Fluorescent immunoprecipitation analysis of cell surface proteins: A methodology compatible with mass-spectrometry

Filatov, Alexander; Krotov, Grigory; Zgoda, Victor G.; Volkov, Yuri

doi:10.1016/j.jim.2006.09.014

Cited by 30 publications

(21 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fluorescent immunoprecipitation analysis (FIPA) used for the detection of proteins in gels was described previously (14). Briefly, 4 ϫ 10 7 Jurkat T cells were pelleted, washed twice with PBS, and labeled with 0.3 g of rhodamine 6G (Syntol, Russia) in 1 ml of buffer containing 150 mM NaCl and 10 mM NaHCO 3 (pH 8.0) for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Role of O-Glycosylation and Expression of CD43 and CD45 on the Surfaces of Effector T Cells in Human T Cell Leukemia Virus Type 1 Cell-to-Cell Infection

Mazurov

Ilinskaya

Heidecker

et al. 2012

J Virol

View full text Add to dashboard Cite

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies ( H uman T cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that causes two major diseases, adult T cell leukemia (ATL) (29) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (1, 51). Unlike HIV-1, which causes fatal immune deficiency-associated diseases in almost all infected individuals if left untreated, only about 5% of HTLV-1-infected people develop disease 10 to 25 years after the initial exposure. The remarkable feature of HTLV-1 is that transmission is 40% effective with blood lymphocytes but never with a patient's plasma (37). The phenomenon was explained by the extremely low infectivity of free viral particles (10, 13) and by the efficient cell-to-cell transmission of HTLV-1. Therefore, HTLV-1 can serve as an excellent model to study the cell-to-cell transmission of retroviruses in vitro. In 2003, Igakura et al. (20) described the formation of a microtubule-dependent cell-cell contact that was induced by HTLV-1. Based on structural and molecular similarity to the immunological synapse (IS), the cell-cell contact zone was named the virological synapse (VS). Later, the formation of the VS was demonstrated for human immunodeficiency virus (HIV) infection (7,21,22,35). Recently, a number of different and remarkable types of cell-cell interactions have been reported to mediate retroviral transmission, particularly nanotubes (43, 45), mono-or polysynapses (42), biofilm-like structures (39), and conduits (52). Nevertheless, our understanding of the mechanisms of viral transmission at the molecular level remains poor.We have recently complemented microscopy studies of cell-tocell transmission by developing inLuc and inYFP reporter vectors for HIV-1 and HTLV-1, which were designed to quantify cell-tocell infection (32). The signal generated by these vectors depends on the completion of a full cycle of virus replication. They are silent in producer cells but generate a readout in target cells. These vectors helped us to clarify the role of the viral protein Tax and different Env proteins in cell-to-cell infection and demonstrated that HTLV-1 is transmitted more efficiently in lymphoid cells than between nonlymphoid cells. In this study, we aimed to define cell surface antigens (Ags) possibly involved in the cell-to-cell infection of HTLV-1 and HIV-1. For that, we generated mouse monoclonal hybridomas against Jurkat cells and screened the monoclonal antibodies (MAbs) by inLuc infectivity assays. Finally, MAbs that decreased cell-to-cell infection were selected. Most of the HTLV-1-inhibitory MAbs recognized the carbohydrate Tn (T-nouvelle) antigen and precipitated the CD43 and CD45 proteins. We found that both the sialophorin CD43 and the phosphatase CD45 were important for HTLV-1 infection. These antigens are heavily O-glycosylated in normal cells, have a rodlike shape, and ext...

show abstract

Section: Methodsmentioning

confidence: 99%

Role of O-Glycosylation and Expression of CD43 and CD45 on the Surfaces of Effector T Cells in Human T Cell Leukemia Virus Type 1 Cell-to-Cell Infection

Mazurov

Ilinskaya

Heidecker

et al. 2012

J Virol

View full text Add to dashboard Cite

show abstract

“…The fluorescent immuno-precipitation assay can also be used to detect all proteins captured by an antibody, even though the method does not provide sequence information [29]. Sample proteins are labelled with fluorescent dyes.…”

Section: Can Monospecific Binders Be Made?mentioning

confidence: 99%

“…Immuno-precipitated proteins are resolved by SDS-PAGE and detected by scanning of the gel with a fluorescence imager (Fig. 1) [19,29]. With this assay, it is realistic to examine up to 200 immuno-precipitates per experiment assuming the use of two colors for sample labelling and a tank that can process 12 gels simultaneously.…”

Section: Can Monospecific Binders Be Made?mentioning

confidence: 99%

Antibody array analysis of labelled proteomes: how should we control specificity?

Holm

Lund-Johansen

2012

New Biotechnology

View full text Add to dashboard Cite

“…Immunoreactive bands were detected with Immobilon Western reagent (Millipore) using ChemiDoc XRS molecular imager (Bio-Rad). Fluorescent immunoprecipitation analysis (FIPA) combined with subsequent mass-spectrometry (MS) analysis was described earlier (Filatov et al, 2007). Briefly, 2x10 7 MT2 cells were pelleted, washed twice with PBS, and labeled with 0.3 mg of rhodamine 6G in 1 ml of buffer containing 150 mmol/l NaCl and 10 mmol/l NaHCO 3 (pH 8.0) for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells

Tarasevich¹,

Filatov²,

Пичугин³

et al. 2015

View full text Add to dashboard Cite

Summary. -Human T lymphotropic virus 1 (HTLV-1) is a pathogenic retrovirus that spreads predominantly via cell-to-cell contact. Two models of cell-to-cell virus transmission are proposed: virological synapse (VS) and viral biofilms (VB). Both infectious structures can be involved in transmission and synergistically enhance HTLV-1 spread between cells. Although transmission of virus via VB has been reported, the molecular composition of VB remains poorly understood. In this study we generated new anti-VB monoclonal antibodies (MAbs) and screened them along with a panel of anti-human cluster of differentiation (CD) MAbs to select antigens associated with VB. Among four MAbs generated against VB, two MAbs were identified as anti-CD25 (IL-2RA). We found that antigens CD4, CD150, CD25, CD70, and CD80 were enriched in VB. We also determined that expression of viral protein Tax, a central molecule in HTLV-1 transmission, upregulates intercellular adhesion molecule 1 (ICAM-1), CD95, CD25, CD70, and CD80. Whether these antigens are essential for VB formation and HTLV-1 infection remains unknown and will be determined in further experiments.Keywords: monoclonal antibody; CD antigen; HTLV-1; biofilms * Corresponding author. E-mail: dvmazurov@yandex.ru; phone: +74996128854. Abbreviations: CD = cluster of differentiation; HIV-1 = human immunodeficiency virus 1; HTLV-1 = human T cell lymphotropic virus 1; ICAM-1 = intercellular adhesion molecule 1; IL-2-RA = interleukin 2 receptor subunit A; IP = immunoprecipitation; MAb(s) = monoclonal antibody(ies); MS = mass-spectrometry; PE = phycoerythrin; TM4SF = transmembrane 4 superfamily proteins; VB = viral biofilms; VS = virological synapse; WB = Western blot; Wt = wild type

show abstract

Fluorescent immunoprecipitation analysis of cell surface proteins: A methodology compatible with mass-spectrometry

Cited by 30 publications

References 23 publications

Role of O-Glycosylation and Expression of CD43 and CD45 on the Surfaces of Effector T Cells in Human T Cell Leukemia Virus Type 1 Cell-to-Cell Infection

Role of O-Glycosylation and Expression of CD43 and CD45 on the Surfaces of Effector T Cells in Human T Cell Leukemia Virus Type 1 Cell-to-Cell Infection

Antibody array analysis of labelled proteomes: how should we control specificity?

Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells

Contact Info

Product

Resources

About