N-glycosylation is required for efficient secretion of a novel human secreted glycoprotein, hPAP21

The present study reported the isolation and characterization of a novel human secreted protein, named as hPAP21 (human protease-associated domain-containing protein, 21 kDa), encoded by the hypothetical gene chromosome 2 open reading frame 7 (C2orf7) that contains signal peptide in its N-terminus, without transmembrane domain, except for PA domain in its middle. Western blotting assay indicated that the c-Myc tagged hPAP21 could be secreted into culture medium in the transfected Chinese hamster ovary cells. However, the molecular weights, whatever intracellular (28 kDa) or extracellular (30 kDa) forms, are larger than that of the prediction. To define whether the glycosylation was important process for its secretion, endoglycosidase H (Endo H) and PNGase F (PNG F) were employed to evaluate the effect of glycosylation types on secretion of hPAP21. Interestingly, the extracellular forms were primarily sensitive to PNG F, not Endo H, implying that complex N-glycosylation could be required for the secretion of hPAP21. Furthermore, N-glycosylation of Asn171 was confirmed as potential crucial process for the secretory protein via site-directed mutagenesis assay. All data will be contributed to the understanding of molecular functions of hPAP21.     

Distinct but dispensable N-glycosylation of human CD69 proteins.

Human CD69 is uniquely glycosylated at typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) motifs, which represents the molecular basis for the formation of CD69 homodimers and heterodimers. Here we examined the importance of N-glycosylation for the assembly and intracellular transport of CD69 proteins using mutant CD69 molecules that specifically lack typical and atypical N-glycan attachment motifs. These studies verify the importance of Cys residues in atypical triplet sequences for N-glycan addition to human CD69 proteins in the endoplasmic reticulum (ER). In addition, these data demonstrate that monoglycosylated CD69 proteins (bearing N-glycans exclusively at atypical or typical sites) and aglycosylated CD69 molecules (lacking N-glycans) efficiently dimerize in the ER and have similar stability as wild-type CD69 molecules. Finally, these results show that CD69 proteins lacking atypical or typical N-glycan addition sites are transported to the plasma membrane.     

CD44 is not required for poliovirus replication.

The identification of a monoclonal antibody, AF3, which recognizes a single isoform of the cell surface protein CD44 and preferentially blocks binding of serotype 2 poliovirus to HeLa cells, suggested that CD44 might be an accessory molecule to Pvr, the cell receptor for poliovirus, and that it could play a role in the function of the poliovirus receptor site. We show here that only AF3 blocks binding of serotype 2 poliovirus to HeLa cells and, in contrast to a previously published report, that the anti-CD44 monoclonal antibodies A3D8 and IM7 are unable to block binding of poliovirus. To determine whether CD44 is involved in poliovirus infection, we analyzed the replication of all three serotypes of poliovirus in human neuroblastoma cells which lack or express CD44 and in mouse neuroblastoma cells which lack Pgp-1, the mouse homolog of human CD44, and which express Pvr. All three poliovirus serotypes replicate with normal kinetics and to normal levels in the absence or presence of CD44 or in the absence of Pgp-1. Furthermore, the binding affinity constants of all three poliovirus serotypes for Pvr are unaffected by the presence or absence of CD44 in the human neuroblastoma cell line. We conclude that CD44 and Pgp-1 are not required for poliovirus replication and are unlikely to be involved in poliovirus pathogenesis.     

Structural and functional characterization of the CD2 immunoadhesion domain. Evidence for inclusion of CD2 in an alpha-beta protein folding class

The T-lymphocyte transmembrane glycoprotein CD2 plays an important physiological role in facilitating adhesion between T-lymphocytes and their cognate cellular partners. This interaction is mediated by binding of CD2 to the broadly distributed surface polypeptide LFA-3 and augments the recognition function of the CD3-Ti antigen-major histocompatibility complex receptor via stabilization of conjugate formation between cells. To define better the structural components of the CD2 extracellular region which are important in contact-mediated cellular adhesion, a single-domain CD2 immunoadhesion protein has been prepared from papain digestion of a soluble two-domain CD2 molecule. This amino-terminal domain fragment binds to LFA-3 on human B-cells with a dissociation constant of 0.4 microM, possesses functional immunoadhesion epitopes as defined by the binding of monoclonal antibodies raised to native CD2, and retains the ability to inhibit sheep erythrocyte rosette formation with human T-cells. Thus, all of the immunoadhesion functions ascribed to CD2 reside within the amino-terminal domain. Circular dichroism analysis of the isolated CD2 adhesion domain suggests the presence of substantial alpha-helical character (22%), consistent with earlier computer modeling analyses that predicted a pattern of alternating alpha-helices and beta-sheets within the extracellular region of CD2. Despite the existence of short stretches of sequence homology between CD2 and immunoglobulin superfamily members, the circular dichroism data provide supporting biophysical evidence for classification of CD2 in an alpha-beta (either alpha/beta or alpha + beta) protein folding class.     

N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells

Human alpha3/4fucosyltransferase (FT3) catalyses the synthesis of fucosylated glycoconjugates involved in cell-cell interactions. FT3 has two potential N-glycosylation sites at Asn(154) and Asn(185). Soluble secretory forms of the enzyme (SFT3) and mutant forms with the first, second and both glycosylation sites (SFT3DN1, SFT3DN2, SFT3DN) mutated have been expressed in baby hamster kidney (BHK) and Spodoptera frugiperda (Sf9) cells. Deletion of the first or both sites caused total enzyme inactivation. Deletion of the second site caused 99% and 75% decrease of secretory enzyme expression in BHK and Sf9 cells, respectively. Sf9 cells produced 1 mg/l SFT3 and 0.3 mg/l SFT3DN2; these values were 175- and 3750-fold higher, respectively, than those observed for BHK cells. A significant amount of protein was accumulated intracellularly in Sf9 cells which for SFT3 was active and for SFT3DN2 was inactive, indicating the importance of the glycans from the second glycosylation site for protein folding. The corresponding full-length forms FT3, FT3DN1 and FT3DN2 associated with calnexin as observed by immunoprecipitation studies, which indicated the possible role of this chaperon in the folding of glycosylated glycosyltransferases.     

CD95 tyrosine phosphorylation is required for CD95 oligomerization

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95^Y232,291F), revealed that at least one tyrosine (Y^232,291)-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.     

N-glycosylation of human nicastrin is required for interaction with the lectins from the secretory pathway calnexin and ERGIC-53

The gamma-secretase complex, composed of four non-covalently bound transmembrane proteins Presenilin, Nicastrin (NCT), APH-1 and PEN-2, is responsible for the intramembranous cleavage of amyloid precursor protein (APP), Notch and several other type I transmembrane proteins. gamma-Secretase cleavage of APP releases the Abeta peptides, which form the amyloid plaques characteristic of Alzheimer's disease brains, and cleavage of Notch releases an intracellular signalling peptide that is critical for numerous developmental processes. NCT, a type I membrane protein, is the only protein within the complex that is glycosylated. The importance of these glycosylation sites is not fully understood. Here, we have observed that NCT N-linked oligosaccharides mediated specific interactions with the secretory pathway lectins calnexin and ERGIC-53. In order to investigate the role played by N-glycosylation, mutation of each site was performed. All hNCT mutants interacted with calnexin and ERGIC-53, indicating that the association was not mediated by any single N-glycosylation site. Moreover, the interaction with ERGIC-53 still occurred in PS1/2 double knockout cells as detected in immunoprecipitation as well as confocal immunofluorescence microscopy studies, which indicated that NCT interacted with ERGIC-53 prior to its association with the active gamma-secretase complex.     

Co-stimulation of PAFR and CD36 is required for oxLDL-induced human macrophages activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, Gαi-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a Gαi-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and Gαi-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.     

Vav2 is required for cell spreading.

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.     

CD63 is not required for production of infectious human immunodeficiency virus type 1 in human macrophages

During the assembly of human immunodeficiency virus type 1 (HIV-1) particles, the tetraspanin CD63 can be incorporated into the viral membrane. Indeed, cell surface tetraspanin microdomains that include CD63 have been proposed as sites for virus release. In addition, antibodies against CD63 can inhibit HIV infection of macrophages. In this cell type, HIV assembles into intracellularly sequestered plasma membrane domains that contain several other tetraspanins, including CD81, CD9, and CD53. CD63 is recruited to this domain following HIV infection. Together, these observations suggest that CD63 may have some function in the assembly of infectious virus particles and/or the infectivity of assembled virions. Here we have used RNA interference to knock down CD63 expression in monocyte-derived primary macrophages. We show that in the absence of CD63, HIV assembly is quantitatively comparable to that seen in CD63-expressing macrophages and that virus assembly occurs on compartments positive for CD81, CD9, and CD53. Moreover, the infectivity of macrophage-derived virus is unaffected by the loss of CD63. Together, our results indicate that at least in tissue culture, CD63 expression is not required for either the production or the infectivity of HIV-1.     

Mouse CD24 is required for homeostatic cell renewal

Under physiological conditions, some adult tissues retain a capacity for self-renewal. This property is attributable to the proliferation and differentiation of stem, transit-amplifying, and differentiating cells, which are regulated by cell-cell or cell-matrix interactions or by secreted factors. By gain and loss of function experiments, we demonstrate the involvement of mouse CD24 (mouse cluster of differentiation 24), which is a glycosyl phosphatidylinositol (GPI)-anchored cell-surface glycoprotein, in the regulation of homeostatic cell renewal. BrdU incorporation observations, at optical and electron-microscopic levels, have revealed increased cell proliferation in the developing brain and in the epithelia of mCD24-deleted mice. We have observed ectopic proliferative cells in the suprabasal layers of the mutant skin leading to a general disruption of basal and suprabasal layers. By contrast, ectopic mCD24 expression mediated by retroviral infection of the embryonic brain leads to a decreased number of clusters of cells generated in the progeny. Together, these results and our previous published data indicate that mCD24 contributes to the regulation of the production of differentiated cells by controlling the proliferation/differentiation balance between transit-amplifying and committed differentiated cells.     

The FGFR pathway is required for the trunk-inducing functions of Spemann's organizer.

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. Here, we report that the fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway was required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. Since the organizer influences the differentiation of these tissues by emitting signals that pattern the mesodermal germ layer, our data indicate that the FGFR regulates the production of these signals. In addition, the FGFR pathway was required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway had a minimal effect on the function of the head organizer. We propose that the FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells. The implications of our findings for the evolutionarily conserved role of the FGFR pathway in the functions of Spemann's organizer and other vertebrate-signaling centers are discussed.     

PKCtheta is required for the activation of human T lymphocytes induced by CD43 engagement

The turnover of phosphoinositides leading to PKC activation constitutes one of the principal axes of intracellular signaling. In T lymphocytes, the enhanced and prolonged PKC activation resulting from the engagement of the TcR and co-receptor molecules ensures a productive T cell response. The CD43 co-receptor promotes activation and proliferation, by inducing IL-2 secretion and CD69 expression. CD43 engagement has been shown to promote phosphoinositide turnover and DAG production. Moreover, PKC activation was found to be required for the activation of the MAP kinase pathway in response to CD43 ligation. Here we show that CD43 engagement led to the membrane translocation and enzymatic activity of specific PKC isoenzymes: cPKC (alpha/beta), nPKC (epsilon and theta;), aPKC (zeta) and PKCmu. We also show that activation of PKCtheta; resulting from CD43 ligation induced CD69 expression through an ERK-dependent pathway leading to AP-1, NF-kappaB activation and an ERK independent pathway promoting NFAT activation. Together, these data suggest that PKCtheta; plays a critical role in the co-stimulatory functions of CD43 in human T cells.     

Importin KPNA2 is required for proper nuclear localization and multiple functions of NBS1

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1, is a component of the MRE11-RAD50-NBS1 (MRN) complex, a central player associated with double strand break (DSB) repair. In response to radiation, NBS1 is phosphorylated by ATM, and the MRN complex relocalizes to form punctate nuclear foci for DNA repair. NBS1 controls both the nuclear localization of the MRN complexes and radiation-induced focus formation. We report here that the KPNA2 (importin alpha1) is important for the normal nuclear localization of the MRN complex and its proper formation of the nuclear foci. KPNA2 is the only member of the importin alpha family that physically interacts with NBS1, and the KPNA2-mediated nucleus localization sequence (NLS) is mapped to amino acid residues 461-467 of NBS1 that is sufficient for both the interaction with KPNA2 and the proper nuclear localization. Inhibition of KPNA2 or blockage of the KPNA2 interaction with NBS1 results in a reduction of radiation-induced nuclear focus accumulation, DSB repair, and cell cycle checkpoint signaling of NBS1. Collectively, our results strongly suggest that an interaction with KPNA2 contributes to nuclear localization and multiple tumor suppression functions of the NBS1 complex.