Deletion of Specific Glycan Chains Affects Differentially the
Stability, Local Structures, and Activity of Lecithin-cholesterol
Acyltransferase*
Jeffrey
Kosman and
Ana
Jonas
From the Department of Biochemistry, College of Medicine at
Urbana-Champaign, University of Illinois, Urbana, Illinois 61801
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 
-helical content of N384Q LCAT and in the 
-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.
*
This work was supported by National Institutes of Health
Grant HL-29939 (to A. J.) and by a predoctoral fellowship from the American Heart Association, Illinois Affiliate (to J. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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