Search

The regulation of cell surface receptor expression is essential for immune cell differentiation and function. At the plasma membrane ubiquitination is an important post-translational mechanism for regulating expression of a wide range of surface proteins. MARCH9, a member of the RING-CH family of transmembrane E3 ubiquitin ligases, down-regulates CD4, major histocompatibility complex-I (MHC), and ICAM-1 in lymphoid cells. To identify novel MARCH9 substrates, we used high throughput flow cytometry and quantitative mass spectrometry by stable isotope labeling by amino acids in cell culture (SILAC) to determine the differential expression of plasma membrane proteins in a MARCH9-expressing B cell line. This combined approach identified 13 potential new MARCH9 targets. All of the SILAC-identified targets for which antibodies were available were subsequently confirmed by flow cytometry, validating the proteomics results. A close correlation (r(2) = 0.93) between -fold down-regulation as determined by SILAC and flow cytometry was found, with no false positive hits detected. The potential new MARCH9 substrates cover a wide range of functions and include receptor-type protein-tyrosine phosphatases (e.g. PTPRJ/CD148) as well as Fc gamma receptor IIB (CD32B), HLA-DQ, signaling lymphocytic activation molecule (CD150), and polio virus receptor (CD155). The identification of plasma membrane targets by SILAC with confirmation by flow cytometry represents a novel and powerful approach to analyze changes in the plasma membrane proteome.

The cellular members of the interleukin-10 (IL-10) cytokine family share sequence homology with IL-10, whereas their sites of expression and their functions are divergent. One of these factors, AK155 or IL-26, was discovered because of its overexpression in human T lymphocytes after growth transformation by the simian rhadinovirus herpesvirus saimiri. In addition, the gene is transcribed in various types of primary and immortalized T-cells. Here we describe epithelial cells, namely colon carcinoma cells and keratinocytes, as targets of this T-cellular lymphokine. Purified recombinant IL-26 induced the rapid phosphorylation of the signal transducer and activator of transcription factors 1 and 3. As a result, secretion of IL-10 and IL-8, as well as cell surface expression of CD54 were enhanced. Moreover, we show that the IL-26 protein binds to heparin, is released from the cell surface, and can be functionally inhibited by heparin. The sensitivity to recombinant IL-26 of various cell lines strictly correlated with the expression of the long chain of the IL-20 receptor. Because blocking antibodies against either the short chain of the IL-10 receptor or the long chain of the IL-20 receptor inhibited IL-26-dependent signal transduction, and transient expression of these receptor chains induced IL-26 responsivity in non-sensitive cells, we propose that the IL-20 receptor 1 and IL-10 receptor 2 chains participate in forming the IL-26 receptor. Targeting epithelial cells, the T-cell lymphokine IL-26 is likely to play a role in local mechanisms of mucosal and cutaneous immunity.

A family of interleukin-10 (IL-10)-related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence paralogs, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads, through the activation of signal transducer and activator of transcription (STAT) factors, to diverse biological effects. For example, whereas IL-10 is a well-studied pleiotropic immunosuppressive and immunostimulatory cytokine, IL-22/IL-TIF mediates acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes, which has been proposed as a pathogenic mechanism of psoriasis.

Herpesvirus saimiri can be used as an efficient gene expression vector for human T lymphocytes and thus may allow applications in experimental leukemia therapy. We constructed recombinant viruses for the functional expression of the thymidine kinase (TK) of herpes simplex virus type 1 (HSV) as a suicide gene. These viruses reliably allowed the targeted elimination of transduced nonpermissive human T cells in vitro after the administration of ganciclovir. To test the reliability of this function under the most stringent permissive conditions, in this study we analyzed the influence of the prodrugs ganciclovir and acyclovir in common marmosets on the acute leukemogenesis induced by either wild-type herpesvirus saimiri C488 or by a recombinant derivative expressing TK of HSV. Antiviral drug treatment did not influence the rapid development of acute disease. In contrast, the presence of the HSV tk gene resulted in a faster disease progression. In addition, HSV TK-expressing viruses showed faster replication than wild-type virus in culture at low serum concentrations. Thus, HSV TK accelerates the replication of herpesvirus saimiri and enhances its pathogenicity. This should be generally considered when HSV TK is applied as a transgene in replication-competent DNA virus vectors for gene therapy.

Although herpesvirus saimiri-transformed T lymphocytes retain multiple normal T-cell functions, only a few changes have been described. By subtractive hybridization, we have isolated a novel cellular gene,ak155, a sequence homolog of the interleukin-10 gene. Specifically herpesvirus saimiri-transformed T cells overexpressak155and secrete the protein into the supernatant. In other T-cell lines and in native peripheral blood cells, but not in B cells,ak155is transcribed at low levels. AK155 forms homodimers similarly to interleukin-10. As a lymphokine, AK155 may contribute to the transformed phenotype of human T cells after infection by herpesvirus saimiri.

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

The aim of this project was to identify the best method for the enrichment of plasma membrane (PM) proteins for proteomics experiments. Following tryptic digestion and extended liquid chromatography-tandem mass spectrometry acquisitions, data were processed using MaxQuant and Gene Ontology (GO) terms used to determine protein subcellular localization. The following techniques were examined for the total number and percentage purity of PM proteins identified: (a) whole cell lysate (total number, 84–112; percentage purity, 9–13%); (b) crude membrane preparation (104–111; 17–20%); (c) biotinylation of surface proteins with N-hydroxysulfosuccinimydyl-S,S-biotin and streptavidin pulldown (78–115; 27–31%); (d) biotinylation of surface glycoproteins with biocytin hydrazide and streptavidin pulldown (41–54; 59–85%); or (e) biotinylation of surface glycoproteins with amino-oxy-biotin (which labels the sialylated fraction of PM glycoproteins) and streptavidin pulldown (120; 65%). A two- to threefold increase in the overall number of proteins identified was achieved by using stop and go extraction tip (StageTip)-based anion exchange (SAX) fractionation. Combining technique (e) with SAX fractionation increased the number of proteins identified to 281 (54%). Analysis of GO terms describing these proteins identified a large subset of proteins integral to the membrane with no subcellular assignment. These are likely to be of PM location and bring the total PM protein identifications to 364 (68%). This study suggests that selective biotinylation of the cell surface using amino-oxy-biotin in combination with SAX fractionation is a useful method for identification of sialylated PM proteins.

Efficiency of lymphoma induction by herpesvirus saimiri (HVS) isolates correlates with the genetically defined viral subgroups A, B, and C. To compare subgroup-specific effects, highly susceptible tamarins were infected with HVS strain A-11, B-SMHI, or C-488. All animals developed T-cell lymphomas indistinguishable with respect to clinical, pathological, and virological parameters. Ex vivo T-cell lines were established readily from the HVS C-488 animal, less efficiently in the presence of HVS A-11, and from only a single HVS B-SMHI sample. These cultivated cells revealed strain-specific biochemical characteristics. HVS A-11 strongly induced the expression of tyrosine kinase Lyn. HVS C-488 led to the activation of STAT3, which is most likely linked to the association of virus-encoded Tip with tyrosine kinase Lck. The lack of these activities in HVS B-SMHI-transformed cells may correlate with the reduced oncogenic phenotype of this virus in species other than tamarins.

Subgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.