Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward α- And β-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via α-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the β-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Δ1-Δ5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Δ1 mutant) resulted in a dramatic loss of α-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full β-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Δ2 mutant) abolished α-LCAT activity and reduced β-LCAT activity to 12% of wild type. Nevertheless, LCATΔ1 and Δ2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the α-LCAT cofactor apoA-I (in Δ1) and/or loss of enzyme activity (in Δ1-Δ5). Since the Δ1 and Δ2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.