Residues in domain III of the dengue virus envelope glycoprotein involved in cell-surface glycosaminoglycan binding

The dengue virus (DENV) envelope (E) protein mediates virus entry into cells via interaction with a range of cell-surface receptor molecules. Cell-surface glycosaminoglycans (GAGs) have been shown to play an early role in this interaction, and charged oligosaccharides such as heparin bind to the E protein. We have examined this interaction using site-directed mutagenesis of a recombinant form of the putative receptor-binding domain III of the DENV-2E protein expressed as an MBP (maltose-binding protein)-fusion protein. Using an ELISA-based GAG-binding assay, cell-based binding analysis and antiviral-activity assays, we have identified two critical residues, K291 and K295, that are involved in GAG interactions. These studies have also demonstrated differential binding between mosquito and human cells.

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Role of Asparagine-Linked Glycosylation in Cell Surface Expression and Function of the Human Adrenocorticotropin Receptor (Melanocortin 2 Receptor) in 293/FRT Cells

Endocrinology ◽

10.1210/en.2009-0826 ◽

2010 ◽

Vol 151 (2) ◽

pp. 660-670 ◽

Cited By ~ 27

Author(s):

Simon Roy ◽

Benoît Perron ◽

Nicole Gallo-Payet

Keyword(s):

Cell Surface ◽

Fold Increase ◽

Surface Expression ◽

Site Directed Mutagenesis ◽

Cell Surface Receptor ◽

Cell Surface Expression ◽

Camp Production ◽

Glycosylation Sites ◽

Surface Receptor

Asparagine-linked glycosylation (N-glycosylation) of G protein-coupled receptors may be necessary for functions ranging from agonist binding, folding, maturation, stability, and internalization. Human melanocortin 2 receptor (MC2R) possesses putative N-glycosylation sites in its N-terminal extracellular domain; however, to date, the role of MC2R N-glycosylation has yet to be investigated. The objective of the present study is to examine whether N-glycosylation is essential or not for cell surface expression and cAMP production in native and MC2R accessory protein (MRAPα, -β, or -dCT)-expressing cells using 293/FRT transfected with Myc-MC2R. Western blot analyses performed with or without endoglycosidase H, peptide:N-glycosidase F or tunicamycin treatments and site-directed mutagenesis revealed that MC2R was glycosylated in the N-terminal domain at its two putative N-glycosylation sites (Asn12-Asn13-Thr14 and Asn17-Asn18-Ser19). In the absence of human MRAP coexpression, N-glycosylation of at least one of the two sites was necessary for MC2R cell surface expression. However, when MRAP was present, cell surface expression of MC2R mutants was either rescued entirely with the N17-18Q (QQNN) and N12-13Q (NNQQ) mutants or partially with the unglycosylated N12-13, 17-18Q (QQQQ) mutant. Functional and expression analyses revealed a discrepancy between wild-type (WT) and QQQQ cell surface receptor levels and maximal cAMP production with a 4-fold increase in EC50 values. Taken together, these results indicate that the absence of MC2R N-glycosylation abrogates to a large extent MC2R cell surface expression in the absence of MRAPs, whereas when MC2R is N-glycosylated, it can be expressed at the plasma membrane without MRAP assistance.

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Mutation analysis of the cross-reactive epitopes of Japanese encephalitis virus envelope glycoprotein

Journal of General Virology ◽

10.1099/vir.0.040238-0 ◽

2012 ◽

Vol 93 (6) ◽

pp. 1185-1192 ◽

Cited By ~ 21

Author(s):

Shyan-Song Chiou ◽

Yi-Chin Fan ◽

Wayne D. Crill ◽

Ruey-Yi Chang ◽

Gwong-Jen J. Chang

Keyword(s):

Amino Acid ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Recombinant Expression ◽

Encephalitis Virus ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Virus Envelope ◽

E Protein ◽

Domain Iii

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.

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Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host

Microbiology ◽

10.1099/mic.0.028795-0 ◽

2009 ◽

Vol 155 (10) ◽

pp. 3294-3303 ◽

Cited By ~ 70

Author(s):

Marco Candela ◽

Elena Biagi ◽

Manuela Centanni ◽

Silvia Turroni ◽

Manuela Vici ◽

...

Keyword(s):

Cell Surface ◽

Glycolytic Enzyme ◽

Site Directed Mutagenesis ◽

Cell Surface Receptor ◽

Surface Receptor ◽

A Cell ◽

Human Plasminogen ◽

Equilibrium Dissociation ◽

Nanomolar Range ◽

Positively Charged

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.

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The Combination of Type I IFN, TNF-α, and Cell Surface Receptor Engagement with Dendritic Cells Enables NK Cells To Overcome Immune Evasion by Dengue Virus

The Journal of Immunology ◽

10.4049/jimmunol.1302240 ◽

2014 ◽

Vol 193 (10) ◽

pp. 5065-5075 ◽

Cited By ~ 17

Author(s):

Daniel Say Liang Lim ◽

Nobuyo Yawata ◽

Kevin John Selva ◽

Na Li ◽

Chen Yu Tsai ◽

...

Keyword(s):

Dendritic Cells ◽

Cell Surface ◽

Dengue Virus ◽

Nk Cells ◽

Immune Evasion ◽

Cell Surface Receptor ◽

Type I ◽

Type I Ifn ◽

Tnf Α ◽

Surface Receptor

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SARS-COV2 Envelope Protein (E) interacts with the Lysophosphatidic Acid Receptor 1 (LPAR1) from humans

10.21203/rs.3.rs-42592/v1 ◽

2020 ◽

Author(s):

GIRISH NALLUR

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Protein Interactions ◽

Biological Significance ◽

Cell Surface Receptor ◽

Proteomic Screen ◽

E Protein ◽

Cell Surface Staining ◽

Surface Receptor ◽

Strong Candidate

Abstract A proteomic screen of human proteins interacting with the SARS-COV2 Envelope (E) protein identified LPAR1 as a strong candidate. Physical association of E protein and LPAR1 was confirmed by co-immunoprecipitation and cell surface staining. LPAR1-E protein interaction was confirmed in all eight human cell lines tested. Many additional proteins participating in the E protein interactions network were also enriched from each of the cell lines, some of which were cell type specific. These findings suggest that LPAR1 is likely a cell surface receptor for the E protein, and pave the way for follow-on studies aimed at understanding the biological significance of the interactions in SARS-COV disease, including the signaling mechanisms.

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A Functional Epitope Determinant on Domain III of the Japanese Encephalitis Virus Envelope Protein Interacted with Neutralizing-Antibody Combining Sites

Journal of Virology ◽

10.1128/jvi.77.4.2600-2606.2003 ◽

2003 ◽

Vol 77 (4) ◽

pp. 2600-2606 ◽

Cited By ~ 68

Author(s):

Cheng-Wen Lin ◽

Suh-Chin Wu

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Spatial Configuration ◽

Neutralizing Antibody ◽

Encephalitis Virus ◽

Molecular Structures ◽

Site Directed Mutagenesis ◽

Virus Envelope ◽

E Protein ◽

Domain Iii

ABSTRACT The envelope (E) protein of Japanese encephalitis virus (JEV) is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing-antibody responses in hosts. Most previous studies have not provided detailed molecular information about the spatial configuration of the functional epitopes on domain III of the E protein. Here site-directed mutagenesis was performed to demonstrate that the functional epitope determinants at Ser331 and Asp332 on domain III of the JEV E protein interacted with neutralizing monoclonal antibody (MAb) E3.3. Bacterial expression of the recombinant Fab E3.3 confirmed the molecular interactions of Arg94 in complementary determining region H3 with Ser331 and Asp332 on domain III. This study elucidates the detailed molecular structures of the neutralizing epitope determinants on JEV domain III, which can provide useful information for designing new vaccines.

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Structure-function studies on human retinol-binding protein using site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3000437 ◽

1994 ◽

Vol 300 (2) ◽

pp. 437-442 ◽

Cited By ~ 24

Author(s):

A Sivaprasadarao ◽

J B Findlay

Keyword(s):

Cell Surface ◽

Specific Binding ◽

Binding Protein ◽

Specific Interaction ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Directed Mutagenesis ◽

Surface Receptor

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

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