Effects of Glycosylation on Lecithin -Cholesterol Acyltransferase
LCAT glycosylation mutants N84Q and N384Q were examined to determine the effects of deleting individual glycan chains. Purified N84Q LCAT did not possess measurable enzymatic activity or interfacial binding affinity for rHDL. Purified N384Q was more enzymatically active than WT LCAT, but lost all activity within months, whereas WT LCAT activity was constant for years under the same conditions. In thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than WT LCAT. Large changes were detected in the alpha helical content of N384Q LCAT and in the beta-sheet content of N84Q LCAT by CD, compared to WT LCAT. Fluorescence measurements of the binding of the probe ANS suggested that in both mutants, the active site cavities became inaccessible with time. In conclusion, both mutants lost catalytic activity---N84Q shortly after purification and N384Q more gradually---and were destabilized, probably because the removal of the glycan chains altered key structural elements. These results support the hypothesis that glycosylation is responsible for the stabilization of only a localized region of protein structure.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign