Preservation of enzyme activity and antigenicity after mutagenesis of the membrane anchoring domain of GAD65.
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 difference was detected using antibodies specific for the PLP-binding site in the middle part of GAD65 or the C-terminal region. Taken together, these data suggest that no major conformational changes have been introduced by mutating the membrane-anchoring domain of GAD65.
Plesner
A
A
Department of Medicine, University of Washington, Seattle 98195-7710, USA. plesner@interchange.ubc.ca
Hampe
C S
CS
Daniels
T L
TL
Hammerle
L P
LP
Lernmark
A
A
D015551:Autoimmunity; D002462:Cell Membrane / Q000201:enzymology; D003922:Diabetes Mellitus, Type 1 / Q000209:etiology / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D016296:Mutagenesis; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2