Endolysosomal Vesicles Research Articles

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Imaging lysosomal enzyme activity in live cells using self-quenched substrates

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.

Endo-Lysosomal Vesicles Positive for Rab7 and LAMP1 Are Terminal Vesicles for the Transport of Dextran

The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.

Dendritic cell maturation and chemotaxis is regulated by TRPM2‐mediated lysosomal Ca2+release

Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.

Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes

The optimal ratio of the polycation’s amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.

Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

Phosphoinositides differentially regulate bacterial uptake and Nramp1-induced resistance toLegionellainfection inDictyostelium

Membrane phosphatidylinositides recruit cytosolic proteins to regulate phagocytosis, macropinocytosis and endolysosomal vesicle maturation. Here, we describe effects of inactivation of PI3K, PTEN or PLC on Escherichia coli and Legionella pneumophila uptake by the professional phagocyte Dictyostelium discoideum. We show that L. pneumophila is engulfed by macropinocytosis, a process that is partially sensitive to PI3K inactivation, unlike phagocytosis of E. coli. Both processes are blocked by PLC inhibition. Whereas E. coli is rapidly digested, Legionella proliferates intracellularly. Proliferation is blocked by constitutively expressing Nramp1, an endolysosomal iron transporter that confers resistance against invasive bacteria. Inactivation of PI3K, but not PTEN or PLC, enhances Legionella infection and suppresses the protective effect of Nramp1 overexpression. PI3K activity is restricted to early infection and is not mediated by effects on the actin cytoskeleton; rather L. pneumophila, in contrast to E. coli, subverts phosphoinositide-sensitive fusion of Legionella-containing macropinosomes with acidic vesicles, without affecting Nramp1 recruitment. A model is presented to explain how Legionella escapes fusion with acidic vesicles and Nramp1-induced resistance to pathogens.

Coxiella burnetii Phase I and II Variants Replicate with Similar Kinetics in Degradative Phagolysosome-Like Compartments of Human Macrophages

Coxiella burnetii infects mononuclear phagocytes, where it directs biogenesis of a vacuolar niche termed the parasitophorous vacuole (PV). Owing to its lumenal pH (approximately 5) and fusion with endolysosomal vesicles, the PV is considered phagolysosome-like. However, the degradative properties of the mature PV are unknown, and there are conflicting reports on the maturation state and growth permissiveness of PV harboring virulent phase I or avirulent phase II C. burnetii variants in human mononuclear phagocytes. Here, we employed infection of primary human monocyte-derived macrophages (HMDMs) and THP-1 cells as host cells to directly compare the PV maturation kinetics and pathogen growth in cells infected with the Nine Mile phase I variant (NMI) or phase II variant (NMII) of C. burnetii. In both cell types, phase variants replicated with similar kinetics, achieving roughly 2 to 3 log units of growth before they reached stationary phase. HMDMs infected by either phase variant secreted similar amounts of the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha. In infected THP-1 cells, equal percentages of NMI and NMII PVs decorate with the early endosomal marker Rab5, the late endosomal/lysosomal markers Rab7 and CD63, and the lysosomal marker cathepsin D at early (8 h) and late (72 h) time points postinfection (p.i.). Mature PVs (2 to 4 days p.i.) harboring NMI or NMII contained proteolytically active cathepsins and quickly degraded Escherichia coli. These data suggest that C. burnetii does not actively inhibit phagolysosome function as a survival mechanism. Instead, NMI and NMII resist degradation to replicate in indistinguishable digestive PVs that fully mature through the endolysosomal pathway.

Uptake of Functionalized Mesoporous Silica Nanoparticles by Human Cancer Cells

Mesoporous silica nanoparticles (MSN) were functionalised by aminofluorescein (AMF) with diethylenetriaminepentaacetic acid spacer molecules which provide free carboxylic groups for binding cell-specific ligands such as folate. AMF allowed the exploration of cellular uptake by HeLa cells using confocal microscopy and flow cytometry. The functionalized nanoparticles (MSN-AMF) penetrated efficiently into HeLa cell cytoplasm through a clathrin dependent endocytosis mechanism. The number of endocytosed MSN-AMF was enhanced when using folate as a targeting molecule. Uptake kinetics revealed that most of MSN-AMF were internalized within 4 h of incubation. Moreover, we found that MSN-AMF were capable of escaping the acidic endolysosomal vesicles of HeLa cells. Cytotoxicity studies suggested that these nanoparticles are non-toxic to HeLa cells up to a dose level of 50 microg/ml.

Transient Receptor Potential Melastatin 2 (TRPM2) is a Calcium Release Channel Essential for Chemotaxis of Dendritic Cells (44.17)

Abstract Chemokines induce Ca2+ signaling and chemotaxis in dendritic cells (DCs) but the molecular players involved in shaping the changes in intracellular Ca2+ remain to be characterized. Using siRNA and knock-out mice, we show that in addition to IP3-mediated Ca2+ release and store-operated Ca2+ entry the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca2+ release, but not Ca2+ influx in mouse DCs. Consistent with this observation, TRPM2 expression in DCs cells is restricted to endolysosomal vesicles, whereas in neutrophils the channel localizes to the plasma membrane and does not participate in Ca2+ release. While TRPM2 is not involved in DCs maturation, TRPM2-deficient DCs show severely compromised chemotaxis regardless of maturation stage. This is due to perturbed Ca2+ signaling manifested through suppression of specific TRPM2-mediated Ca2+-release events and an additional secondary modification of store-operated Ca2+ entry. DCs deficient in both TRPM2 and inositol-1,4,5-trisphosphate receptor signaling (InsP3R) lose their ability to perform chemotaxis entirely. These findings confirm ADPR as a novel second messenger and highlight TRPM2 as a key player regulating DCs chemotaxis through its function as Ca2+ release channel.

Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system

For luminescent quantum dots (QDs) to realize their full potential as intracellular labeling, imaging and sensing reagents, robust noninvasive methods for their delivery to the cellular cytosol must be developed. Our aim in this study was to explore a range of methods aimed at delivering QDs to the cytosol. We have previously shown that QDs functionalized with a polyarginine 'Tat' cell-penetrating peptide (CPP) could be specifically delivered to cells via endocytic uptake with no adverse effects on cellular proliferation. We began by assessing the long-term intracellular fate and stability of these QD-peptide conjugates. We found that the QDs remained sequestered within acidic endolysosomal vesicles for at least three days after initial uptake while the CPP appeared to remain stably associated with the QD throughout this time. We next explored techniques designed to either actively deliver QDs directly to the cytosol or to combine endocytosis with subsequent endosomal escape to the cytosol in several eukaryotic cell lines. Active delivery methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol as aggregates. Delivery of QDs using a variety of transfection polymers also resulted in primarily endosomal sequestration of QDs. However, in one case the commercial PULSin reagent did facilitate a modest cytosolic dispersal of QDs, but only after several days in culture and with significant polymer-induced cytotoxicity. Finally, we demonstrated that an amphiphilic peptide designed to mediate cell penetration and vesicle membrane interactions could mediate rapid QD uptake by endocytosis followed by a slower efficient endosomal release which peaked at 48 h after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested.

Cysteine protease‐mediated cytoskeleton interactions with LFA‐1 promote T‐cell morphological changes

T cells migrate through restrictive barriers in a protease-independent, amoeboid fashion that is characterized by morphological cell polarization. The interaction of cysteine-dependent carboxypeptidase cathepsin X with beta(2) integrin LFA-1 (lymphocyte function associated antigen 1) induces T-cell morphological changes, displaying into a 3D extracellular matrix a cytoplasmic projection termed a uropod. In the present study we show that inhibition of cathepsin X and a cysteine-dependent endopeptidase, cathepsin L, markedly inhibits T-cell actin polymerization, shape polarization, and chemotaxis. We propose that cathepsin L promotes T-cell migration associated processes by activating procathepsin X in the endolysosomal vesicles near the cell membrane and at the peak of the uropod, where both proteases were colocalized. We show that active cathepsin X modifies the beta(2) cytoplasmic tail of LFA-1 in the uropod, promoting its high affinity conformation. We suggest that LFA-1 cleavage contributes to the conformational change in the cytoplasmic tail, promoting the binding of the cytoskeletal protein talin. This interaction is restricted to the uropod and results in the stabilization of this region, promoting LFA-1-mediated cell uropod elongation.

The Role of Cathepsins in Involution and Breast Cancer

Cysteine cathepsins are proteolytic enzymes that reside in endolysosomal vesicles. Some are expressed constitutively while others are transcriptionally regulated. However, the expression and subcellular localization of cathepsins changes during cancer progression and cathepsins have been shown to be causally involved in various aspects of tumorigenesis including metastasis. The use of mouse models of breast cancer genetically ablated for cathepsin B has shown that both the growth of the primary tumor and the extend of lung metastasis is reduced by the loss of cathepsin B. The role of cathepsins in involution of the mammary gland has received little attention although it is clear that cathepsins are involved in tissue remodeling in the second phase of involution. We discuss here the roles of cathepsins and their endogenous inhibitors in breast tumorigenesis and post-lactational involution.

Surfactant−Polymer Nanoparticles Enhance the Effectiveness of Anticancer Photodynamic Therapy

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.

Highly efficient cellular labeling of mesoporous nanoparticles in human mesenchymal stem cells: implication for stem cell tracking

Tracking the distribution of stem cells is crucial to their therapeutic use. However, the usage of current vectors in cellular labeling is restricted by their low internalizing efficiency. Here, we reported a cellular labeling approach with a novel vector composed of mesoporous silica nanoparticles (MSNs) conjugated with fluorescein isothiocyanate in human bone marrow mesenchymal stem cells and 3T3-L1 cells, and the mechanism about fluorescein isothiocyanate-conjugated MSNs (FITC-MSNs) internalization was studied. FITC-MSNs were efficiently internalized into mesenchymal stem cells and 3T3-L1 cells even in short-term incubation. The process displayed a time- and concentration-dependent manner and was dependent on clathrin-mediated endocytosis. In addition, clathrin-dependent endocytosis seemed to play a decisive role on more internalization and longer stay of FITC-MSNs in mesenchymal stem cells than in 3T3-L1 cells. The internalization of FITC-MSNs did not affect the cell viability, proliferation, immunophenotype, and differentiation potential of mesenchymal stem cells, and 3T3-L1 cells. Finally, FITC-MSNs could escape from endolysosomal vesicles and were retained the architectonic integrity after internalization. We conclude that the advantages of biocompatibility, durability, and higher efficiency in internalization suit MSNs to be a better vector for stem cell tracking than others currently used.

Nuclear Targeting of Macromolecular Polyanions by an HIV-Tat Derived Peptide: ROLE FOR CELL-SURFACE PROTEOGLYCANS

New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The transactivator of HIV transcription (Tat) peptide stimulated cellular uptake of both DNA and heparan sulfate in a time-, concentration-, and temperature-dependent manner. Peptide-polyanion complexes accumulated in large, acidic, cytoplasmic vesicles formed de novo. This was followed by transfer of polyanion into the nuclear compartment and subsequent disappearance of the endolysosomal vesicles. In the absence of polyanion the Tat peptide displayed rapid accumulation in the nuclear compartment. However, in the presence of polyanion the peptide was almost exclusively retained in cytoplasmic vesicles. Cell-surface proteoglycans played a pivotal role in the uptake of complexes exhibiting a relatively high peptide to polyanion ratio, corresponding to a net positive charge of the complexes. Uptake of polyanions per se or complexes with a relatively low peptide to polyanion ratio was favored by proteoglycan deficiency in the recipient cells, indicating the existence of distinct transport mechanisms. Moreover, expression of full-length HIV-Tat as well as exogenous addition of HIV-Tat peptide resulted in cellular accumulation of endogenous proteoglycans. We conclude that an HIV-Tat derived peptide efficiently targets extraneous DNA and glycosaminoglycans to the nuclear compartment and that proteoglycans serve a regulatory role in these processes, which may have implications for directed gene and drug delivery in vivo.

Analysis of differential lipofection efficiency in primary and established myoblasts.

In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis.

Down-regulation of the cell surface display of class I MHC proteins is an important mechanism of immune evasion by human and animal viruses. Herpesviruses in particular encode a variety of proteins that function to lower MHC I display by several mechanisms. These include binding and retention of MHC I chains in the endoplasmic reticulum, dislocation of class I chains from the ER, inhibition of the peptide transporter (TAP) involved in antigen presentation, and shunting of newly assembled chains to lysosomes. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human herpesvirus strongly linked to the development of KS and to certain AIDS-associated lymphoproliferative disorders. Here we show that KSHV encodes two distinctive gene products that function to dramatically reduce cell surface MHC I expression. These viral proteins are localized predominantly to the ER. However, unlike previously described MHC I inhibitors, they do not interfere with the synthesis, translocation, or assembly of class I chains, nor do they retain them in the ER. Rather, they act to enhance endocytosis of MHC I from the cell surface; internalized class I chains are delivered to endolysosomal vesicles, where they undergo degradation. These KSHV proteins define a mechanism of class I down-regulation distinct from the mechanisms of other herpesviruses and are likely to contribute importantly to immune evasion during viral infection.

UDP-GlcNAc:Ser-ProteinN-Acetylglucosamine-1-Phosphotransferase fromDictyostelium discoideum Recognizes Serine-containing Peptides and Eukaryotic Cysteine Proteinases

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.