Disturbed vesicular trafficking of membrane proteins in prion disease

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.     

Viral proteins expressed on the surface of murine leukemia cells.

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.     

The surface protein superfamily of Trypanosoma cruzi stimulates a polarized Th1 response that becomes anergic

Trypanosoma cruzi is an obligate intracellular parasite that chronically infects mammals. Extracellular mammalian stage trypomastigotes simultaneously express and release multiple members of the parasite's surface protein superfamily; these extracellular proteins should stimulate MHC class II-restricted CD4 T cells. The surface protein superfamily, however, encodes variant epitopes that may inhibit this CD4 response. In this report the surface protein-specific CD4 response was investigated. CD4 cells isolated from acutely and chronically infected mice did not proliferate when stimulated with surface proteins. Adoptive transfer of surface protein-specific CD4 clones or immunization with a peptide encoding a surface protein T cell epitope protected mice during T. cruzi infection. These data strongly suggested that surface proteins were expressed and presented to CD4 cells during infection. Limiting dilution analysis identified an expanded population of surface protein-specific CD4 cells during the acute and chronic infection. These surface protein-specific CD4 cells did not produce IL-2 or IL-4, but did produce IFN-gamma. Enzyme-linked immunospot analyses confirmed that many of the surface protein-specific CD4 cells produce IFN-gamma. Together these results suggest that during T. cruzi infection a potentially protective CD4 response becomes anergic. It is possible that this anergy is induced by variant T cell epitopes encoded by the surface protein superfamily.     

Radioiodination of L 1210 cells.

The lymphoid leukaemia L 1210 cells of mice were labelled with 125I. The cell homogenates were fractionated and from the microsomal fraction 90 per cent of the radioactive material could be precipitated with perchloric acid, whereas only 4 per cent was precipitated from the soluble fraction. Papain bound with Enzacryl AH released 31 per cent of radioactivity. It was concluded therefrom that the surface proteins of the cells were labelled. Electrophoretic separation of these proteins in polyacrylamide gel with sodium dodecyl sulphate was performed and 6--8 radioactive fractions of surface peptides were found.     

Monoclonal antibodies to rat brain astroglial cells—Their characterization and application for isolation of astroglial subpopulations

Three monoclonal antibodies (MAbs) specifically recognizing rat astrocyte cell surface proteins have been characterized and their antigen binding specificities determined. One of these MAbs has been employed to isolate a distinct subpopulation of astroglial cells using immunoaffinity chromatography.

MAbs to rat astroglial cell surface proteins, generated by fusion of mouse Sp2/O-Ag 14 myeloma cells and spleen cells from Balb/C mice immunized with purified astroglial cells, were screened for their cell binding specificities using ELISA and indirect immunofluorescence assay. The antigen binding specificity of three of these clones, which displayed specific binding to astrocytes, was determined by radioiodination of whole astrocytes and precipitation of the iodinated surface proteins by the MAbs. Immunoaffinity chromatography, using IgG from one of the clones coupled to CNBr activated Sepharose 6MB, demonstrated the potential usefulness of such MAbs in isolating a specific subpopulation of astroglial cells.     

The role of Staphylococcus aureus sortase A and sortase B in murine arthritis

Gram-positive pathogenic bacteria display proteins on their surface that play important roles during infection. In Staphylococcus aureus, these surface proteins are anchored to the cell wall by two sortase enzymes, SrtA and SrtB, that recognize specific surface protein sorting signals. The role of sortase enzymes in bacterial virulence was examined using a murine septic arthritis model. Intravenous inoculation with any of the Delta(srtA), Delta(srtB) or Delta(srtAB) mutants resulted in significantly increased survival and significantly lower weight loss compared with the parental strain. Mice inoculated with the Delta(srtA) mutant did not express severe arthritis, while arthritis in mice inoculated with the Delta(srtB) mutant was not different from that seen in mice that were infected with the wild-type parent strain. Furthermore, persistence of staphylococci in kidneys and joints following intravenous inoculation of mice was more pronounced for wild-type and Delta(srtB) mutant strains than for Delta(srtA) or Delta(srtAB) variants. Together these results indicate that sortase B (srtB) plays a contributing role during the pathogenesis of staphylococcal infections, whereas sortase A (srtA) is an essential virulence factor for the establishment of septic arthritis.     

Expression of Three Gastrula Cell Surface Glycoproteins during Embryonic and Larval Development in the Amphibian Pleurodeles waltlii

We have previously reported the identification of cell surface glycoproteins in Pleurodeles waltlii gastrulae. In an attempt to study the expression of three of these cell surface glycoproteins (proteins referred to 1, 11 and 14), we have produced monoclonal and polyclonal antibodies by immunizing mice with the spots of the three selected glycoproteins excised from 2D-gels. Expression of the three glycoproteins was detected on the surfaces of all cells during embryonic development. Before hatching, proteins 1, 11 and 14 become expressed in a limited number of tissues.     

Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense

Surface membrane proteins and glycoproteins of procyclic Trypanosoma rhodesiense were labeled with 125I by the use of the insoluble catalyst Iodo-Gen. Autoradiography of whole solubilized procyclic trypanosomes after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a minimum of 25 surface components to have incorporated radioactivity. These components ranged in m.w. from approximately 10,000 to approximately 285,000. Immunoprecipitation with rabbit antisera of Triton X-100 extracts of radiolabeled trypanosomes revealed a subset of at least 14 surface antigens. Two of these antigens (m.w. of 63,000 and 96,000) showed heavy incorporation of label and may be major proteins of the procyclic membrane. Sera from trypanosome-infected mice recognized an overlapping but different subset of surface antigens, including a doublet of very high m.w. Lectin precipitation using antilectin conjugates or bead-bound lectins indicated that many of the labeled surface components are glycoproteins including the two major proteins precipitated by rabbit antisera. Radiolabeled glycoproteins identified by these methods bear alpha-methyl-mannopyranoside and/or galactose residues but not N-acetyl glucosamine or fucose residues in quantity. The use of these methods in identifying potentially pathogenic trypanosomal antigens is suggested.     

Immunity induced by Staphylococcus aureus surface protein A was protective against lethal challenge of Staphylococcus aureus in BALB/c mice

Staphylococcus aureus is the most common cause of hospital-acquired bacteremia. Due to emergence of antibiotic-resistant strains, these infections present a serious public health threat. In this study, to develop a broadly protective vaccine, we tested whether immune responses induced by several proteins associated with S. aureus toxicity could protect mice from lethal challenge with human clinical S. aureus isolate USA300. We found that the surface protein A (SasA) of S. aureus could protect mice from lethal challenge of the bacteria.     

Virus-induced immune complex disease: identification of specific viral antigens and antibodies deposited in complexes during chronic lymphocytic choriomeningitis virus infection.

Structural proteins of LCMV were identified and their role in the immune complex glomerulonephritis of LCMV carrier mice was examined. Purified LCMV contained three major polypeptides, a single nonglycosylated nucleoprotein with an estimated m.w. of 63,000, and two surface glycoproteins of 54,000 and 35,000. Deposition of nucleoprotein antigen in the glomeruli of LCMV carrier mice of several strains was demonstrated by immunofluorescent staining with a monospecific antibody. In addition, Ig eluted from kidneys of three strains of LCMV carrier mice was shown by immune precipitation to react against all of major viral polypeptides of LCMV. Antibody from normal mice, and from mice with immune complex disease unrelated to LCMV did not show deposition of LCMV antigen in glomeruli, and Ig eluted from the kidneys of these mice did not react against LCMV antigens. Hence, mice infected at birth with LCMV and persistently infected throughout their life make antibodies to all the known structural polypeptides of the virus.     

Declining Levels of Specialized Synaptic Surface Proteins in nNOS-Expressing Interneurons in Mice Treated Prenatally with Valproic Acid

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorder characterized by impaired social interaction, and repetitive or restricted interests and behaviors. Membrane proteins are a significant part of the proteins in cell and play key functions in synaptic transmission. We have recently shown that neuronal nitric oxide synthase (nNOS) expression was reduced in the basolateral amygdala (BLA) of mice following postnatal valproic acid (VPA) exposure. In the current study, we utilized a label-free proteomics approach to identify and quantify surface protein expression in nNOS-positive interneurons between VPA-treated and control mice. Western blot was used to confirm the expression of selected membrane proteins. Our proteomics data revealed differentially expressed surface proteins in nNOS interneurons, e.g. Narp, AMPA-type glutamate (AMPA) receptor subunit GluA4 and Protein kinase C gamma (PKCγ), which were validated by Western blotting in mice treated with VPA. This work will pave the way for further elucidation of the mechanisms of these differentially membrane proteins in nNOS interneurons-medicated ASD.

     

幽门螺杆菌表面抗原免疫保护作用的体外与活体研究

目的：调查幽门螺杆菌（Hp)几种表面蛋白体外对T细胞增殖的影响和在小鼠体内的免疫保护作用。方法：评价Hp全菌抗原、尿原酶（Urease)、黏附素（hpaA)、外膜蛋白25（Hop25)和38（Hop38)对人外周血T细胞及小鼠CD4^ T细胞增殖的影响;与佐剂合用,评价上述重组蛋白对小鼠Hp感染的免疫预防作用。结果：Urease和Hop25可刺激人及鼠T细胞增殖,hapA只能刺激Hp^ PBL增殖,而Hop38则有毒性作用;Hop25和Hop38均可产生60％的完全保护,hpaA可产生100％的部分保护即降低细菌定植密度,而Urease只能产生40％的部分保护。结论：外膜蛋白可能是一组高效的Hp疫苗免疫原;其长期免疫效果及对T细胞功能的活体调节作用尚需进一步评价。国际上尚未见相关报道。     

Unravelling the chemical basis of competitive scent marking in house mice

Major urinary proteins (MUPs) in the urine of male house mice, Mus domesticus, bind the male signalling volatiles 2- sec -butyl-4,5-dihydrothiazole (thiazole) and 3,4-dehydro- exo -brevicomin (brevicomin) and slowly release these volatiles from urinary scent marks. To examine the role of urinary proteins and volatiles, either attached or unattached to the proteins, in competitive scent marking, we fractionated urine from isolated male BALB/c laboratory mice, Mus musculus, by size-exclusion chromatography into three pools. Pool I contained all of the urinary proteins and their bound ligands while pools II and III contained lower molecular weight components including unbound signalling volatiles. In experiment 1, pools I-III were streaked out on to absorbent paper (Benchkote) and introduced into enclosures housing single wild-caught male mice, together with a clean control surface. Each male was tested with fresh stimuli and with aged stimuli deposited 24 h previously. Only pool I stimulated significantly more countermarking and investigation than the control, attracting mice to investigate from a distance even when the rate of ligand release was considerably reduced after 24 h. Experiment 2 examined responses to pool I when this was fresh, aged by 7 days, or had been mixed with menadione to displace ligands from the proteins. Although all three protein stimuli were investigated and countermarked more than a clean control, the aged and menadione-treated pool I stimulated the strongest responses, despite containing the lowest levels of thiazole and brevicomin. Thus competitive countermarking is stimulated by proteins or by nonvolatile protein-ligand complexes in male urine, while release of volatile ligands attracts attention to a competitor's scent marks. Copyright 1999 The Association for the Study of Animal Behaviour.     

Driving adenovirus type 12-transformed BALB/c mouse cells to express high levels of class I major histocompatibility complex proteins enhances, rather than abrogates, their tumorigenicity.

The tumorigenicity of adenovirus type 12 (Ad12)-transformed cells has been attributed to the low levels of class I major histocompatibility complex (MHC) protein expression by these cells. These levels of class I proteins are thought to be below the threshold critical for cytotoxic T-lymphocyte recognition, a process that may be involved in tumor cell immunosurveillance. We have used gene transfer experiments to investigate the role played by class I protein expression in the tumorigenicity of Ad12-transformed BALB/c mouse cells in naive, syngeneic adult mice. Our Ad12-transformed mouse cells were tumorigenic in adult mice and were similar to other Ad12-transformed mammalian cells in that they expressed low levels of class I MHC mRNA and cell surface proteins. Despite these low levels of expression, the cells were highly immunogenic in syngeneic mice and were rejected as allografts by allogeneic mice. Transfection of genomic H-2Dd or H-2Ld fragments into these cells produced a variety of cell clones that expressed increased levels of cell surface class I proteins. These cells expressing high levels of class I protein were up to 16-fold more tumorigenic than the parental cells in syngeneic adult mice. Thus, by quantitative assays, the tumorigenicity of Ad12-transformed BALB/c mouse cells is not functionally related to the low levels of class I MHC proteins they express. The increased tumorigenicity expressed by H-2Dd- and H-2Ld-transfected cells was not detected in BALB/c nu/nu mice, suggesting that a thymus-dependent mechanism that is not mediated by evasion of cytotoxic T-lymphocyte recognition could contribute to the difference in tumorigenicity of Ad12-transformed BALB/c mouse cells that express low and high levels of class I MHC proteins.     

Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse

Lipid droplets in adipocytes serve as the principal long-term energy storage depot of animals. There is increasing recognition that lipid droplets are not merely a static neutral lipid storage site, but in fact dynamic and multi-functional organelles. Structurally, lipid droplet consists of a neutral lipid core surrounded by a phospholipid monolayer and proteins embedded in or bound to the phospholipid layer. Proteins on the surface of lipid droplets are crucial to droplet structure and dynamics. To understand the lipid droplet-associated proteome of primary adipocyte with a large central lipid droplet, lipid droplets of white adipose tissue from C57BL/6 mice were isolated. And the proteins were extracted and analyzed by liquid chromatography coupled with tandem mass spectrometry. A total of 193 proteins including 73 previously unreported proteins were identified. Furthermore, the isotope-coded affinity tags (ICAT) was used to compare the difference of lipid droplet-associated proteomes between the normal lean and the high-fat diet-induced obese C57BL/6 mice. Of 23 proteins quantified by ICAT analysis, 3 proteins were up-regulated and 4 proteins were down-regulated in the lipid droplets of adipose tissue from the obese mice. Importantly, two structural proteins of lipid droplets, perilipin A and vimentin, were greatly reduced in the lipid droplets of the adipose tissue from the obese mice, implicating reduced protein machinery for lipid droplet stability.     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

Cellular changes in the thymuses of preleukemic AKR mice: correlation with changes in the expression of murine leukemia viruses.

Thymus cells of preleukemic and leukemic AKR mice express on their cell surface elevated levels of antigens associated with the murine leukemia virus (MuLV) proteins gp70 and p30. The gp70 antigenicity is contained in a 70,000 dalton polypeptide that corresponds to the viral envelope protein, while the p30 antigenicity is contained in two polypeptides of 85,000 and 95,000 daltons that correspond to glycosylated forms of the polyprotein product of the gag gene.The expression of these viral coded proteins on the cell surface of thymocytes varies both quantitatively with the age of the mouse and qualitatively with the cellular populations that express these antigens. Four discrete stages in the leukemic pathway can be identified. First, low numbers of cells from the thymuses of young (2 month old) AKR mice express p30 (<0.25%) and gp70 (2–7%) antigens. Expression of gp70 antigen is restricted to large cells in the subcapsular region of the thymus. Second, thymuses of 6 month old AKR mice show a selective depletion of cortical thymocytes with a concomitant increase in the medullary region of the thymus. Thymus cells of these mice contain elevated numbers of cells that express an increased concentration of p30 and gp70 antigens. Viral antigens are found on the surface of all large cells of the subcapsular region of the thymus, and in variable numbers (2–85%) of small cells of the cortical and medullary regions. Third, the thymuses of some 8 month old AKR mice demonstrate selective hypertrophy of a single thymic lobe. The enlarged lobe contains a population of cells that are intermediate in size between the small cortical cells and leukemic blast cells. This new cell population expresses elevated levels of p30 and gp70 viral antigens. These cells, which are not leukemic (since transfer of high numbers of these cells to syngeneic hosts does not induce transplantable disease), may represent preleukemic thymocytes. Fourth, thymuses of mice with overt leukemia contain primarily leukemic blast cells. These cells express extremely high levels of viral antigens on their cell surfaces, and upon transfer of these cells to syngeneic hosts, they rapidly induce transplantable leukemias.The increased expression of viral antigens on the surface of thymus cells is correlated with an increased production of infectious ecotropic and xenotropic MuLV in the thymus. During aging, the percentage of cells producing ecotropic MuLV increases 10-fold, while the percentage of cells producing xenotropic MuLV increases 100 fold.     

Identification and immunotherapeutic targeting of antigens induced by chemotherapy

Cancer cells differ from normal cells in their response to chemotherapy. We exploited this dissimilarity by identifying and targeting tumor-specific, cell-surface proteins whose expression is induced by the chemotherapeutic irinotecan (CPT-11; Camptosar). A cytotoxin-armed antibody reactive with one of these drug-induced surface proteins, the LY6D/E48 antigen, originally identified as the target of a monoclonal antibody reactive with squamous cell carcinomas, caused complete regression of colorectal tumor xenografts in mice treated with CPT-11, whereas either agent alone was less effective. These results suggest that a positive therapeutic index may be generated for other drug combinations by immunotherapeutic targeting of chemotherapy-induced antigens.     

Murine Humoral Immune Response against Recombinant Structural Proteins of Hepatitis C Virus Distinct from Those of Patients

We examined the humoral immune response to recombinant structural proteins of hepatitis C virus (HCV) such as C, E1 and E2 in immunized mice. Mice showed high induction of antibodies against these three structural proteins. Conformational and/or linear epitopes of these regions showed high responses in mice. Comparison with patients revealed higher anti-E1 and anti-E2 responses in mice and 15 immunoreactive peptides which are unique to mice, especially 11 peptides from the E2 region. The hydrophilic regions of these proteins were found to be the most immunogenic. Therefore, the murine immune system against recombinant E1 and E2 glycoproteins was distinct from those of patients in natural infection, and may be a target to find protective activity against HCV infection.