neuron·M G ErlanderA J Tobin
Jan 1, 1985·Methods in Enzymology·J Y WuG X Song
May 1, 1995·The Journal of Experimental Medicine·P Panina-BordignonF Sinigaglia
Jan 1, 1995·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·L LiA Lernmark
Aug 1, 1994·Endocrine Reviews·J F Bach
Apr 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·M SolimenaA Hayday
Dec 22, 1999·Hormone and Metabolic Research = Hormon- Und Stoffwechselforschung = Hormones Et Métabolisme·C S HampeA Lernmark
Jan 4, 2001·Journal of Neuroimmunology·C S HampeA Lernmark

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