Identification of the receptor scavenging hemopexin-heme complexes

The continued success of Leishmania as an intramacrophage parasite is dependent on its ability to survive within an acidic intracellular compartment, resist degradation by lysosomal hydrolases, exploit the host cell as a source of nutrients, and avoid the macrophage9s antigen-presenting capabilities. All these requirements are dependent on the properties of the parasitophorous vacuole in which Leishmania resides. This study shows that the vacuole possesses membrane proteins characteristic of a lysosome, and has MHC class II molecules. The trafficking of a variety of endocytic markers supports this finding. However, a temporal study up to 14 days post-infection indicates that, as it matures, the vacuole gains mannose 6-phosphate receptor, and becomes more accessible to endocytosed ligand, suggesting that the vacuole has functionally translocated from a lysosomal to late endosomal compartment. Endocytosed material was detected in the flagellar pocket and inside the amastigote, demonstrating parasite uptake of intra-vacuolar material. Careful analysis of amastigotes suggests that they avoid antigen presentation by their host cell by limiting the release of potential antigens. These findings significantly extend our understanding of the mechanisms employed by Leishmania to ensure its survival in the macrophage.

Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages.

The α2-macroglobulin receptor and epithelial glycoprotein-330 : two giant receptors mediating endocytosis of multiple ligands

The inventors have discovered a targeting signal that will direct proteins to the endosomal/lysosomal compartment, and they have demonstrated that chimeric proteins containing a luminal antigenic domain and a cytoplasmic endosomal/lysosomal targeting signal will effectively target antigens to that compartment, where the antigenic domain is processed and peptides from it are presented on the cell surface in association with major histocompatibility (MHC) class II molecules. Chimeric DNA encoding the antigen of interst, linked to an endosomal/lysosomal targeting sequence, inserted in an immunization vector, can introduce the chimeric genes into cells, where the recombinant antigens are expressed and targeted to the endosomal/lysosomal compartment. As a result, the antigens associate more efficiently with MHC class II molecules, providing enhanced in vivo stimulation of CD4+ T cells specific for the the recombinant antigen. Delivering antigens to an endosomal/lysosomal compartment by means of chimeric constructs containing such lysosomal targeting signals will be of value in any vaccination or immunization strategy that seeks to stimulate CD4+ MHC class II restricted immune responses.

Lysosomal targeting of immunogens

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.

/pdf/plasticity-of-early-endosomes-2cwhilekp5.pdf

Plasticity of early endosomes

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.

Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1).:

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Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1).: