scholarly journals Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

2022 ◽  
Vol 18 (1) ◽  
pp. e1010169
Author(s):  
Kwok-ho Lam ◽  
Jacqueline M. Tremblay ◽  
Kay Perry ◽  
Konstantin Ichtchenko ◽  
Charles B. Shoemaker ◽  
...  
Keyword(s):  
Single Domain ◽  
Protease Domain ◽  
High Potency ◽  
X Ray Crystallography ◽  
Biochemical Assays ◽  
Variable Heavy ◽  
Botulinum Neurotoxins ◽  
Single Domain Antibodies ◽  
Vhh Antibodies ◽  
And Function

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

scholarly journals A heterohexameric protein consisting of six linked single-domain antibodies is highly protective for BoNT/A, BoNT/B and BoNT/E exposures

2021 ◽  
Author(s):  
Jean Mukherjee ◽  
Jacqueline M. Tremblay ◽  
Michelle Debatis ◽  
Alexa Foss ◽  
Junya Awata ◽  
...  
Keyword(s):  
Single Domain ◽  
Host Cells ◽  
Mammalian Host ◽  
High Potency ◽  
Binding Properties ◽  
Small Pool ◽  
Single Domain Antibodies ◽  
Vhh Antibodies ◽  
High Doses ◽  
Target Binding

AbstractBotulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.

Using llama derived single domain antibodies to target botulinum neurotoxins

2010 ◽  
Author(s):  
Marla D. Swain ◽  
George P. Anderson ◽  
Rachael D. Bernstein ◽  
Jinny L. Liu ◽  
Ellen R. Goldman
Keyword(s):  
Single Domain ◽  
Botulinum Neurotoxins ◽  
Single Domain Antibodies

scholarly journals Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Obinna C. Ubah ◽  
Eric W. Lake ◽  
Gihan S. Gunaratne ◽  
Joseph P. Gallant ◽  
Marie Fernie ◽  
...  
Keyword(s):  
Phage Display Library ◽  
Crystallographic Analysis ◽  
Single Domain ◽  
Antigen Receptors ◽  
Protein Motifs ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Naturally Occurring ◽  
Single Domain Antibodies ◽  
Phylogenetic Lineages

AbstractSingle-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

X-Ray Crystallography of Botulinum Neurotoxins

2000 ◽  
Author(s):  
Martin Sax ◽  
W. Furey ◽  
S. Swaminathan
Keyword(s):  
X Ray ◽  
X Ray Crystallography ◽  
Botulinum Neurotoxins

Engineering of single-domain antibodies for next generation snakebite antivenoms

2021 ◽  
Author(s):  
Carla F.C. Fernandes ◽  
Soraya S. Pereira ◽  
Marcos B. Luiz ◽  
Nauanny K.R.L. Silva ◽  
Marcela Cristina da Silva ◽  
...  
Keyword(s):  
Single Domain ◽  
Next Generation ◽  
Single Domain Antibodies

scholarly journals PDGFRα: Expression and Function during Mitral Valve Morphogenesis

2021 ◽  
Vol 8 (3) ◽  
pp. 28
Author(s):  
Kelsey Moore ◽  
Diana Fulmer ◽  
Lilong Guo ◽  
Natalie Koren ◽  
Janiece Glover ◽  
...  
Keyword(s):  
Mitral Valve ◽  
Primary Cilia ◽  
Growth Factor Receptor ◽  
Akt Phosphorylation ◽  
Valve Development ◽  
Biochemical Assays ◽  
Factor Receptor Alpha ◽  
Mesenchymal Transformation ◽  
And Function

Mitral valve prolapse (MVP) is a common form of valve disease and can lead to serious secondary complications. The recent identification of MVP causal mutations in primary cilia-related genes has prompted the investigation of cilia-mediated mechanisms of disease inception. Here, we investigate the role of platelet-derived growth factor receptor-alpha (PDGFRα), a receptor known to be present on the primary cilium, during valve development using genetically modified mice, biochemical assays, and high-resolution microscopy. While PDGFRα is expressed throughout the ciliated valve interstitium early in development, its expression becomes restricted on the valve endocardium by birth and through adulthood. Conditional ablation of Pdgfra with Nfatc1-enhancer Cre led to significantly enlarged and hypercellular anterior leaflets with disrupted endothelial adhesions, activated ERK1/2, and a dysregulated extracellular matrix. In vitro culture experiments confirmed a role in suppressing ERK1/2 activation while promoting AKT phosphorylation. These data suggest that PDGFRα functions to suppress mesenchymal transformation and disease phenotypes by stabilizing the valve endocardium through an AKT/ERK pathway.

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