科研之友
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摘要
Lipoprotein lipase (LPL) is a complex enzyme consisting of multiple functional domains essential for the initial hydrolysis of triglycerides present in plasma lipoproteins. Previous studies have localized the catalytic domain of LPL, responsible for the hydrolytic function of the enzyme, to the N-terminus whereas the C-terminal end may play a role in lipid and heparin binding. To date, most described missense mutations resulting in a nonfunctional LPL have been located in the N-terminal region of the enzyme. In this manuscript we describe the defect in the LPL gene of a patient with triglycerides ranging from normal to 12,000 mg/dl, low LPL mass, and no LPL activity in postheparin plasma. Sequencing of patient PCR-amplified DNA identified two separate mutations in the C-terminal domain of LPL: an A --> T transversion at nucleotide 1484 resulting in a Glu(410) --> Val substitution and a C --> G mutation at position 1595 that introduces a premature stop codon at position 447. Digestion with MaeIII and MnII established that the patient is a true homozygote for both mutations. In order to investigate the functional significance of these defects, mutant enzymes containing either the Val(410) or the Ter(447) mutations as well as both Val(410) and Ter(447), were expressed in vitro. Compared to the wild-type enzyme, LPL(447) demonstrated a moderate reduction of specific activity using triolein (70% of normal) and tributyrin (74% of normal) substrates, while LPL(410) had a significant (11% and 23% of normal) reduction of the normal lipase and esterase specific activities, respectively. Mutant-LPL(410/447) was virtually inactive using either triolein or tributyrin substrates establishing the functional significance of this combined defect. When analyzed by heparin-Sepharose affinity chromatography, a small fraction of LPL(410), like the native LPL dimer, eluted at an NaCl concentration of 1.3 M and had a normal specific activity. However, most of the LPL(410) mass was detected in an inactive peak that, like the normal LPL-monomer, eluted at 0.8 M NaCl, indicating that the Glu(410) --> Val substitution may alter the stability of the LPL dimer. In summary, we have identified a unique mutation in the C-terminal domain of LPL that significantly affects the function of the mutant enzyme. Despite its location in the C-terminal domain of LPL, this mutation does not directly disrupt the heparin-binding properties of the mutant enzyme or its ability to interact with or hydrolyze either lipid or water-soluble substrates. Instead, the loss of enzyme activity appears to be related to a change in the monomer-dimer equilibrium of the mutant enzyme. Our studies indicate that, in addition to the proposed role in the lipid binding function, the C-terminal domain may play an important role in the formation of the active LPL dimer.
- 出版日期1994-9
