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 acyltransferase (LCAT) is affected differentially by the location and extent of its glycosylation. Two LCAT glycosylation-deficient mutants, N84Q and N384Q, 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 studied by spectroscopic structural methods and enzymatic and binding assays to develop a molecular rationale for the relationship between LCAT glycosylation and activity. The N84Q LCAT mutant did not possess measurable enzymatic activity or interfacial binding affinity for reconstituted high-density lipoproteins. In addition, in thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than wild-type LCAT. The N384Q variant was initially more enzymatically active than wild-type LCAT, but gradually lost activity within months; however, it retained full interfacial binding activity. Significant changes were detected over time by circular dichroism in the alpha-helical content of N384Q LCAT and in the beta-sheet content of N84Q LCAT, compared with wild-type LCAT. Fluorescence measurements with the probe 1-anilinonapthalene-8-sulfonate suggested an alteration of the active site cavity in both mutants. In conclusion, both mutants lost catalytic activity, N84Q shortly after purification and N384Q more gradually, 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.
Kosman
J
J
Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, Urbana, Illinois 61801, USA.
Jonas
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004795:Enzyme Stability; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D011134:Polysaccharides; D007862:Phosphatidylcholine-Sterol O-Acyltransferase