Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.1.34 (lipoprotein lipase)
7,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Most missense mutations of the lipoprotein lipase (LPL) gene identified among LPL-deficient subjects cluster in a segment of the sequence that encodes the catalytic triad as well as functional elements involved in the activation of the lipase at lipid-water interfaces. Consequently, loss of activity may result either from direct alterations of such functional elements or from less specific effects on protein folding and stability. This issue was addressed by examining biochemical properties of four such variants (A176T, G188E, G195E, and S244T) in a heterologous expression system (COS-1 cells). Variant G195E (GGA----GAA) was previously unreported. In all instances, inactive enzyme was recovered in medium, albeit at reduced levels. Cellular synthesis and extracellular degradation were similar to those for wild type, suggesting that reduced secretion resulted from increased intracellular degradation. When cell extracts were subjected to heparin-Superose affinity chromatography followed by elution on a linear salt gradient, all variants exhibited a single, inactive, low affinity immunoreactive peak. By contrast, wild-type enzyme presented an additional, high affinity, active species, which we interpret as homodimeric enzyme. Substitution of the active-site serine (S132A) led to loss of activity but maintenance of the high affinity species. When large amounts of the G188E variant were applied to the column, small but significant amounts of high affinity, active enzyme were recovered. Systematic substitutions at residue 188 showed that only glycine could accommodate structural constraints at this position. We conclude that the mutations examined did not impart lipase deficiency by affecting specific functional elements of the enzyme. Rather, they appear to affect protein folding and stability, and thereby formation and maintenance of subunit assembly.
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PMID:Missense mutations in exon 5 of the human lipoprotein lipase gene. Inactivation correlates with loss of dimerization. 140 Mar 31

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.
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PMID:A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity. 152 25

For studying the role of Ser132 in the putative catalytic site of human lipoprotein lipase (LPL), mutant LPL cDNAs expressing LPLs with amino acid substitutions of Gly or Asn for Ser132 were obtained by site-directed mutagenesis, and were expressed in COS-1 cells. Considerable amounts of LPL enzyme protein mass were detected in the culture medium of COS-1 cells transfected with wild-type LPL, LPL-Gly132, or LPL-Asn132. LPL-Gly132 hydrolyzed Triton X-100-triolein and tributyrin as effectively as wild-type LPL, whereas LPL-Asn132 showed no activity. LPL-Asn132 bound to very low density lipoproteins as effectively as wild-type LPL.
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PMID:Effects of substitutions of glycine and asparagine for serine132 on activity and binding of human lipoprotein lipase to very low density lipoproteins. 154 20

Studies on the molecular biology of lipoprotein lipase (LPL) deficiency have been facilitated by the availability of LPL gene probes and the recent characterization of gene mutations underlying human LPL deficiency. Typically, missense mutations have predominated and show a preferential localization to exons 4 and 5. This distribution supports earlier studies attributing functional significance to residues encoded by these exons. We now report a further missense mutation within exon 5 of the LPL gene in three unrelated patients. Amplification of individual exons by the polymerase chain reaction and direct sequencing revealed a T----C transition at codon 194 of the LPL cDNA which results in a substitution of threonine for isoleucine at this residue. The catalytic abnormality induced by this mutation was confirmed through in vitro mutagenesis studies in COS-1 cells. Transfection with a LPL cDNA containing the codon 194 transition resulted in the synthesis and secretion of a catalytically defective protein. The Thr194 substitution was associated with two different DNA haplotypes, consistent with a multicentric origin for this mutation.
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PMID:Amino acid substitution (Ile194----Thr) in exon 5 of the lipoprotein lipase gene causes lipoprotein lipase deficiency in three unrelated probands. Support for a multicentric origin. 167 45

We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.
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PMID:Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis. 173 Jul 27

The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.
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PMID:Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency. 175 47

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.
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PMID:Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis. 190 87

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)
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PMID:Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family. 190 78

Cloning and sequencing of lipoprotein lipase (LPL) cDNA prepared from the adipose tissue of a patient with classical LPL deficiency revealed a G to A transition at nucleotide 818 in all sequenced clones, leading to the substitution of glutamic acid for glycine at residue 188 of the mature protein. Hybridization of genomic DNA with allele-specific oligonucleotides confirmed that the patient was homozygous for this mutation and revealed that carrier status for this mutation among relatives of the patient was significantly associated with hypertriglyceridemia. Assay of the patient's plasma for immunoreactive enzyme and activity demonstrated the presence of a circulating inactive enzyme protein, the concentration of which was further increased by injection of heparin. The mutant sequence was produced by oligonucleotide-directed mutagenesis, and both normal and mutant sequences were cloned into the expression vector pSVL and transfected into COS-1 cells. The normal sequence led to the in vitro expression of an enzyme that bound to heparin-Sepharose and had a specific catalytic activity similar to that of normal postheparin plasma enzyme. By contrast, the mutant enzyme expressed in vitro was catalytically inactive and displayed a lower affinity for heparin than the normal enzyme. We conclude that this single amino acid substitution leads to the in vivo expression of an inactive enzyme accounting for the manifestations of LPL deficiency noted in the patient.
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PMID:Missense mutation (Gly----Glu188) of human lipoprotein lipase imparting functional deficiency. 196 8

Complete deficiency of lipoprotein lipase (LPL) causes the chylomicronemia syndrome. To understand the molecular basis of LPL deficiency, two siblings with drastically reduced postheparin plasma lipolytic activities were selected for analysis of their LPL gene. We used the polymerase chain reaction to examine the nine coding LPL exons in the two affected siblings and three relatives. DNA sequence analysis revealed a single nucleotide change compared with the normal LPL cDNA: a G----A substitution at nucleotide position 680. This transition caused a replacement of glutamic acid for glycine at amino acid residue 142 of the mature LPL protein. Amino acid sequence comparisons of the region surrounding glycine-142 indicated that it is highly conserved among lipases from different species, suggesting a crucial role of this domain for the LPL structure. Expression studies of the mutant LPL cDNA in COS-7 cells produced normal amounts of enzyme mass. However, the mutated LPL was not catalytically active, nor was it efficiently secreted from the cells. This established that the Gly----Glu substitution at amino acid 142 is sufficient to abolish enzymatic activity and to result in the chylomicronemia syndrome observed in these patients.
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PMID:Familial chylomicronemia (type I hyperlipoproteinemia) due to a single missense mutation in the lipoprotein lipase gene. 201 May 33


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