GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.

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Exosome-Driven Antigen Transfer for MHC Class II Presentation Facilitated by the Receptor Binding Activity of Influenza Hemagglutinin

The Journal of Immunology ◽

10.4049/jimmunol.1001768 ◽

2010 ◽

Vol 185 (11) ◽

pp. 6608-6616 ◽

Cited By ~ 37

Author(s):

James S. Testa ◽

Geraud S. Apcher ◽

Joseph D. Comber ◽

Laurence C. Eisenlohr

Keyword(s):

Mhc Class Ii ◽

Receptor Binding ◽

Class Ii ◽

Binding Activity ◽

Influenza Hemagglutinin ◽

Receptor Binding Activity ◽

Antigen Transfer

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A Mutant Influenza Virus That Uses an N1 Neuraminidase as the Receptor-Binding Protein

Journal of Virology ◽

10.1128/jvi.01889-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 12531-12540 ◽

Cited By ~ 52

Author(s):

Kathryn A. Hooper ◽

Jesse D. Bloom

Keyword(s):

Influenza Virus ◽

Receptor Binding ◽

Influenza A ◽

Binding Pocket ◽

Binding Activity ◽

Dependent Manner ◽

Influenza A Viruses ◽

Cell Agglutination ◽

Binding Function ◽

Receptor Binding Activity

In the vast majority of influenza A viruses characterized to date, hemagglutinin (HA) is the receptor-binding and fusion protein, whereas neuraminidase (NA) is a receptor-cleaving protein that facilitates viral release but is expendable for entry. However, the NAs of some recent human H3N2 isolates have acquired receptor-binding activity via the mutation D151G, although these isolates also appear to retain the ability to bind receptors via HA. We report here the laboratory generation of a mutation (G147R) that enables an N1 NA to completely co-opt the receptor-binding function normally performed by HA. Viruses with this mutant NA grow to high titers even in the presence of extensive mutations to conserved residues in HA's receptor-binding pocket. When the receptor-binding NA is paired with this binding-deficient HA, viral infectivity and red blood cell agglutination are blocked by NA inhibitors. Furthermore, virus-like particles expressing only the receptor-binding NA agglutinate red blood cells in an NA-dependent manner. Although the G147R NA receptor-binding mutant virus that we characterize is a laboratory creation, this same mutation is found in several natural clusters of H1N1 and H5N1 viruses. Our results demonstrate that, at least in tissue culture, influenza virus receptor-binding activity can be entirely shifted from HA to NA.

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Chemical deglycosylation of ovine pituitary lutropin. A study of the reaction conditions and effects on biochemical, biophysical and biological properties of the hormone

Biochemical Journal ◽

10.1042/bj2070011 ◽

1982 ◽

Vol 207 (1) ◽

pp. 11-19 ◽

Cited By ~ 16

Author(s):

P. Manjunath ◽

M. R. Sairam ◽

P. W. Schiller

Keyword(s):

Receptor Binding ◽

Conformational Changes ◽

Concanavalin A ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Binding Activity ◽

Glycoprotein Hormone ◽

Reaction Conditions ◽

Receptor Binding Activity

The oligomeric glycoprotein hormone, ovine lutropin was treated with anhydrous HF at 0°C for 30, 60 and 180 min and at 23°C for 60 and 180 min. The products, designated deglycosylated lutropin 1 (DGLH-1) to deglycosylated lutropin 5 (DGLH-5) respectively, were characterized by gel filtration, concanavalin A–Sepharose binding, disc electrophoresis, amino acid analysis, carbohydrate composition and spectral properties. The preparations were also evaluated for receptor binding activity and immunological activity and bioassayed in vitro in collagenase-dispersed rat interstitial cells. In DGLH-1, fucose and galactosamine were removed completely, and there was a 94% decrease in hexoses and 39% decrease in N-acetylglucosamine. Reaction with HF at 0°C for 1 or 3h led to removal of all hexoses and additional loss of hexosamines. Reactions at 23°C for either 1 or 3h were not of additional value in deglycosylation and none of the reaction conditions yielded the apohormone. All the five deglycosylated hormone preparations were not retained on immobilized-concanavalin A columns and on Sephadex G-100 they were eluted with an increased Ve/V0 ratio consistent with the loss of carbohydrate residues. Loss of all but the last of the N-acetylglucosamine residues decreased the abnormality of lutropin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but did not eliminate it. Receptor binding activities of DGLH-1 and DGLH-2 were not different from that of the native hormone, but that of DGLH-3 was slightly decreased and the products obtained at 23°C (DGLH-4 and DGLH-5) had lower activity. Immunoreactivities followed a similar pattern. None of the derivatives had activity in the bioassay in vitro. All of the five derivatives inhibited the action of the native hormone in the bioassay in vitro. Their hormonal antagonistic activity was consistent with the receptor binding activity, with DGLH-5 being the least potent in this respect. The DGLH-4 and DGLH-5 preparations had undergone conformational changes as revealed by 8-anilinonaphthalene-1-sulphonate fluorescence, but this did not result in loss of quaternary structure.

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