Disulfide Bond Introduction for General Stabilization of Immunoglobulin Heavy-Chain Variable Domains

Saerens, Dirk; Conrath, Katja; Govaert, Jochen; Muyldermans, Serge

doi:10.1016/j.jmb.2008.01.022

Cited by 128 publications

(98 citation statements)

References 35 publications

(52 reference statements)

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“…Chemical and Thermal Stability-The GdmCl-induced and heat-induced unfolding of different VHHs was determined according to Dumoulin et al (19). Fluorescence at a single wavelength and the center of spectral mass of the intrinsic fluorescence emission spectra were used as chemical unfolding parameters (34). The intrinsic fluorescence of the protein at 25 g/ml in 50 mM sodium phosphate, pH 7.0 and variable concentration of GdmCl was monitored with the excitation wavelength at 280 nm, and emission spectra were recorded from 300 to 420 nm.…”

Section: Generation Of Codon-randomized Libraries and Selection Ofmentioning

confidence: 99%

“…This set of VHHs covers a broad range of CDR3 loop lengths, various locations of Cys in CDR1/CDR3 (Fig. 1), different loop structures (32,41), and a variety of thermal and chemical stabilities (31,34). Randomization of the extra Cys residues in cAbAn33, cAbPSA-N7, and cAbLys3 was performed by substituting the TGY codons with NNN.…”

Section: Replacing Amino Acids Forming Interloop Disulfide Bond Withimentioning

confidence: 99%

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Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

Govaert

Pellis

Deschacht

et al. 2012

Journal of Biological Chemistry

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Background:The presence of cystines connecting antigen-binding loops in single domain antibodies is puzzling. Results: Cysteines forming such cystine are substituted, and the performance of functional antibody fragments is determined. Conclusion: An interloop disulfide bond stabilizes the domain and rigidifies the long third antigen-binding loop, leading to stronger antigen interaction. Significance: This beneficial effect explains in vivo antibody maturation favoring antibodies with an interloop disulfide bond.

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Section: Generation Of Codon-randomized Libraries and Selection Ofmentioning

confidence: 99%

Section: Replacing Amino Acids Forming Interloop Disulfide Bond Withimentioning

confidence: 99%

Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

Govaert

Pellis

Deschacht

et al. 2012

Journal of Biological Chemistry

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124

100

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“…The same inhibition was observed for cAb-HuL6 and the I56T variant [82], and for cAb-HuL22 and both the I56T and D67H variants [44]. On the other hand, these yellow peaks are observed in the mass spectra of the D67H variant in the presence of cAb-HuL5; the binding of cAb-HuL5 does therefore not inhibit the was added between the two b-sheets thanks to the S54C and I78C mutations (IMGT numbering) [70,71]. The variant obtained (named cAb-HuL22-S54C/I78C) is significantly more thermostable (i.e.…”

Section: Human Lysozyme and Systemic Amyloidosismentioning

confidence: 53%

“…The function of conventional antibodies or antibody fragments inside the cell is generally compromised due to the inability of the disulphide bonds to form in the reducing environment of the cytoplasmic environment and thus the inability of the protein to fold or remain folded in its native state. Due to the high stability of nanobodies [37], their conserved disulphide bridge can in many cases be deleted without significantly affecting their functionality [71] and nanobodies fold well into functional entities in the reducing intracellular environment [172]. Given this intracellular robustness, combined to the easiness with which they can be fused to signal peptides to direct them to specific cellular compartments, nanobodies have been used for a variety of purposes in cells including imaging [172e174].…”

Section: Prospects For Diagnostic and Therapeutic Applicationsmentioning

confidence: 99%

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

2015

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Keywords:Variable domain of heavy-chain antibody Inhibition of protein misfolding and aggregation Protein misfolding diseases Amyloidoses V H H Nanobody a b s t r a c tThe deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how V H Hs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, b2-microglobulin, a-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid b-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated with protein misfolding and aggregation.© 2015 Elsevier B.V. and Societe Française de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.Abbreviations: Ab, antibody; Ab, amyloid b-peptide; AD, Alzheimer's disease; ANS, anilino naphthalene-sulfonic acid; AP, alkaline phosphatase; APP, amyloid precursor protein; aSyn, a-synuclein; b2m, b2-microglobulin; BBB, bloodebrain barrier; CDR, complementarity determining region; C m , concentration of mid-denaturation; CR, Congo red; CSF, cerebrospinal fluid; DN6b2m, a truncated form of b2m lacking the six N-terminal amino acids; DLS, dynamic light scattering; DRA, dialysis-related amyloidosis; FR, framework; FTIR, Fourier transform infrared spectroscopy; Fv, variable fragment made of the VH and VL domains of conventional antibodies; GFP, green fluorescent protein; HCAb, heavy-chain antibody; H/D, hydrogen/deuterium; HSQC, heteronuclear s...

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“…The introduction of cysteines at position 54 and 78 result in an extra stabilizing disulfide bond at the buried hydrophobic region. 72,73 These proteolysis-resistant platforms can be utilized in oral immunotherapy. Trypsinresistant Nbs for oral delivery have been obtained after random mutagenesis through DNA shuffling and selection for trypsin-resistant variants.…”

Section: Therapeutic Applications Safety and Tolerabilitymentioning

confidence: 99%

Nanobodies as novel agents for disease diagnosis and therapy

Siontorou¹

2013

IJN

123

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Disulfide Bond Introduction for General Stabilization of Immunoglobulin Heavy-Chain Variable Domains

Cited by 128 publications

References 35 publications

Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

Nanobodies as novel agents for disease diagnosis and therapy

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