CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is ...thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO4(2-) radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.
We have previously demonstrated hyposialylation of the two major CD45 and leukosialin (CD43) molecules at the surface of latently human immunodeficiency virus type 1-infected CEM T cells (CEMLAI/NP), ...(Lefebvre, J. C., Giordanengo, V., Doglio, A., Cagnon, L., Breittmayer, J. P., Peyron, J. F., and Lesimple, J. (1994) Virology 199, 265–274; Lefebvre, J. C., Giordanengo, V., Limouse, M., Doglio, A., Cucchiarini, M., Monpoux, F., Mariani, R., and Peyron, J. F. (1994) J. Exp. Med. 180, 1609–1617). Searching to clarify mechanism(s) of hyposialylation, we observed two sulfated secreted glycoproteins (molecular mass ∼47 and ∼40 kDa) (P47 and P40), which were differentially sulfated and/or differentially secreted in the culture supernatants of CEMLAI/NP cells when compared with parental CEM cells. A hybridoma clone (7H1) resulting from the fusion between CEMLAI/NP and human embryonic fibroblasts MRC5 cells produced very large amounts of P47 that was purified using Jacalin lectin (specific for O-glycans) and microsequenced. Cloning of P47 was achieved using a CEMLAI/NP cDNA library screened with a degenerate oligonucleotide probe based on its NH2-terminal amino acid sequence. A single open reading frame encoding a protein of 323 amino acids was deduced from the longest isolated recombinant (1.4 kilobase). P47 is a secreted sulfated protein. It carries an NH2-terminal RGD (Arg-Gly-Asp) triplet, a striking α-helical leucine zipper composed of six heptads, and a C-terminal C-type lectin domain. The NH2-terminal portion is rich in glutamic acids with a predicted pI of 3.9. In addition, a hinge region with numerous condensed potential sites forO-glycan side chains, which are also the most likely sulfation sites, is located between the RGD and leucine zipper domains. Transcripts were detected in lymphoid tissues (notably bone marrow) and abundantly in T and B lymphoblastoid but very faintly in monocytoid cell lines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The synthesis of Galalpha1-3Gal-terminated oligosaccharides (alpha-Gal) epitopes has been interrupted during the course of evolution, starting with Old World primates. Partial sequences similar to ...the alpha1,3-galactosyltransferase (alpha1,3GalT) gene, which governs the synthesis of alpha-Gal epitopes, have been detected in the human genome and were found to correspond to pseudogenes. We completed the sequence of the human alpha1,3GalT pseudogene present on chromosome 9 and found it to be organized like the murine alpha1,3GalT gene. In human cell lines and several normal and tumor tissues we detected truncated transcripts corresponding to this pseudogene. Considering these mRNAs, translation of an open reading frame containing the first four translated exons but missing the two catalytic exons could predict a truncated alpha1,3GalT polypeptide that should be enzymatically inactive. We show that transcription of human alpha1,3GalT is prematurely terminated at the level of a strong transcriptional stop signal in the middle of intron VII. We were able to reproduce this effect in vitro by subcloning the implicated DNA region upstream from a reporter cDNA. The premature transcriptional arrest of human alpha1,3-GalT gene leads to an ectopic splicing event and to the connection of a short intronic sequence downstream from translated exons. Finally, we show that these truncated transcripts are overexpressed in cell lines with modifications of O-glycans.
In vitro, LSLCL is expressed by numerous myeloid, promyelocytic, and T or B lymphoblastoid cell lines. In vivo, LSLCL is strongly expressed in bone marrow and only faintly in lymphoid organs. We show ...here that, in bone marrow, LSLCL is detected: (i) concentrated in the cytoplasm of immature neutrophils but not in myeloblasts nor in mature neutrophils, (ii) in extracellular bone marrow fluid. Besides, numerous cDNAs, similar to LSLCL (identity of 93–99 %), are found in ‘expressed sequence tags’ databases from various origins, mostly fetal and undifferentiated tumour tissues. Since LSLCL and various closely related cDNAs are expressed at definite stages of cellular maturation processes, we hypothesize that this class of proteins could play an important role in the control of cellular differentiation.
In vitro, la LSLCL est exprimée par de nombreuses lignées : myélocytaires, promyélocytaires, cellules T ou B. In vivo, la LSLCL est fortement exprimée dans la moelle osseuse, uniquement par les neutrophiles immatures et non pas par les myéloblastes ni par les neutrophiles matures. La LSLCL est également détectée dans le milieu extracellulaire médullaire. Par ailleurs, des ADNc très proches de la LSLCL (avec 93 à 99 % d’identités) sont retrouvés dans les banques de ‘séquences exprimées’ (EST) et proviennent principalement de tissus fœtaux ou de tumeurs indifférenciées. Constatant que la LSLCL et des ADNc pratiquement identiques sont détectés à des stades bien précis de maturation cellulaire, nous émettons l’hypothèse d’une possible implication de cette nouvelle classe de protéines dans le contrôle de la différenciation cellulaire.
ABSTRACT
The mechanisms by which caspases are stimulated following T cell receptor engagement are presently unknown. We show here that TCR cross‐linking induces caspase activation, annexin V binding, ...loss of cell viability, Fyn cleavage, and apoptosis in a T cell hybridoma. All these events are increased in T cells overexpressing wild‐type Fyn and are inhibited in cells expressing a kinase dead mutant or a soluble form of Fyn. Moreover, DNA fragmentation mediated by various proapoptotic stimuli and anti‐CD3‐induced caspase activation and DNA fragmentation are reduced significantly in mouse embryo fibroblasts and thymocytes from Fyn knockout mice respectively. These results indicate that an active and membrane‐anchored Fyn is required for T cell receptor‐induced caspase activation and apoptosis in T lymphocytes and point out a specific role of Fyn in caspase activation and apoptosis in T cells.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Immunodeficiency caused by HIV infection probably results from profound dysregulation of normal T lymphocyte properties by the virus. Despite description of the virus cytopathicity and numerous ...modifications in T cell functions, such as perturbation of antigen receptor signaling, CD4 downregulation, and induction of apoptosis, the precise mechanisms underlying the disruption of normal immune responses have not yet been elucidated. In the present study, we show that HIV-1-infected lymphocytes of the CEM cell line (either latent or virus-producing) and HIV-1-infected CD4+ lymphocytes have several membrane proteins with altered glycosylation patterns. Using lectins with specificity for different carbohydrate moieties, we could demonstrate the presence of two exposed nonsialylated disaccharides: a terminal Galβ1 → 3GalNAc and a terminal Galβ1 → 4GlcNAc. In particular, CD45, one of the major T cell glycoproteins, appeared to be partially sialylated on N- and O-linked carbohydrate moieties. Concerning the latter, PNA lectin which recognizes nonsialylated terminal Galβ1 → 3GalNAc might precipitate up to 75% of the total tyrosine phosphatase activity displayed by CD45 molecules from one latently HIV-1-infected CEM cell line. Since CD45 glycoproteins are thought to play an important regulatory role in cell-to-cell interactions owing to their variable extracellular region and because they may regulate membrane signaling through their intracellular phosphatase domains, we suggest that these altered CD45 molecules may present an abnormal signal for natural ligands such as the B-cell-specific surface receptor CD22, thus perturbing the normal immune response in HIV-1-infected individuals.
CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal ...endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.
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