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1.
Front Cell Infect Microbiol ; 11: 664221, 2021.
Article in English | MEDLINE | ID: mdl-33854985

ABSTRACT

Human lymphocytes exposed to Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3ß. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3ß. Since non-phosphorylated GSK3ß is the active form of this kinase, we compared Cdts for dependence on GSK3ß activity. Two GSK3ß inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity (and presumably internalization of CdtB); CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt's molecular mode of action are discussed.


Subject(s)
Campylobacter jejuni , Haemophilus ducreyi , Bacterial Toxins , Humans , Phosphatidylinositols , Phosphoric Monoester Hydrolases , Polyphosphates
2.
Protein Sci ; 25(7): 1290-8, 2016 07.
Article in English | MEDLINE | ID: mdl-27334407

ABSTRACT

Engineering monovalent Fab fragments into bivalent formats like IgGs or F(ab')2 can lead to aggregation presumably because of nonspecific off-target interactions that induce aggregation. In an effort to further understand the molecular determinants of nonspecific interactions for engineered antibodies and natively folded proteins in general, we focused on a synthetic Fab with low nanomolar affinity to histone chaperone Anti-silencing factor 1 (Asf1) that demonstrates off-target binding through low solubility (∼5 mg/mL) in the multivalent F(ab') 2 state. Here, we generated phage display-based shotgun scanning libraries to introduce aspartate as a negative design element into the antibody paratope. The antibody-combining site was amenable to aspartate substitution at numerous positions within the antigen binding loops and one variant, Tyr(L93) Asp/His(L94) Asp/Thr(H100b) Asp, possessed high solubility (>100 mg/ml). Furthermore, the mutations decreased nonspecific interactions measured by column interaction chromatography and ELISA in the multivalent antibody format while maintaining high affinity to the antigen. Structural determination of the antibody-antigen complex revealed that the aspartate-permissive residues formed a polar ring around the structural and functional paratope, recapitulating the canonical feature of naturally occurring protein-protein interactions. This observation may inform future strategies for the design and engineering of molecular recognition.


Subject(s)
Amino Acid Substitution , Cell Cycle Proteins/metabolism , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Antibody Affinity , Aspartic Acid , Binding Sites, Antibody , Cell Cycle Proteins/chemistry , Cell Surface Display Techniques , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/chemistry , Models, Molecular , Molecular Chaperones , Protein Conformation , Protein Engineering
3.
J Immunol Methods ; 415: 24-30, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25450256

ABSTRACT

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (K(D) ~10 nM) for the Fc region of the IgG, but a much lower affinity (KD~low µM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 1,000-fold change in affinity from pH7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.


Subject(s)
Bacterial Proteins/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Amino Acid Sequence , Antibody Affinity , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Hydrogen-Ion Concentration , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Kinetics , Molecular Sequence Data , Mutation , Peptide Library , Protein Binding , Protein Engineering/methods , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology
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