Deletion of specific glycan chains affects differentially the stability, local structures, and activity of lecithin-cholesterol acyltransferase

The enzymatic and interfacial binding activity of lecithin-cholesterol acyl transferase (LCAT) is affected differentially by the location and extent of its glycosylation. Two LCAT glycosylation-deficient mutants, N84Q and N384 Q, were constructed, permanently expressed in Chinese hamster ovary cells, and purified to determine the effects of deleting individual glycan chains on its stability, structure, and function. These purified mutants were stud ied by spectroscopic structural methods and enzymatic and binding assays to develop a molecular rationale for the relationship between LCAT glycosylat ion and activity. The N84Q LCAT mutant did not possess measurable enzymatic activity or interfacial binding affinity for reconstituted high-density li poproteins. In addition, in thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than wild-type LCAT. The N3 84Q variant was initially more enzymatically active than wild-type LCAT, bu t gradually lost activity within months; however, it retained full interfac ial binding activity. Significant changes were detected over time by circul ar dichroism in the alpha -helical content of N384Q LCAT and in the beta -s heet content of N84Q LCAT, compared with wild-type LCAT. Fluorescence measu rements with the probe 1-anilinonapthalene-8-sulfonate suggested an alterat ion of the active site cavity in both mutants. In conclusion, both mutants lost catalytic activity, N84Q shortly after purification and N384Q more gra dually, and were destabilized, probably because the deletion of the glycan chains altered local structural elements near the active site cavity and/or the interfacial binding regions.