Lipoprotein lipaseBethesda: a single amino acid substitution (Ala-176----Thr) leads to abnormal heparin binding and loss of enzymic activity.

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Abstract
The molecular defect that leads to a deficiency of lipoprotein lipase (LDL) activity in the proband from a Bethesda kindred has been identified. The pre- and post-heparin plasma LDL mass in the proband was elevated when compared to controls; however, there was no detectable LPL activity, indicating the presence of a defective enzyme (termed LPLBethesda). Analysis of the patient''s post-heparin plasma by heparin-Sepharose affinity chromatography demonstrated that the mutant LPL had an altered affinity for heparin. Southern blot hybridization of the gene for LPLBethesda revealed no major rearrangements. Northern blot analysis of LPLBethesda mRNA from patient monocyte-derived macrophages revealed normal-sized mRNAs (3.4 and 3.7 kilobases) as well as normal cellular mRNA levels when compared to control macrophages. Sequence analysis of polymerase chain reaction-amplified LPL cDNA revealed a G .fwdarw. A substitution at position 781 of the normal LPL gene that resulted in the substitution of an alanine for a threonine at residue 176 and the loss of an SfaNI site present in the normal LPL gene. Amplification of cDNA by the PCR followed by digestion with SfaNI established that the patient was a true homozygote for the mutation. Expression of LPL cDNA in COS-7 cells resulted in the synthesis of a nonfunctional LPL enzyme establishing that the Ala .fwdarw. Thr substitution was the mutation responsible for the inactive LPL. The identification of this mutation in the LPL gene defines a region of the LPL enzyme, at Ala-176, that is essential for normal heparin-binding and catalytic activity. We propose that an amino acid substitution in this critical region of LPLBethesda results in the synthesis of a nonfunctional enzyme that leads to the chylomicronemia syndrome expressed in this proband.