scholarly journals Recruitment of Complement Factor H-Like Protein 1 Promotes Intracellular Invasion by Group A Streptococci

2003 ◽  
Vol 71 (12) ◽  
pp. 7119-7128 ◽  
Author(s):  
Vinod Pandiripally ◽  
Lin Wei ◽  
Christine Skerka ◽  
Peter F. Zipfel ◽  
David Cue
Keyword(s):  
Surface Plasmon Resonance ◽  
Epithelial Cells ◽  
Binding Site ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Coiled Coil ◽  
Factor H ◽  
Group A Streptococci ◽  
Coiled Coil Domain ◽  
Group A

ABSTRACT Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium, Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells by S. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba+ group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenic fba mutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.

scholarly journals Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Tina M. Cairns ◽  
Noah T. Ditto ◽  
Doina Atanasiu ◽  
Huan Lou ◽  
Benjamin D. Brooks ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Binding Site ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Geographical Area ◽  
Direct Interaction ◽  
Viral Envelope ◽  
Simplex Virus

ABSTRACTHerpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support of this, we have shown that gD is structurally divided into two “faces”: one for the binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when (i) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip, (ii) gD is bound to either one of its receptors on a chip, and (iii) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding, and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.IMPORTANCEHSV entry is a multistep process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, until now we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

Role of the mobility of antigen binding site in high affinity antibody elucidated by surface plasmon resonance

2016 ◽  
Vol 161 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Natsuki Fukuda ◽  
Yoshiaki Suwa ◽  
Makiyo Uchida ◽  
Yoshihiro Kobashigawa ◽  
Hideshi Yokoyama ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
High Affinity

Kinetic Study on the Formation of a de Novo Designed Heterodimeric Coiled-Coil:  Use of Surface Plasmon Resonance To Monitor the Association and Dissociation of Polypeptide Chains†

Biochemistry ◽  
1996 ◽  
Vol 35 (37) ◽  
pp. 12175-12185 ◽  
Author(s):  
Heman Chao ◽  
Michael E. Houston, ◽  
Suzanne Grothe ◽  
Cyril M. Kay ◽  
Maureen O'Connor-McCourt ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Kinetic Study ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
De Novo ◽  
Coiled Coil ◽  
Polypeptide Chains

Bivalent and co-operative binding of complement Factor H to heparan sulfate and heparin

2012 ◽  
Vol 444 (3) ◽  
pp. 417-428 ◽  
Author(s):  
Sanaullah Khan ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Barbara Mulloy ◽  
Stephen J. Perkins
Keyword(s):  
Surface Plasmon Resonance ◽  
Heparan Sulfate ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Inflammatory Diseases ◽  
Factor H ◽  
Radius Of Gyration ◽  
Complement Factor ◽  
X Ray ◽  
Kd Values

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant Kd of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH–heparin oligomers. The X-ray radius of gyration, RG, of FH in the presence of heparin fragments 18–36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with full-length FH gave Kd values of 1–3 μM, and similar but weaker Kd values of 4–20 μM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces.

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