Wolfgang J. Schneider

The low density lipoprotein receptor

The study of familial hypercholesterolemia at the molecular level has led to its advancement from a clinical syndrome to a fascinating experimental system. FH was first described 50 years ago by Carl Müller who concluded that the disease produces high plasma cholesterol levels and myocardial infarctions in young people, and is transmitted as an autosomal dominant trait determined by a single gene. The existence of two forms of FH, namely heterozygous and homozygous, was recognized by Khachadurian and Fredrickson and Levy much later. The value of FH as an experimental model system lies in the availability of homozygotes, because mutant genes can be studied without interference from the normal gene. The first and most important breakthrough was the realization that the defect underlying FH could be studied in cultured skin fibroblasts. Rapidly, the LDL receptor pathway was conceptualized and its dysfunction in cells from FH homozygotes was demonstrates. Isolation of the normal LDL receptor protein and studies on the biosynthesis and structure of abnormal receptors in mutant cell lines provided essential groundwork for elucidation of defects at the DNA level. The power of the experimental system, FH, became nowhere more obvious than in work that correlated structural information at the protein level with the elucidation of defined defects in the LDL receptor gene. In addition to revealing important structure-function relationships in the LDL receptor polypeptide and delineating mutational events, studies of FH have established several more general concepts. First, the tight coupling of LDL binding to its internalization suggested that endocytosis was not a non-specific process as suggested from early observations. The key finding was that LDL receptors clustered in coated pits, structures that had been described by Roth and Porter 10 years earlier. These investigators had demonstrated, in electron microscopic studies on the uptake of yolk proteins by mosquito oocytes, that coated pits pinch off from the cell surface and form coated vesicles that transport extracellular fluid into the cell. Studies on the LDL receptor system showed directly that receptor clustering in coated pits is the essential event in this kind of endocytosis, and thus established receptor-mediated endocytosis as a distinct mechanism for the transport of macromolecules across the plasma membrane. Subsequently, many additional systems of receptor-mediated endocytosis have been defined, and variations of the overall pathway have been described.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of oogenesis : the piscine receptor for vitellogenin

The receptor-mediated uptake of vitellogenin (VTG), a plasmatic lipophosphoglycoprotein, is crucial for oocyte growth in egg-laying animals. The plasma membrane receptor for VTG was characterized from oocytes of coho salmon, Oncorhynchus kisutch. In direct binding studies, the receptor exhibited high affinity (Kd, 180 nM) for salmonid VTG, and by ligand blotting with radiolabelled VTG it was visualized as a protein with an apparent Mr of 100,000, under non-reducing conditions. The fish VTG receptor was shown to share key structural elements with VTG receptors from chicken and Xenopus laevis. Namely, cross-reactivity at the level of ligand recognition was observed among VTG receptors from these species and immunological relatedness was demonstrated by immunoblotting with anti-chicken VTG receptor antibodies. In addition, as in chicken and Xenopus, binding of VTG to fish oocyte receptors was shown to be mediated by the lipovitellin domain of VTG. These results clearly indicate that regulation of oocyte growth at the level of yolk formation has been accomplished by the conservation of structural features of receptors required for internalization of VTG.

Transport and uptake of retinol during chicken oocyte growth

Most, if not all, components found in the yolk of a chicken egg are extracted from the plasma compartment during the rapid growth phase of the oocyte. Uptake of the major yolk constituents, very-low-density lipoprotein and vitellogenin, has been shown to be mediated by a specific receptor in the plasma membrane of the oocyte (Barber, D.L., Aebersold, R., Sanders, E.J. and Schneider, W.J. (1991) J. Biol. Chem. 266, 18761-18770). In the current study, we sought biochemical evidence for the uptake into oocytes of a minor but biologically very important component, the vitamin retinol. For transport in serum, retinol is bound to retinol-binding protein (RBP), which in turn forms a complex with transthyretin (TTR). In order to gain insight into the biochemical details of transport of the vitamin, we have identified, purified and characterized RBP, TTR, and RBP-TTR complexes from chicken serum and yolk. The results demonstrate that both serum and yolk contain the tertiary retinol-RBP-TTR complexes as well as free RBP and TTR. Western blots of yolk collected from oocytes at different stages of growth show that both RBP and TTR, but not albumin, are more abundant at early stages relative to total yolk protein. In addition, we find both RBP and TTR in endocytic clathrin-coated vesicles of the oocyte. Our results support the hypothesis that retinol, which must be imported by the oocyte for proper embryonic development, is internalized by the chicken oocyte bound to its serum protein-transport complex.

Control of platelet glycogenolysis activation of phosporylase kinase by calcium

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels; Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca-2+ markedly activate phosphorylase b kinase from human platelets, with a Ka of 0i muM Ca-2+, which is similar to that for the skeletal muscle enzyme; The kinase activity is unstable, and on enzyme ageing is a 50% loss in activity with the Ka decreasing to 0.33 muM Ca-2+. In unstilulated platelets, phosphorylase a was 13.3% of toal measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes; These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca-2+, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved;

Development of an avian model for restenosis

Recurrence of atherosclerotic plaque growth after interventional therapy, restenosis, is a significant clinical problem occurring in 20%-50% of cases. We have developed a new avian model for the investigation of restenosis after arterial injury in cholesterol fed White Leghorn roosters. Atherosclerotic plaque growth 1-30 weeks after angioplasty balloon mediated endothelial injury in the abdominal aorta was studied in 37 roosters. Roosters were maintained on either normal poultry diet or high cholesterol diet. Twelve cholesterol fed roosters were also fed a hormone supplemented diet in order to modify plaque morphology. The procedural success rate was high. Angiographic stenoses (mean 36% with maximum of 74%) were detectable in cholesterol fed roosters after balloon angioplasty with associated histological evidence of plaque growth (P < 0.017). Cholesterol feeding enhanced fatty plaque growth; hormone manipulation increased calcific and ulcerated plaque but with high associated morbidity. Three interventional devices were subsequently examined in 32 roosters (16 laser angioplasty, 7 atherectomy, and 9 stent implant). Plaque development was again assessed by contrast angiography and histological analysis. We conclude that balloon mediated arterial injury in cholesterol fed roosters produces early proliferative and late, complex atherosclerotic lesions providing an inexpensive model for plaque development after intimal injury.

The gastrulating chick blastoderm contains 16-kDa and 14-kDa galactose-binding lectins possibly associated with an apolipoportein

Extracts from chick blastoderms were subjected to affinity chromatography on lactoside-Sepharose. Lactose-eluted fractions were examined by gradient SDS-PAGE with silver staining, as well as by immunoblot analysis using antibodies to the chicken galactose-binding lectins of 14 kDa and 16 kDa and to an apolipoprotein of chicken very low density lipoprotein (Apo-VLDL-II). Fractions containing the highest lectin activity contained four main bands. One, unidentified, comigrated with albumin; two bands were identified by immunoblotting as the 14-kDa and 16-kDa lectins. The fourth band comigrated with Apo-VLDL-II and in immunoblot analysis reacted with antibodies to this apolipoprotein. In our electrophoretic system this protein migrates close to bovine trypsin inhibitor and has an apparent molecular weight of 6500 +/- 500. The present studies establish the identity of this previously described 6.5 kDa protein (Zalik et al. J. Cell. Sci. 88, 483, 1987) as Apo-VLDL-II. While the 16-kDa lectin was present consistently in all the affinity-purified preparations, the relative frequencies of the 14-kDa lectin and Apo-VLDL-II varied. In sections of primitive streak blastoderms, lectin immunofluorescence was present in the lowest, most ventral area of the primitive groove and in the cells emerging laterally from the groove to form the endoderm. Cells of the extraembryonic endoderm also displayed high lectin immunoreactivity. The localization of the lectins is similar to the one described previously for Apo-VLDL-II. Double immunofluorescence staining indicates that Apo-VLDL-II and the lectin(s) colocalize. The copurification and colocalization of Apo-VLDL-II and the lectins in the chick blastoderm suggest that this apolipoprotein may associate with the galactose-binding lectins or may display lectin activity.

The endogenous lectins of the chick blastoderm are present in association with an apolipoprotein in distinct organelles and in the extracellular matrix

SummaryAffinity purified preparations of the galactose-binding lectin from gastrulating chick blastoderms consist of three main polypeptides. Two of these have been identified as the 14 kD and 16 kD galactose-binding lectins. A third one migrates in SDS-PAGE gels with a relative molecular weight of 6,500±500 and has been identified as an apolipoprotein (Apo) of plasma very low density lipoproteins, Apo-VLDL-II. We have studied the localization of these polypeptides using immunofluorescence and ultrastructural immunocytochemistry with peroxidase and protein-A gold. The 14 kD lectin occurs in the intracellular yolk where it is mainly present within the electron lucent component. The 16 kD is also present in the intracellular yolk platelets, but tends to predominate in the electron-dense component. In addition, the 16 kD lectin is also present in pleiomorphic yolk-associated organelles and in the extracellular matrix. Apo-VLDL-II is also localized in the electron-lucent component of the yolk platelet and in the extracellular matrix. Our results suggest that the lectin(s) are associated with Apo-VLDL-II in the yolk platelet, and may subsequently become externalized.

Laser-induced fluorescence identification of intimal hyperplasia after intravascular stent implantation

Laser-induced fluorescence spectroscopy has been developed as a guidance system for laser angioplasty. We have investigated fluorescence spectroscopic detection of neo-intimal formation from the endoluminal surface of stent implanted arteries. Nine White Leghorn roosters had Palmaz-Schatz intra-abdominal aortic stent implantation, nine had aortic balloon angioplasty. Five roosters with stent implantation and four roosters with balloon angioplasty were on a high cholesterol diet and the remainder were on a normal diet. Roosters were sacrificed at intervals of 1, 2, 4, 8, and 12 weeks after intervention. Fluorescence emission spectra were recorded from aortic segments during excimer laser excitation at 308 nm (XeCl, 1.5 - 2.0 mJ/pulse, 5 Hertz). Spectra were then correlated with histology. Fluorescence emission intensity recorded from abdominal aortic segments with stent implantation was higher than that of adjacent segments (p < 0.002 at 440 - 460 nm). Abdominal aortic segments of roosters on normal diets with stent implantation and balloon angioplasty were similar (p equals NS). With cholesterol feeding, aortic spectra from roosters with stent implant had higher intensity at 440 - 460 nm than spectra from aortic segments with balloon angioplasty (p < 0.004). Morphometric analysis demonstrated a twofold increase in intimal thickness in stent segments from cholesterol fed roosters when compared to similar segments of roosters on normal diets (p < 0.005). Our conclusion is: (1) Fluorescence emission spectra can be used to detect native artery fluorescence in stent implant areas. (2) Neo-intimal thickening at the stet implant site can be distinguished from adjacent areas by fluorescence emission spectra. (3) Cholesterol feeding increased neo-intimal thickening.

Receptor-Mediated Low Density Lipoprotein Metabolism

The concept of receptor-mediated metabolism of lipoproteins emerged 17 years ago from studies on human skin fibroblasts grown in culture1. These experiments were designed to elucidate the normal function of low density lipoprotein (LDL), about which little was known at that time. Biochemical studies showed that a specific cell surface receptor, the LDL receptor, mediates the binding, uptake and degradation of LDL, thus supplying almost all cells in the body with cholesterol. Detailed insight into the molecular mechanisms underlying this complex process was obtained from studies with fibroblasts derived from patients with the phenotype of homozygous familial hypercholesterolemia (FH). As in many other biological systems, the expression of a disease state in a defined cellular system was essential to the discovery of the causal factor: FH is now one of the best characterized genetic diseases at the molecular level. As will be outlined below, several groups of mutations occur naturally in the structural gene for the LDL receptor which disrupt its normal function and lead to severe hypercholesterolemia, myocardial infarctions and premature atherosclerosis. Thus, the important role of lipoprotein receptors in normal physiology is underscored by the dramatic consequences of their functional absence.

The LDL Receptor: Structural Insight from Human Mutations

The cell surface receptor for low density lipoprotein (LDL) plays an important role in the regulation of cholesterol metabolism in humans and animals (Brown and Goldstein, 1986). This receptor binds LDL, the major plasma cholesterol transport protein and carries it into cells. Since its discovery 13 years ago, the LDL receptor has proven to be an extremely useful system for the study of receptor-mediated endocytosis, the process by which macromolecules enter cells after binding to receptors in coated pits. This receptor is also of interest because its expression is regulated at the transcriptional level by a feedback mechanism that is responsive to the level of cholesterol in the cell (Russell et al., 1983). A third and distinctive feature of the LDL receptor is the frequent occurrence of mutations that disrupt its structure and function. These mutations occur in patients with the genetic disease, familial hypercholesterolemia (FH) (Goldstein et al., 1985). When mutant receptor genes are present in the heterozygous or homozygous state, the plasma level of LDL rises and atherosclerosis ensues.