The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.

Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E−/−) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E−/−hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E−/−, and E−/−hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in E−/−hTgE+/0 plasma. Depending on incubation time, E−/− plasma released 48–74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E−/−hTgE+/0 plasma was only 11–25% lower than into E+/+ plasma. E−/−hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E−/− plasma. E+/+ and E−/−hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E−/− plasma. Moreover, E−/− plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic γ and α mobility than E+/+ and E−/−hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

In situ synthesis of β-glucan microfibrils on tobacco plasma membrane sheets

A major concern in plant morphogenesis is whether cortical microtubules are responsible for the arrangement and action of β-glucan synthases in the plasma membrane. We prepared isolated plasma membrane sheets with cortical microtubules attached and tested whether β-glucan synthases penetrated through the membrane to form microfibrils and whether these synthases moved in the fluid membrane along the cortical microtubules. This technique enabled us to examine synthesis of β-glucan as a fiber with a two-dimensional structure. The synthesis of β-glucan microfibrils was directed in arrays by cortical microtubules at many loci on the membrane sheets. The microfibrils were mainly arranged along the microtubules, but the distribution of microfibrils was not always parallel to that of the microtubules. The rate of β-glucan elongation as determined directly on the exoplasmic surface was 620 nm per min. When the assembly of microtubules was disrupted by treatment with propyzamide, the β-glucans were not deposited in arrays but in masses. This finding shows that the arrayed cortical microtubules are not required for β-glucan synthesis but are required for the formation of arranged microfibrils on the membrane sheet.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

Inhibition of advanced glycation endproduct formation by acetaldehyde: Role in the cardioprotective effect of ethanol

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called “French paradox,” (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells1 : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387–392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.

Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma.

High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse.

Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide.

Pancreatic islet amyloid deposits are a characteristic pathologic feature of non-insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin-dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia.

Suppression of synapsin II inhibits the formation and maintenance of synapses in hippocampal culture.

Numerous synaptic proteins, including several integral membrane proteins, have been assigned roles in synaptic vesicle fusion with or retrieval from the presynaptic plasma membrane. In contrast, the synapsins, neuron-specific phosphoproteins associated with the cytoplasmic surface of synaptic vesicles, appear to play a much broader role, being involved in the regulation of neurotransmitter release and in the organization of the nerve terminal. Here we have administered antisense synapsin II oligonucleotides to dissociated hippocampal neurons, either before the onset of synaptogenesis or 1 week after the onset of synaptogenesis. In both cases, synapsin II was no longer detectable within 24-48 h of treatment. After 5 days of treatment, cultures were analyzed for the presence of synapses by synapsin I and synaptophysin antibody labeling and by electron microscopy. Cultures in which synapsin II was suppressed after axon elongation, but before synapse formation, did not develop synapses. Cultures in which synapsin II was suppressed after the development of synapses lost most of their synapses. Remarkably, with the removal of the antisense oligonucleotides, neurons and their synaptic connections recovered. These studies lead us to conclude that synapsin II is involved in the formation and maintenance of synapses in hippocampal neurons.

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.

The very low density lipoprotein (VLDL) receptor is a recently cloned member of the low density lipoprotein (LDL) receptor family that mediates the binding and uptake of VLDL when overexpressed in animal cells. Its sequence is 94% identical in humans and rabbits and 84% identical in humans and chickens, implying a conserved function. Its high level expression in muscle and adipose tissue suggests a role in VLDL triacylglycerol delivery. Mutations in the chicken homologue cause female sterility, owing to impaired VLDL and vitellogenin uptake during egg yolk formation. We used homologous recombination in mouse embryonic stem cells to produce homozygous knockout mice that lack immunodetectable VLDL receptors. Homozygous mice of both sexes were viable and normally fertile. Plasma levels of cholesterol, triacylglycerol, and lipoproteins were normal when the mice were fed normal, high-carbohydrate, or high-fat diets. The sole abnormality detected was a modest decrease in body weight, body mass index, and adipose tissue mass as determined by the weights of epididymal fat pads. We conclude that the VLDL receptor is not required for VLDL clearance from plasma or for ovulation in mice.