Preparation and properties of fluorescent polysaccharides

A new method for preparing fluorescein derivatives of polysaccharides is described. These derivatives are prepared by activation of the polysaccharide with cyanogen bromide and subsequent reaction with fluoresceinamine. The optimum conditions for coupling have been established in this report. Using this procedure, we have prepared fluorescein derivatives of a wide variety of polysaccharides. Degrees of substitution in the range of 3.0 X 10(-3) to 2.4 X 10(-2) mol of fluorescein per mole of monosaccharide equivalent were obtained. The fluorescent derivatives are stable: no free fluorescein was detected after incubation at 22 degrees C for 48 h or at -10 degrees C for 4 months. The fluorescein-derivatized polysaccharides were found to have the same potency in inhibiting lectin-mediated hemagglutination as the underivatized polysaccharide. In addition, these fluorescent polysaccharides can be radioiodinated to specific activities exceeding 10(6) dpm/micrograms due to incorporation of 125I into fluorescein. The cell binding properties of 125I-fucoidin and 125I-heparin are indistinguishable from the corresponding underivatized polysaccharides. This general approach for preparing fluorescent polysaccharides should produce useful reagents for localizing and quantifying cell surface carbohydrate-binding proteins (lectins).     

Duration of infection and antigen display have minimal influence on the kinetics of the CD4+ T cell response to Listeria monocytogenes infection

The T cell response to infection consists of clonal expansion of effector cells, followed by contraction to memory levels. It was previously thought that the duration of infection determines the magnitude and kinetics of the T cell response. However, recent analysis revealed that transition between the expansion and contraction phases of the Ag-specific CD8+ T cell response is not affected by experimental manipulation in the duration of infection or Ag display. We studied whether the duration of infection and Ag display influenced the kinetics of the Ag-specific CD4+ T cell response to Listeria monocytogenes (LM) infection. We found that truncating infection and Ag display with antibiotic treatment as early as 24 h postinfection had minimal impact on the expansion or contraction of CD4+ T cells; however, the magnitudes of the Ag-specific CD4+ and CD8+ T cell responses were differentially affected by the timing of antibiotic treatment. Treatment of LM-infected mice with antibiotics at 24 h postinfection did not prevent generation of detectable CD4+ and CD8+ memory T cells at 28 days after infection, vigorous secondary expansion of these memory T cells, or protection against a subsequent LM challenge. These results demonstrate that events within the first few days of infection stimulate CD4+ and CD8+ T cell responses that are capable of carrying out the full program of expansion and contraction to functional memory, independently of prolonged infection or Ag display.     

Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein

Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.     

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.     

In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-gamma receptor

The precise mechanisms that govern the commitment of CD4 T cells to become Th1 or Th2 cells in vivo are incompletely understood. Recent experiments demonstrate colocalization of the IFN-gammaR chains with the TCR during activation of naive CD4 T cells, suggesting that association of these molecules may be involved in determining lineage commitment. To test the role of IFN-gamma and its receptor in the generation of Th1 Ag-specific CD4 T cells, we analyzed mice after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. In the absence of IFN-gamma, Ag-specific CD4 T cells were generated in response to both these infections. In addition, IFN-gamma-producing (Th1) Ag-specific CD4 T cells were generated in mice lacking the ligand-binding chain of the IFN-gammaR (IFN-gammaR1-/-) or the signaling chain (IFN-gammaR2-/-). There was no increase in the number of IL-4-producing Ag-specific CD4 T cells, nor was there a decrease in the expression of T-bet in the absence of functional IFN-gamma signaling, indicating that the cells were committed Th1 cells. Thus, both chains of the IFN-gammaR are dispensable for the generation of Th1 Ag-specific CD4 T cells after infection in vivo.     

L-domain flanking sequences are important for host interactions and efficient budding of vesicular stomatitis virus recombinants

Vesicular stomatitis virus (VSV) possesses a PPPY and a PSAP motif within the matrix (M) protein. The PPPY motif has significant L-domain activity in BHK-21 cells, whereas the PSAP motif does not. Since the core PSAP motif alone is insufficient to provide L-domain activity, we modified upstream or downstream amino acids flanking the PSAP core motif to determine their effect on L-domain activity. VSV recombinants were recovered that contained single or multiple amino acid mutations in upstream or downstream sequences flanking the PSAP core. Recombinant viruses were examined for growth kinetics, budding efficiency, and functional interactions with host proteins. We demonstrate that the composition of amino acids surrounding the L-domain core motifs are critical for efficient L-domain activity and for interactions with host proteins in the context of a VSV infection.     

T cells undergo rapid ON/OFF but not ON/OFF/ON cycling of cytokine production in response to antigen

Inflammatory cytokines such as IFN-gamma and TNF produced by Ag-stimulated CD4(+) and CD8(+) T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8(+) T cells maintain IFN-gamma production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8(+) T cells could rapidly cycle IFN-gamma production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8(+) T cells, our results show that Listeria monocytogenes-specific CD4(+) T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-gamma and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4(+) T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8(+) T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8(+) T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-gamma after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-gamma secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.     

O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p). O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.