The testosterone binding mechanism of an antibody derived from a naïve human scFv library

Niemi, Merja; Takkinen, Kristiina; Amundsen, Lotta K.; Rouvinen, Juha; Höyhtyä, Matti

doi:10.1002/jmr.1039

Cited by 12 publications

(7 citation statements)

References 38 publications

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“…Sequences in VH regions were compared with the sequences from cDNA encoding monoclonal IgG antibodies specific for Gd-IgA1. 6 Chimera Homology Modeling PDB ID 3KDM 30 was chosen as the homologymodeling template after NCBI BLAST against the PDB databank of full-length aa sequence of IgG 1123 VH indicated 86% identity to that of 3KDM. Chimeric aa sequences for the VH CDR3 sequences from a patient with IgAN and a healthy control were made by using the corresponding VH CDR3 from the patient's and the control's IgG VH regions (Supplemental Table 1).…”

Section: Statistical Analysesmentioning

confidence: 99%

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy

Huang

Raška

Stewart

et al. 2016

JASN

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Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, YCS, in the complementarity-determining region 3 of the heavy chain variable region compared with a YCA sequence in similar isotype-matched IgG from healthy controls. We previously found that the S residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the YC(A/V) amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent β-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

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Section: Statistical Analysesmentioning

confidence: 99%

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy

Huang

Raška

Stewart

et al. 2016

JASN

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“…Data were indexed and scaled to 2.8 Å resolution using HKL2000 [19]. The structure was solved by the molecular replacement method using Balbes [17] automatically using 2JB5 [20] for light chain, 3KDM [21] for heavy chain, 1B5L [22] for IFNα1b as search models. The initial phases were improved with OASIS [23].…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

Structural insights into a human anti-IFN antibody exerting therapeutic potential for systemic lupus erythematosus

Ouyang

Gong

Li³

et al. 2012

J Mol Med

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Increasing evidences suggest that the type I interferon α (IFN α) plays a critical role in the etiopathogenesis of systemic lupus erythematosus (SLE), which makes it a promising therapeutic target for the treatment of the disease. By screening a large size non-immune human antibody library, we have developed a human single-chain antibody (ScFv) AIFN α 1bScFv01 and corresponding whole antibody AIFN α 1bIgG01 to human interferon α 1b (IFN α 1b) with high specificity and high affinity. The IgG antibody could down-regulate the expression of ISG15 and IFIT-1 induced by either recombinant IFN α 1b or naïve IFN α from SLE patients' sera, and reduced total serum IgG and IgM antibodies level in a pristane-primed lupus-like mouse model. The crystal structure of AIFN α 1bScFv01-IFN α 1b complex solved to 2.8 Å resolution revealed that both Pro26-Gln40 region in loop AB and Glu147-Arg150 region in helix E of IFN α 1b contribute to binding with AIFN α 1bScFv01. Four residues of above two regions (Leu30, Asp32, Asp35 and Arg150) are critical for the formation of antigen-antibody complexes. AIFN α 1bScFv01 shares partial epitopes of IFN α 1b with its receptor IFNAR2 but with much higher binding affinity to IFN α 1b than IFNAR2. Thus, AIFN α 1bIgG01 exhibits its neutralizing activity through competition with IFNAR2 to bind with IFN α and prevents the activation of IFN α-mediated signaling pathway. Our results highlight the potential use of the human antibody for modulating the activity of IFN α in SLE.

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“…This phenomenon can be summarized as deep binding pockets for anti-hapten antibodies, and binding grooves for linear peptide antibodies, or antibodies that bind proteins possessing relatively flat binding sites [ 20 ]. The development of cavity-like binding sites that recognize haptens has been reported for a number of species including: human [ 21 ], mouse [ 22 ], llama [ 23 ], and rabbit [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

Qaraghuli

Palliyil

Broadbent³

et al. 2015

BMC Biotechnol

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BackgroundLow molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes.MethodsDifferent hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential.ResultsThe simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL.ConclusionsThe best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

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The testosterone binding mechanism of an antibody derived from a naïve human scFv library

Cited by 12 publications

References 38 publications

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy

Structural insights into a human anti-IFN antibody exerting therapeutic potential for systemic lupus erythematosus

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

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