Multivalent sialic acid binding proteins as novel therapeutics for influenza and parainfluenza infection

Carrier proteins;Multivalent molecules;Sialic acids;Influenza viruses--Antagonists;Parainfluenza viruses--Antagonists

Alias, Nadiawati ; Taylor, Garry L. ; Taylor, Garry L.

University:University of St Andrews

Department:Biology (School of)

关键词: Carrier proteins;    Multivalent molecules;    Sialic acids;    Influenza viruses--Antagonists;    Parainfluenza viruses--Antagonists;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/4479/NadiawatiAliasPhDThesis.pdf?sequence=3&isAllowed=y

来源: DR-NTU

【 摘 要 】

In nature, proteins with weak binding affinity often use a multivalency approach toenhance protein affinity via an avidity effect. Interested in this multivalency approach,we have isolated a carbohydrate binding module (CBM) that recognises sialic acid(known as a CBM40 domain) from both Vibrio cholerae (Vc) and Streptococcuspneumoniae (Sp) NanA sialidases, and generated multivalent polypeptides from themusing molecular biology. Multivalent CBM40 constructs were designed either using atandem repeat approach to produce trimeric or tetrameric forms that we call Vc3CBMand Vc4CBM, respectively, or through the addition of a trimerization domain derivedfrom Pseudomonas aeruginosa pseudaminidase to produce three trimeric forms ofproteins known as Vc-CBMTD (WT), Vc-CBMTD (Mutant) and Sp-CBMTD). Due tothe position and flexibility of the linker between the trimerization domain and theCBM40 domain, site directed mutagenesis was employed to introduce a disulphidebond between the monomers at positions S164C and T83C of the CBM40 domain inorder to promote a stable orientation of the binding site for easier access of sialicacids.Data from isothermal titration calorimetry (ITC) reveals that interaction of multivalentCBM40 proteins with α(2,3)-sialyllactose was mainly enthalpy driven with entropycontributing unfavorably to the interaction suggesting that these proteins establish astrong binding affinity to their ligand minimizing dissociation to produce stablemultivalent molecules. However, using surface plasmon resonance (SPR), a mixedbalance of entropy and enthalpy contributions was found with all constructs asdetermined by Van’t Hoff plots. This proved that binding does not occur through asimple protein-ligand interaction but through disruption of hydrophobic and/or ionichydration that provide the driving force to the process. Interestingly, the valency of multiple-linked polypeptides also plays an important part in the protein stabilization.However, little is known about their detailed structure when in multivalent form, asattempts to crystallize the whole protein molecule of Vc-CBMTD (WT) failed due tolinker and domain flexibility. Only the trimerization domain (TD) part fromPseudomonas aeruginosa pseudaminidase was successfully crystallized andstructure was determined to 3.0 Å without its CBM40 domain attached.In this thesis, we have also reported on the potential anti-influenza and anti-parainfluenza properties of these proteins, which were found to block attachment andinhibit infection of several influenza A and parainfluenza virus strains in vitro. Aswidely mentioned in literature, terminal sialic acids on the cell surface of mammalianhost tissue provide a target for various pathogenic organisms to bind. Levels of viralinhibition were greatest against A/Udorn/72 H3N2 virus for Vc4CBM and Vc3CBMconstructs with the lowest EC50 of 0.59 µM and 0.94 µM respectively, however most ofthe multivalent proteins tested were also effective against A/WSN/33 H1N1 andA/PR8/34 H1N1 subtypes. For parainfluenza virus, all constructs containing V.cholerae sialidase CBM40 domain showed great effect in inhibiting virus infectionduring cell protection assay. The best EC50 values were 0.2 µM from Vc-CBMTD (WT)followed by 1.17 µM from Vc4CBM and 1.78 µM from Vc-CBMTD (Mutant) which wasagainst hPIV2, hPIV3 and hPIV5 infections respectively. Only a construct from S.pneumoniae sialidase known as Sp-CBMTD showed negligible effect on cellprotection. All constructs were further tested for cytotoxicity in mammalian cell cultureas well as undergoing an inhibition study on viral replication proteins. For the in vivostudy, we also demonstrated the effectiveness of Vc4CBM to protect cotton rats andmice from hPIV3 and Streptococcus pneumoniae infections, when given intranasallyin advance or on the day of infection. Therefore, these novel multivalent proteinscould be promising candidates as broad-spectrum inhibitors or as a prophylactictreatment for both influenza and parainfluenza associated diseases.