Pierre Baudhuin

A model of protein-colloidal gold interactions.

We prepared homogeneous populations of colloidal gold particles of various sizes. These were analyzed for size distribution and number of particles per unit volume. On exposure to increasing concentrations of insulin, myoglobin, protein A, peroxidase, serum albumin, galactosylated serum albumin, lactoferrin, transferrin, catalase, low-density lipoprotein, ferritin, and polymeric IgA, protein binding was a saturable process. Using serum albumin, we verified that a reversible equilibrium was reached within 15 minutes. Scatchard analysis of the interactions between all of these proteins and the gold particles resulted in a single component, linear relation. For a given particle size, the number of binding sites for various proteins was inversely proportional to their molecular weight. Conversely, when the size of particles was varied, the number of binding sites was directly proportional to the average area of each gold particle. All results are compatible with a monomolecular shell of protein surrounding the particle at saturation, the binding capacity being inversely proportional to the projection area of the protein. We present direct morphological evidence for this model. The affinity of the various proteins for the colloid also increased with molecular weight, and was not related to the protein isoelectric point. For globular proteins, the monomolecular shell model makes possible prediction of the number of molecules that will saturate a gold particle, if the average diameter of the gold particles and the molecular weight of the protein are known.

LIVER PEROXISOMES, CYTOLOGY AND FUNCTION

Since the occurrence of a special type of cytoplasmic particle containing hydrogen peroxide-producing oxidases and catalase was first reported some nine years ago,l this new type of organelle has been extensively investigated and characterized, both biochemically and morphologically. It is the aim of this article to provide a summary of the information presently available on the properties of mammalian liver peroxisomes and to discuss some of the still unanswered problems. Emphasis will be mostly on rat liver particles, since peroxisomes have been most extensively studied in this species; some of the differences occurring between liver peroxisomes of various sources will, however, be mentioned.

Biogenesis of the glycosome in Trypanosoma brucei: the synthesis, translocation and turnover of glycosomal polypeptides.

Glycosomes, the microbodies of Trypanosoma brucei, contain a number of enzymes involved in glucose and glycerol metabolism. The biogenesis of three of these enzymes has been studied. Aldolase, D‐glyceraldehyde‐3‐phosphate dehydrogenase and NAD‐linked glycerol‐3‐phosphate dehydrogenase are all synthesized in the cytosol on free rather than on membrane‐bound polysomes. In vitro, as well as in vivo, these polypeptides are synthesized at their mature size, and no evidence was found for any processing upon entry into the glycosomes. Continuous and pulse‐chase labelling experiments with procyclic trypomastigotes revealed that the enzymes have a half‐life in the cytosol of approximately 3 min or less, and then turn over rapidly in the glycosomes, with half‐lives as short as 30 min.

Role of acidic compartments in Trypanosoma brucei, with special reference to low-density lipoprotein processing.

In the bloodstream form of Trypanosoma brucei, specific receptors mediate the endocytosis of host low-density lipoprotein particles. We have explored the fate of ligand and receptor with a biochemical approach, using inhibitors of the endocytotic apparatus. The weak base chloroquine rapidly accumulates in trypanosomes, its uptake is prevented by the proton ionophore monensin, and it induces the swelling of intracellular vacuoles, indicating that their content is acidic. Cell-associated LDL is rapidly degraded into intermediately sized fragments and TCA-soluble material that can be recovered in cell extracts and extracellular medium. Chloroquine, leupeptin and E64, but not PMSF, efficiently prevent LDL proteolysis, suggesting that degradation occurs in those acidic compartment(s) and is mediated by thiol-protease(s). Both monensin and chloroquine decrease the number of LDL receptors exposed at the cell surface, a phenomenon amplified in the presence of LDL. This provides indirect evidence that internalised LDL receptors are recycled at a rate which is slowed down by receptor occupancy and by agents that impair acidification of the endocytic organelles. Finally, chloroquine decreases by half the growth rate of procyclic trypanosomes in vitro at 5 micrograms ml-1. At 40 mg kg-1 per day, it also slows down the increase in parasitaemia and prolongs the survival time of infected mice by up to 2 days.

Tissue Distribution of [actinomycin-h-3 D Adsorbed On Polybutylcyanoacrylate Nanoparticles

Abstract Suspensions of polybutylcyanoacrylate nanoparticles have been characterized morphologically. The influence of the adsorption of [ 3 H]actinomycin D on these particles upon the tissue distribution of the drug has been studied in rats. It has been demonstrated that 24 h after injection, adsorbed [ 3 H]actinomycin D is 5.6-, 44- and 64-fold more concentrated than the free drug in muscle, spleen and liver respectively. Furthermore, the urinary excretion of [ 3 H]actinomycin D is diminished when the drug is bound to polybutylcyanoacrylate nanoparticles.

A rapid method purifies a glycoprotein of Mr 145,000 as the LDL receptor of Trypanosoma brucei brucei.

The trypanosome LDL receptor has been isolated from bloodstream form and cultured insect-stage trypanosomes as a protein of Mr 145,000, using a rapid purification procedure in the presence of a cocktail of protease inhibitors, whereas previously a polypeptide of Mr 86,000 was purified as the LDL receptor. Both the 145,000 and the 86,000 polypeptides are glycosylated and recognized by a monospecific antibody raised against the 86,000 species. This antibody inhibits LDL binding to the intact trypanosomes, to the isolated 145,000 receptor and to the 86,000 species. Hence, the previously isolated 86,000 polypeptide is a degradation product probably representing the cleaved-off ectodomain of the trypanosome LDL receptor.

Polymeric IgA and Galactose-Specific Pathways in Rat Hepatocytes: Evidence for Intracellular Ligand Sorting

In 1974 three papers appeared that triggered the investigations reported in this chapter. Ashwell and Morell (1974), respectively at the NIH and at Albert Einstein, New York, described the specific uptake by hepatocytes of galactose-exposing proteins and their rapid degradation in lysosomes. The same year, Brandtzaeg (1974), at the Institute of Pathology of the Rikshospitalet, Oslo, Norway, proposed a model for the selective transfer of polymeric IgA (pIgA) across mucous and glandular epithelia. Brandtzaeg’s proposal implied the selective binding of pIgA to a receptor called secretory component (SC), exposed at the basolateral surface of epithelial cells. SC would mediate pIgA translocation to the apical surface and into secretion, and protect pIgA from lysosomal degradation. This model, “based on test tube experiments with purified proteins and on immunofluorescence studies on dead tissues” (Brandtzaeg, 1981), was essentially correct but called for further work on living epithelia. Yet the same year, Heremans’ laboratory at the University of Louvain in Belgium reported the selective secretion of IgA into dog bile (Dive et al., 1974). Later, pIgA was also shown to be rapidly and actively secreted from blood to bile by rat liver (Jackson et al., 1978; Lemaitre-Coelho et al., 1978; Orlans et al., 1978) and the role of SC as pIgA receptor on rat hepatocytes was demonstrated (Fisher et al., 1979; Orlans et al., 1979; Socken et al., 1979).

TRANSFER OF IgA INTO RAT BILE: ULTRASTRUCTURAL DEMONSTRATION

Several ligands are internalized in rat hepatocytes by receptor-mediated endocytosis. Whereas most of them (eg. galactose-exposing proteins) will eventually be digested in lysosomes, polymeric IgA (pIgA) is selectively transferred into bile.’.‘ In order to investigate by electron microscopy the transepithelial transfer of pIgA with a direct probe, we have prepared conjugates of horseradish peroxidase (HRP) with monoclonal human pIgA and examined their fate in rat liver after i.v. injection. The HRP-pIgA conjugate behaved like native pIgA with respect to in vitro, binding to cultured rat hepatocytes and in vivo, transfer into bile’ and subcellular distribution in liver upon differential and isopyknic centrifugation. HRP-pIgA was mostly found in hepatocytes and to a much lesser extent in sinusoidal cells. One minute after injection, numerous small pits and profiles (-100 nm) were labeled along the sinusoidal and lateral plasmalemma of the hepatocytes. These profiles were generally embedded in a dense microfilamentous meshwork. Where this matrix was less prominent, the profiles were covered by a coat suggestive of clathrin, surrounded by a pale halo. In less than five minutes, HRP-pIgA was, in addition, found in larger irregular vesicles (>zoo nm) with no apparent coat. Labeling was seen on the internal aspect of their membrane, and their content was otherwise electron-lucent (FIGURE 1). Hence, these structures resemble the “receptosomes” described by Willingham and Pastan! These findings are similar to those observed with HRP-pIgA in cultured rat hepatocytes.’ Within 15 minutes, labeled vesicles selectively migrated to the biliary pole, where they clustered around bile canaliculi. Some lysosomes were also labeled, but never Golgi stacks and rims. By 30 minutes, these vesicles could still be observed around bile canaliculi that also became labeled at this time (FIGURE 2). In conclusion, transepithelial transfer of pIgA into bile includes (1) selective internalization through coated pits, (2) transfer into larger vesicles that may be related to the “receptosomes.” (3) migration of the vesicles to the biliary pole, (4) clustering around bile canaliculi, and (5) membrane fusion with the plasmalemma enclosing bile canaliculi and the discharge into bile. Hence, the initial steps of this pathway are identical to those described for ligands that will be digested in lyso~omes.~ Possible involvement of lysosomes in pIgA transfer a s well as the sorting mechanism of the two classes of ligands are presently under investigation.

Trypanosoma brucei brucei: Antigenic stability of its LDL receptor and immunological cross-reactivity with the LDL receptor of the mammalian host

The rapid growth of Trypanosoma brucei brucei in the blood and tissue fluids of vertebrates requires the receptor-mediated endocytosis of LDL from the host (Coppens et al. 1987; Gillett and Owen 1987) and is slowed by a monospecific rabbit antiserum against the purified LDL receptor of the parasite. We have used this antiserum in combination with several well-characterized antigenic variants (originating from stock 427: MITat 1.1a, 1.3a, 1.4a, 1.5a, 1.5d, 1.8b) to examine whether the LDL receptor of T. b. brucei is a stable surface antigen, common to all parasite variants despite antigenic variation of the major surface glycoprotein, and whether it is immunologically distinct from the LDL receptor of the host. At low concentrations, binding at 4 degrees C of rat LDL to several variants of T. b. brucei and to isolated rat hepatocytes was inhibited to a similar extent by the antiserum. In double immunodiffusion, a single precipitation line was observed, showing continuity between the extracts of all variants as well as between that of trypanosomes and of mammalian tissues. In Western blots of trypanosome extracts, the LDL receptor was strongly labeled as a single band of Mr 145,000, whereas with a rat liver extract, a single band of similar electrophoretic mobility was weakly labeled at a high concentration of the antiserum. In conclusion, the LDL receptor occurred in all variants of T. b. brucei, was a stable surface antigen despite variation of the major surface glycoprotein, and displayed biochemical and immunological similarities with the LDL receptor of the rat host.

Identification of a specific epitope on the extracellular domain of the LDL-receptor of Trypanosoma brucei brucei.

We have previously shown that an antiserum raised against the 86-kDa fragment of the low-density lipoprotein-receptor (LDL-receptor) of bloodstream Trypanosoma brucei brucei shows extensive cross-reactivity with the mammalian LDL-receptor. Here we report on the production and characterisation of 30 monoclonal antibodies (mAbs) raised against the same 86-kDa fragment of the T. b. brucei LDL-receptor. Of these, only 8 mAbs recognise in an ELISA test the purified (presumably intact) 145-kDa LDL-receptor. Seven of them also recognise the LDL-receptors isolated from rat and rabbit, whereas one mAb (1A9) is specific for the trypanosome LDL-receptor. A pool of several mAbs inhibits by 90% the specific binding of 125I-LDL to trypanosomes at 4 degrees C, but does not interfere with binding of 125I-LDL to rat fibroblasts. 125I-mAb 1A9 is efficiently taken up by T. b. brucei at 30 degrees C and its uptake is inhibited by an excess of unlabelled LDL particles, indicating that mAb 1A9 follows the LDL-receptor pathway. Uptake of 125I-mAb 1A9 by rat fibroblasts is less efficient and is not significantly reduced by an excess of unlabelled LDL. MAb 1A9 as well as other pooled mAbs activate rabbit complement, leading to lysis of trypanosomes in vitro. We conclude that the T. b. brucei LDL-receptor contains at least one specific epitope that is accessible on live cells to antibodies and which can activate the complement system.