Characterization of the Structural Features and Interactions of Sclerostin

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Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1␤. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals.The ability of antibodies to bind to an almost unlimited number of target proteins with high specificity makes them one of the fastest growing classes of therapeutics in the biological drugs market (1). Since the first description of monoclonal antibodies (2), dramatic progress has been made in the expression, engineering, humanization, and applications of antibodies as therapeutics. A wide variety of antibody fragments have been evaluated as potential therapeutics including the well characterized antigen binding fragment (Fab), which contains the light chain (V L and C L domains) and N-terminal portion of the heavy chain (V H and C H domains). The smallest fragment to retain full binding activity has also attracted considerable interest, with the so-called single chain variable fragment (scFv) 3 (3) consisting of the two variable domains joined by a short peptide.A detailed understanding of the interactions between candidate therapeutic antibodies and target proteins is key to further progress in rational design and humanization. Currently, identification of the binding sites for antibodies on target proteins is achieved via one or a combination of indirect methods such as protease protection, peptide scanning, site-directed mutagenesis, or analysis of backbone amide exchange (4 -6). Although providing valuable information, each of these approaches has drawbacks; in particular, they may not detect discontinuous epitopes and do not provide information on the spatial organization of ep...

Section: Discussionmentioning

confidence: 94%

High Resolution NMR-based Model for the Structure of a scFv-IL-1β Complex

Wilkinson

Hall

Veverka

et al. 2009

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“…Furthermore, discordant correlations between the levels of mRNA and protein have been observed (52)(53)(54), and the correlations between mRNA and secreted protein levels for various human genes are low (52,55). Because the primary structure of sclerostin is known to contain a positively charged basic region (32), it is likely that this region strongly interacts with negatively charged proteoglycans present in dHOB multilayer cultures (56). For this reason we measured the level of sclerostin extracted from the dHOB multilayer.…”

Section: Discussionmentioning

confidence: 99%

“…Solubilization of Mineralized HOB Multilayers for Protein Detection-Because sclerostin has been reported to have a basic region, which causes it to bind to bone matrix proteins (32), extraction from the multilayers was tested as well as ELISA, because secreted sclerostin levels might be expected to be low. HOBs were grown to confluence on Thermanox TM as described previously, and subsequently grown for a further 7, 14, or 28 days in mineralization medium in the presence or absence of strontium.…”

Section: Hob Culturementioning

confidence: 99%

An Akt-dependent Increase in Canonical Wnt Signaling and a Decrease in Sclerostin Protein Levels Are Involved in Strontium Ranelate-induced Osteogenic Effects in Human Osteoblasts

Rybchyn

Slater

Conigrave

et al. 2011

101

Sclerostin is an important regulator of bone homeostasis and canonical Wnt signaling is a key regulator of osteogenesis. Strontium ranelate is a treatment for osteoporosis that has been shown to reduce fracture risk, in part, by increasing bone formation. Here we show that exposure of human osteoblasts in primary culture to strontium increased mineralization and decreased the expression of sclerostin, an osteocyte-specific secreted protein that acts as a negative regulator of bone formation by inhibiting canonical Wnt signaling. Strontium also activated, in an apparently separate process, an Akt-dependent signaling cascade via the calcium-sensing receptor that promoted the nuclear translocation of ␤-catenin. We propose that two discrete pathways linked to canonical Wnt signaling contribute to strontium-induced osteogenic effects in osteoblasts.Strontium ranelate is a treatment for osteoporosis that decreases the incidence of vertebral and femoral fracture risk in postmenopausal women (1-3). Strontium ranelate has been shown to modulate the physiological processes of bone formation and bone resorption (2), resulting in increased bone apposition rates (3) and bone mineral density (1, 2, 4 -6), while maintaining the quality of bone mineral (6).Sclerostin is exclusively expressed by osteocytes in adult life (7, 8) but is more widely expressed during development (7) and plays a physiological role as a negative regulator of bone formation by repressing bone morphogenic protein-induced osteogenesis (9 -11). The importance of sclerostin in bone-loss disorders has been described in several in vivo studies. Positional cloning studies identified loss of function mutations in the SOST gene that cause sclerostosis and van Buchem disease, bone dysplasias characterized by progressive skeletal overgrowth (12). In contrast, transgenic mice overexpressing sclerostin had significant reductions in bone mass and mineral apposition rate compared with wild type (7). A sclerostin knock-out mouse model was shown to have increased osteoblast activity and enhanced osteoblast/osteocyte viability and was resistant to mechanical unloading-induced bone loss. This phenotype was associated with an increase in canonical Wnt signaling when compared with wild-type mice (8).Sclerostin functions as an antagonist of canonical Wnt signaling, whereby GSK-3␤ 2 -stimulated, ubiquitin-mediated breakdown of ␤-catenin is alleviated, resulting in its nuclear translocation, and binding to transcription factors of the T-cell factor/lymphoid enhancer factor family, to induce the transcription of growth-associated genes (13). Non-canonical Wnt signaling does not involve ␤-catenin translocation to the nucleus (11). Sclerostin binds to the extracellular domains of the Wnt co-receptors LRP5, LRP6, and LRP4 and disrupts extracellular Wnt-induced Frizzled/LRP complex formation thus providing a molecular mechanism by which loss of sclerostin function may lead to conditions such as sclerostosis (12,14). In addition to Frizzled/LRP-mediated activation of canonical Wnt s...

“…Chemical Shift-based Mapping of Interaction Sites-Backbone chemical shift ( 15 N, 13 C, and 1 H) comparisons between free and bound antibody fragments and antigens were made using the minimal shift method (7)(8)(9) and by direct comparison of assigned backbone signals for the free and bound proteins where available. For the anti-IL-1␤ Fab, peaks from the comprehensively assigned HNCO spectrum of the free protein were compared with the unassigned HNCO spectrum of the Fab⅐IL-1␤ complex.…”

Section: Methodsmentioning

confidence: 99%

Conformational Heterogeneity in Antibody-Protein Antigen Recognition

Addis

Hall

Bruton

et al. 2014

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Background: Antibodies are essential components of the immune system which recognize specific antigens with high affinity. Results: Protein antigen binding sites on antibodies show conformational exchange on a millisecond to second timescale. Conclusion: Conformational heterogeneity at high affinity protein-protein interaction sites may be common and facilitate efficient protein complex formation. Significance: High affinity protein-protein interactions are critical for many biological processes.