No abstract

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r=-0.81 or higher, p<0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r=0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL, and HDL,) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p<0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet. These studies show that expression of primate CETP in the C57BL/6 mouse has a marked effect on the mouse's apo B-and apo A-I-containing lipoproteins, and they establish a metabolic basis for the previously reported (Arterioscler Thromb 1991;11:1759-1771) negative correlation between hepatic CETP mRNA levels and plasma apo A-I levels in the cynomolgus monkey. In addition, they suggest that increased CETP activity is associated with an atherogenic shift in the lipoprotein profile. ( can carry cholesterol into and out of the interstitium, 1 are believed to play a key role in the interchange of cholesterol between the plasma and the tissues. In addition, because of the strong negative correlation between plasma HDL cholesterol levels and the incidence of coronary artery disease, 2 -6 it is thought that perturbations in the metabolism of HDL may significantly influence the atherogenic process. That is especially true in experimental atherosclerosis. In most models of that disease, the diet-induced changes in the lipoprotein profile include not only a marked increase of the apolipoprotein (apo) B-containing lipoproteins, but also a severe reduction of the HDL levels. Although early studies focused primarily on the increased levels of the apo B-containing lipoproteins, it is now becoming clear that these diet-induced alterations in HDL metabolism are also contributing significantly to the development of the disease. An example of the magnitude of

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6±0.4 pg//ig total RNA (mean±SEM) to 4.1 ±0.8 pg//ig (p<0.005); mesenteric fat CETP mRNA increased from 0.4±0.1 pg/ftg total RNA to 5.3±2.2 pg/ftg (p<0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r=-0.85, p<0.001) and apoprotein A-I (r= -0.84,p<0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a functional CETP gene. (Arteriosclerosis and Thrombosis 1991;11:1759-1771)

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35 S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His 297 3 Gln, Asp 384 3 Ala, and Arg 540 3 His. Transient expression of each mutant showed that only the Arg 540 3 His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg 540 3 His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg 540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation. The microsomal triglyceride transfer protein (MTP)1 is an endoplasmic reticulum resident protein that catalyzes the transfer of lipids between phospholipid surfaces. Although MTP can transfer most classes of lipids, it shows a distinct preference for triglyceride and cholesteryl esters. MTP is a heterodimer composed of the multifunctional protein, protein disulfide isomerase (PDI) and a unique large subunit of 97 kDa (1). PDI is a ubiquitous protein found at high levels in many different tissues (2). The 97-kDa subunit of MTP is expressed primarily in hepatocytes and intestinal enterocytes (3). Thus, the active MTP complex is found predominantly in the liver and small intestine. The subcellular localization, tissue distribution, and activity of MTP all suggest a role for this protein in the assembly and secretio...

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.