Molecular Biological Study on Regulation and Expression of Physiological Function of Human Lipoprotein Lipase

点击翻译按钮获取中文摘要

Lipoprotein lipase (LPL) is a key enzyme to catalyze triglyceride-rich lipoprotein at the first step of their metabolism in circulation. LPL enzyme takes on a functionally active form at capillary endothelium through three processes such as (1) expression of LPL gene, and systhesis and secretion of LPL (2) transport of LPL into capillary endothilium (3) binding of LPL on the surface of capillary endothelium. The study on these processes has been delayed by a lack of monospecific antibody directed to human LPL, LPL gene from a patient with LPL deficiency, and binding of LPL into bovine endothelim.(1) Biosynthesis and secretion of LPL : Macrophage-like cells (Mphi) derived from THP-1 cells were cultured with "protein labeling medium (PLM)" containing ^<35>S-methionine. The labeled LPL protein was immunoprecipitated from medium and Mphi cells by anti-human PHP-LPL antibody. The percipitated LPL protein was analyzed by 9% SDS-Page. LPL was newly synthesized in a 55 KDa protein, processed to be 60 KDa protein by N-glycosylation, secreted in a form of 61 KDa protein. The secreted LPL protein could bind on the surface of bovine endothelium.(2) Study on LPL molecule in a patient with LPL deficiency : A patient TN was judged to be LPL deficiency by a lack of LPL activity and mass in postheparin plasma. We examined LPL molecule which was newly synthesized in monocyte-derived macrophages from patient TN, and also analyzed LPL mRNA by Norther blot with a probe of THP-1 LPL cDNA. LPL protein and LPL mRNA were not detected from patient TN. Analyzing LPL gene of patient TN, we found one base deletion in LPL coding region following by a premature terminal codon by frame shift. This mutation leads no detectable LPL protein due to the absence of LPL mRNA transcript.(3) Binding of LPL in endothelium : we studied the binding of human LPL molecule on bovine endothelium. Maximum binding of LPL was estimated to be 3.4 mu mol FFA/h/cm^2 of endothelium.