Journal article
Analysis of human lecithin–cholesterol acyltransferase activity by carboxyl-terminal truncation
Biochimica et biophysica acta. Lipids and lipid metabolism, Vol.1344(3), pp.250-261
1997
DOI: 10.1016/S0005-2760(96)00149-X
PMID: 9059515
Abstract
Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in reverse cholesterol transport and catalyzes the esterification of cholesterol in human plasma. Human LCAT is a glycosylated protein, containing 416 amino acids and a proline-rich region at the C-terminus. To address the function of the C-terminal region of LCAT as well as that of the proline-rich region, we constructed and expressed LCAT mutants with C-terminal truncations at different positions. The expression of wild-type LCAT in COS-1 cells resulted in an enzymatically active protein that was secreted by the cells. The mutants lacking the proline-rich region at the C-terminus were expressed and secreted at levels comparable to those of wild-type (∼50% of wild-type concentrations in cell media). The proline-deletion mutants were similar to wild-type LCAT in terms of phospholipase or transferase activities with various interfacial substrates, including reconstituted HDL, proteoliposomes, LDL, and micelles of platelet activating factor. Thus, the binding of LCAT to the diverse interfaces is not affected by the removal of its C-terminal region. Also, the activation by apolipoproteins and access of water-insoluble substrates to the active site are not significantly affected by the deletion of the proline-rich region. However, deletions of the proline-rich region, including the five amino acids nearest to the C-terminus, resulted in approximately an 8-fold increase in the specific activity of LCAT towards the water-soluble substrate,
p-nitrophenylbutyrate. This suggests that the C-terminal proline-rich region may interfere with the access of this water-soluble substrate to the active site of LCAT, and may form part of a protective covering of the active site of LCAT while in solution. Further deletions at the C-terminus, beyond the proline-rich region, impaired the secretion of the enzyme, implying that this region may play a critical role in either the secretion or folding of LCAT in COS-1 cells.
Details
- Title: Subtitle
- Analysis of human lecithin–cholesterol acyltransferase activity by carboxyl-terminal truncation
- Creators
- Young-Phil Lee - Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 190 Medical Sciences Building, 506 South Mathews Avenue, Urbana, IL 61801, USAShanthi Adimoolam - Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 190 Medical Sciences Building, 506 South Mathews Avenue, Urbana, IL 61801, USAMing Liu - Department of Medicine and Biochemistry, Rush Medical College, Chicago, IL 60612, USAPapasani V Subbaiah - Department of Medicine and Biochemistry, Rush Medical College, Chicago, IL 60612, USAKevin Glenn - Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 190 Medical Sciences Building, 506 South Mathews Avenue, Urbana, IL 61801, USAAna Jonas - Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 190 Medical Sciences Building, 506 South Mathews Avenue, Urbana, IL 61801, USA
- Resource Type
- Journal article
- Publication Details
- Biochimica et biophysica acta. Lipids and lipid metabolism, Vol.1344(3), pp.250-261
- DOI
- 10.1016/S0005-2760(96)00149-X
- PMID
- 9059515
- NLM abbreviation
- Biochim Biophys Acta
- ISSN
- 0005-2760
- eISSN
- 1879-145X
- Publisher
- Elsevier B.V
- Language
- English
- Date published
- 1997
- Academic Unit
- General Internal Medicine; Internal Medicine
- Record Identifier
- 9984094705502771
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