Determinants of CD81 dimerization and interaction with hepatitis C virus glycoprotein E2

The tetraspanin CD81 plays an essential role in diverse cellular processes. CD81 also acts as an entry receptor for HCV through an interaction between the large extracellular loop (LEL) of CD81 and HCV glycoprotein E2. The E2-CD81 interaction also results in immunomodulatory effects in vitro. In this study, we examined the relationship between the dimeric crystal structure of the CD81 LEL and intact CD81. Using random mutagenesis, amino acids were identified that abolished dimerization of recombinant LEL in regions that were important for intermonomer contacts (F150S and V146E), salt bridge formation (K124T), and intramonomer disulfide bonding (T166I, C157S, and C190R). Two monomeric LEL mutants retained the ability to bind E2, K124T, and V146E, whereas F150S, T166I, C157S, and C190R did not. Introduction of K124T, V146E, and F150S mutations in full-length CD81 did not affect its oligomerization and the effects on E2 binding were less severe than for isolated LEL. These results suggest that the LEL has a more robust structure in the intact tetraspanin with regions outside the LEL contributing to CD81 dimerization.     

Reduced fertility of female mice lacking CD81

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.     

Kinetics of HCV envelope proteins' interaction with CD81 large extracellular loop

We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2(661) HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2(661). The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.     

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.     

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

Trafficking and function of the tetraspanin CD63

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.     

The transferrin receptor and the tetraspanin web molecules CD9, CD81, and CD9P-1 are differentially sorted into exosomes after TPA treatment of K562 cells

Here we show that treatment of K562 cells with the phorbol ester TPA induces the down-modulation of various surface antigens. Among them, the transferrin receptor (TfR), the tetraspanin CD81, and a CD81-associated protein, CD9P-1, were unique in that their expression levels were lower after 24 h incubation than after 3 h. We demonstrated that like the TfR, CD81 was internalized at early times, and was less synthesized at latter times. Despite the association of a fraction of the TfR with CD81, these two molecules were subjected to different fates. TPA increased targeting of CD81 and CD9P-1 into exosomes but strongly reduced the localization of the TfR in these vesicles. Using this model we have shown that a fraction of CD81 and CD9P-1 in exosomes comes from a surface pool and that these molecules remain associated in exosomes. However, CD9P-1 could be targeted to exosomes in the absence of CD81 and of another tetraspanin, CD9. The targeting of CD9 into exosomes did not require palmitoylation of the protein. J. Cell. Biochem. 102: 650-664, 2007. (c) 2007 Wiley-Liss, Inc.     

跨膜蛋白CD82在流感病毒感染初期的表达

了解细胞跨膜蛋白CD82在流感病毒感染初期的表达变化,分析CD82在流感病毒感染中的作用。

采用逆转录−聚合酶链式反应（RT-PCR）方法对流感病毒感染4 h内的HEP-2细胞中CD82蛋白mRNA表达量进行检测。

流感病毒感染HEP-2细胞后1 h−3 h细胞 CD82的mRNA表达量降低,并且随着流感病毒接种量增加CD82表达量降低持续时间延长。

流感病毒可以在转录水平调节宿主细胞CD82的表达。CD82在流感病毒感染初期表达下降,提示CD82可能在流感病毒与宿主细胞粘附和入侵过程中发挥作用。

     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition

Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the immunogenicity of RV-based vector and enhancing in vivo half-life. Such tools can also be applied to lentiviral or other enveloped viral vectors.     

The tetraspanin CD9 associates with transmembrane TGF-alpha and regulates TGF-alpha-induced EGF receptor activation and cell proliferation

Transforming growth factor-alpha (TGF-alpha) is a member of the EGF growth factor family. Both transmembrane TGF-alpha and the proteolytically released soluble TGF-alpha can bind to the EGF/TGF-alpha tyrosine kinase receptor (EGFR) and activate the EGFR-induced signaling pathways. We now demonstrate that transmembrane TGF-alpha physically interacts with CD9, a protein with four membrane spanning domains that is frequently coexpressed with TGF-alpha in carcinomas. This interaction was mediated through the extracellular domain of transmembrane TGF-alpha. CD9 expression strongly decreased the growth factor- and PMA- induced proteolytic conversions of transmembrane to soluble TGF-alpha and strongly enhanced the TGF- alpha-induced EGFR activation, presumably in conjunction with increased expression of transmembrane TGF-alpha. In juxtacrine assays, the CD9-induced EGFR hyperactivation by transmembrane TGF-alpha resulted in increased proliferation. In contrast, CD9 coexpression with transmembrane TGF-alpha decreased the autocrine growth stimulatory effect of TGF-alpha in epithelial cells. This decrease was associated with increased expression of the cdk inhibitor, p21(CIP1). These data reveal that the association of CD9 with transmembrane TGF-alpha regulates ligand-induced activation of the EGFR, and results in altered cell proliferation.     

CD151及其突变体在人脐静脉内皮细胞系中的稳定表达

目的建立能够稳定高表达四跨膜蛋白CD151及其突变体的人脐静脉内皮细胞系,为研究CD151在内皮细胞血管形成中的作用奠定基础。方法成功构建重组质粒rAAV-CD151,rAAV-CD151-AAA突变体及rAAV-CD151-ARSA突变体,并用rAAV-GFP作对照,应用脂质体介导质粒转染的方法将重组质粒和对照质粒分别转染至人脐静脉内皮细胞中,通过G418进行筛选,建立稳定高表达CD151蛋白的内皮细胞系。Westernblot检测目的蛋白表达,对构建好的稳定转染细胞系进行筛选和鉴定。结果rAAV-CD151,rAAV-CD151-AAA突变体,rAAV-CD151-ARSA突变体及rAAV-GFP重组质粒成功整合人人脐静脉内皮细胞,并能够稳定的表达相应的目的蛋白。结论通过本实验能够获得稳定高表达野生型CD151及突变体CD151的内皮细胞系,为研究CD151在调节内皮细胞各种病理生理改变中的作用提供了坚实的实验基础。     

应用水动力转染技术使人低密度脂蛋白受体、CD81和浸入诱导蛋白L同时在小鼠体内表达

为建立人低密度脂蛋白受体（LDLR）、CD81和浸入诱导蛋白L（Sip—L）等多个HCV感染相关分子共表达的转基因小鼠,首先分别构建了白蛋白启动子调控的人LDLR、CD81和Sip-L基因的小鼠肝脏组织特异性表达载体,将3种质粒同时采用水动力转染技术导入小鼠体内,FIT—PCR和免疫组化技术检测转入体内基因的表达及持续时间。结果表明,转染8h后即可检测到3种基因在体内转录,人LDLR和CD81在转染1～4d后可在50％-90％的肝细胞中高效表达,7d后检测不到目的基因表达。为了进一步延长转染基因在小鼠体内的表达时间,又分别构建了人LDLR、CD81和Sip—L的染色体整合型表达载体,将它们与噬菌体ФC31的整合酶表达载体同时水动力转染小鼠,3种基因在体内表达时间可持续到转染后25d。以上结果表明建立了多个HCV感染相关分子共表达的转基因小鼠,为确定这些分子能否使小鼠感染HCV奠定了基础。     

Human CD69 associates with an N-terminal fragment of calreticulin at the cell surface

CD69 is thought to be a pluripotent signaling molecule expressed on the surface of a number of activated leukocytes including B, T, and NK cells, monocytes, neutrophils, and platelets. While some advances have been made regarding the mechanisms by which CD69 may participate in such diverse functions as cell aggregation, cellular cytotoxicity, and release of cytokines and inflammatory mediators, the most proximal links of signal initiation have not been identified. Our study has identified, by immunoprecipitation and direct protein sequencing (LC/MS/MS), binding of CD69 to an N-terminal protein fragment of calreticulin expressed on the cell surface of human PBMCs. Given the recently identified roles calreticulin plays in cell adhesion and angiogensis, the identification of CD69 binding directly to calreticulin may provide insights into mechanism(s) by which CD69 or other CD69 family members, i.e., LLT1 and AICL participates in such diverse functions.     

Characterization of the expression of variant and standard CD44 in prostate cancer cells: Identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4–v10 variant CD44) in prostate cancer cells over‐expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT‐PCR, surface labeling with NHS–biotin, and immunoblotting. Expression of variant CD44 encompassing v4–v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT‐fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock‐down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent. J. Cell. Biochem. 108: 272–284, 2009. © 2009 Wiley‐Liss, Inc.     

Cutting edge: dynamic redistribution of tetraspanin CD81 at the central zone of the immune synapse in both T lymphocytes and APC

The tetraspanin CD81 has been involved in T-dependent B cell-mediated immune responses. However, the behavior of CD81 during immune synapse (IS) formation has not been elucidated. We determined herein that CD81 redistributed to the contact area of T cell-B cell and T cell-dendritic cell conjugates in an Ag-dependent manner. Confocal microscopy showed that CD81 colocalized with CD3 at the central supramolecular activation complex. Videomicroscopy studies with APC or T cells transiently expressing CD81-green fluorescent protein (GFP) revealed that in both cells CD81 redistributed toward the central supramolecular activation complex. In T lymphocytes, CD81-GFP rapidly redistributed to the IS, whereas, in the APC, CD81-GFP formed a large accumulation in the contact area that later concentrated in a discrete cluster and waves of CD81 accumulated at the IS periphery. These results suggest a relevant role for CD81 in the topography of the IS that would explain its functional implication in T cell-B cell collaboration.     

Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules.

mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb.