Abstract
We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the $Departmentsof Cell Biology and Medicine, BaylorCollege of Medicine, and The Methodist Hospital, Houston, Texas 77030,and the §Departmentsof Pediatrics and Clinical Biochemistry, Algemeen Zickhuis St
Direct sequencing Human LPL shows hihgohmology to hepatic triglycof pools of >2000 cDNA clones indicates that their eride lipase and pancreatic lipase
The of genomic DNAs of the patients and their fammielmy - totally inactiveAsp'56 +Gly LPL mutant described in bers by the polymerase chain reaction, restriction en- this study is the first naturally occurring mutant rezyme digestion
Summary
Human LPL cDNA Expression Vector-A cDNA containing 1786 bases extending fromnucleotides -320 to 1466 (counting the first base of the translation initiation codon as nucleotide +1) was subcloned into M13mp and used as a template for site-specific mutagenesis. Mutant anwdild type LPL cDNAs were used to transfectEscherichia coli TG1 cells, and positive clones wereidentified by direct sequencing.Replicative form DNAs were isolated,digested with EcoRI, and inserted into the EcoRI site of p91023(B) (Wong et al, 1985). In Vitro Expression and LPL Enzyme Assay-COS M-6 cells (0.251.0 X IO6)were plated on 100-mm diameter tissue culture dishes and transfected with 20 pg of plasmid DNA/dish by the technique of HDU: PL MOL TO. Cell extracts andmedia were assayed for LPL enzyme activity aspreviously described Second base of the codonTCA for Ser447from a C to a G producing a premature stopcodon TGA (Fig. 2B) thatwould
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have