Reaction of 5-iodonaphthyl-1-nitrene with the IgE receptor on normal and tumour mast cells

Holowka, David; Gitler, Carlos; Bercovici, Tuvia; Metzger, Henry

doi:10.1038/289806a0

Cited by 41 publications

(22 citation statements)

References 14 publications

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“…This finding is consistent with the idea that a substantial portion of the INA-labeled T3 species lies within the plasma membrane, or on its cytoplasmatic side. A similar case was reported for the IgE receptor on mast cells [19]. There, a 50-kDa glycoprotein subunit was found to be associated with a 30-kDa, ['251]INA-labeled subunit.…”

Section: Discussionsupporting

confidence: 79%

The T3 complex on human thymus‐derived lymphocytes contains two different subunits of 20 kDa

1983

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The human cell surface antigen T3 is involved in several T lymphocyte specific functions, as determined by the effect of monoclonal antibodies (OKT3, anti-Leu-4, UCHT1) directed at this molecular structure. The main target antigen of these monoclonal antibodies is a glycoprotein of 20 kDa. It is associated with four, less predominant, structurally distinct glycoproteins of 25-28 kDa, 37 kDa and 44 kDa. Of these molecules only the 20-kDa T3 antigen could be labeled with the hydrophobic reagent 5-iodonaphthyl-1-azide (INA). Here we present evidence that the main 20-kDa T3 antigen is comprised of, in fact, two structurally different molecules. One of these is a glycoprotein with a protein backbone of 14 kDa, the other is an unglycosylated protein of 20 kDa. This unglycosylated protein is labeled specifically with INA. Additional evidence for the existence of two different 20-kDa T3 antigens is provided by studies using the enzymes endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F and the drug tunicamycin. We hypothesize that the specific susceptibility to labeling with INA of the unglycosylated 20-kDa T3 form reflects a positioning in the lipid bilayer different from that of the glycosylated 20-kDa T3 form.

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Section: Discussionsupporting

confidence: 79%

The T3 complex on human thymus‐derived lymphocytes contains two different subunits of 20 kDa

1983

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“…In addition, we wanted to see whether the receptor was associated with other cellular components either before or after aggregation. We used a variety of approaches: crosslinking with bifunctional reagents (37), labeling with a hydrophobic photoactivatable radiolabeled probe (38) and, perhaps most importantly, modifying the conditions by which the receptor was extracted from the cells (39). The last studies arose in part from our first attempts to re‐incorporate the IgE‐binding component into liposomes (40).…”

Section: Setting the Stagementioning

confidence: 99%

Molecular versatility of antibodies

Metzger¹

2002

Immunological Reviews

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As immunology developed into a discrete discipline, the principal experimental efforts were directed towards uncovering the molecular basis of the specificity exhibited by antibodies and the mechanism by which antigens induced their production. Less attention was given to how antibodies carry out some of their effector functions, although this subject presents an interesting protein-chemical and evolutionary problem; that is, how does a family of proteins that can bind a virtually infinite variety of ligands, many of which the species producing that protein has never encountered, reproducibly initiate an appropriate response? The experimental data persuasively suggested that aggregation of the antibody was a necessary and likely sufficient initiating event, but this only begged the question: how does aggregation induce a response? I used the IgE:mast cell system as a paradigm to investigate this subject. Data from our own group and from many others led to a molecular model that appears to explain how a cell 'senses' that antigen has reacted with the IgE. The model is directly applicable to one of the fundamental questions cited above, i.e. the mechanism by which antigens induce the production of antibodies. Although the model is conceptually simple, incorporating the actual molecular events into a quantitatively accurate scheme represents an enormous challenge.

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“…Finally, the iodine on this compound provides a very sensitive detection through its radioactive form. [ 125 I]-INA has been extensively used to exclusively label the portions of membrane proteins that are embedded within the lipid domain (6)(7)(8)(9)(10)(11)(12)(13). We applied hydrophobic labeling to the HIV-1 Env expressed on the virus or on the surface of cells to assess the topology of the Env glycoprotein.…”

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confidence: 99%

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

et al. 2008

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The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.The human immunodeficiency virus (HIV 1 ) gains entry into its target cell through a fusion process driven by its membrane protein gp41 (1). HIV-1 Env (gp120-gp41) is present on the † This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, by a grant from the NIH Intramural AIDS Targeted Antiviral Program (IATAP), and by a grant from the NIAID Intramural Biodefense Research Program. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. INA is composed of three moieties. The core of the compound is a naphthalene ring that confers a very high hydrophobicity. Its partition coefficient in biological membranes of ∼10 5 (5) ensures its selective and exclusive targeting to the lipidic bilayer. Its azido moiety allows its photoactivation. Upon irradiation with near UV light, the azido group undergoes transformation into a highly reactive nitrene that will covalently bind the proteins and lipids in its vicinity (6). Finally, the iodine on this compound provides a very sensitive detection through its radioactive form. [ 125 I]-INA has been extensively used to exclusively label the portions of membrane proteins that are embedded within the lipid domain (6-13). We applied hydrophobic labeling to the HIV-1 Env expressed on the virus or on the surface of cells to assess the topology of the Env glycoprotein. MATERIALS AND METHODS Reagents and CellsDulbecco's modified Eagle medium (DMEM), RPMI, fetal bovine serum (FBS), G418, hygromycin, and antibiotics were obtained from Invitrogen (Carlsbad, CA). 293T and HeLa cells were cultured in DMEM supplemented with 10% FBS and antibiotics (DMEM-10). Different HIV-1 Env were transiently expressed on the surface of HeLa cells using the recombinant vaccinia vectors vSC60 (IIIB/Lai BH8 Env) or vPE17 (14) (IIIB/Lai BH8 Env truncated at AA733). GHOST (3) X4/R5 (G345), a HOS...

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Reaction of 5-iodonaphthyl-1-nitrene with the IgE receptor on normal and tumour mast cells

Cited by 41 publications

References 14 publications

The T3 complex on human thymus‐derived lymphocytes contains two different subunits of 20 kDa

The T3 complex on human thymus‐derived lymphocytes contains two different subunits of 20 kDa

Molecular versatility of antibodies

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

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