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1: JAG1 antibody characterization. 2: antibody inhibition of JAG1 mediated Notch signalling in tumor cell lines. 3: antibody inhibition of JAG1 signalling in vascular cells. 4: antibody inhibition of JAG1 signalling in HUVEC-HUVSMC co-culture. 5: effect of anti-Notch pathway treatment on MDA-MB231 2D growth and J1-65D titration and effect on 3D growth and gene expression. 6: J1-65D treatment of established OVCAR3 xenograft. 7: JAG1 antibodies cross-reactivity with rat Jag1. 8: anti-JAG1 treatment effect in a rat brain metastasis model.

Supplementary material and methods. Supplementary table 1: cell lines and growth conditions. Supplementary table 2: Notch ligands and control protein for plate coating. Supplementary table 3: qPCR primers. Supplementary table 4: flow cytometry antibodies. Supplementary table 5: ICC/IHC antibodies. Supplementary table 6: titration of Notch1-JAG1 binding inhibition by JAG1 antibodies. Supplementary table 7: amino acid sequence of JAG1 antibodies. Supplementary table 8: blood analysis on antibody-treated tumor-bearing rats.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Recent data have expanded our understanding of Notch signaling by identifying a C2 domain at the N-terminus of Notch ligands which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and DLL4 and human Notch-2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signaling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations which affect these loops and are associated with extrahepatic biliary atresia lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signaling in different physiological contexts.

Background: The site of Jagged/Serrate ligand recognition by Notch is unknown. Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central β-sheet of EGF12 form a ligand-binding platform. Conclusion: The ligand-binding region is adjacent to a Fringe-sensitive residue involved in modulating Notch activity. Significance: The results have implications for understanding receptor/ligand recognition, Notch regulation by O-glycosylation, and the development of paralogue-specific antibodies., We have mapped a Jagged/Serrate-binding site to specific residues within the 12th EGF domain of human and Drosophila Notch. Two critical residues, involved in a hydrophobic interaction, provide a ligand-binding platform and are adjacent to a Fringe-sensitive residue that modulates Notch activity. Our data suggest that small variations within the binding site fine-tune ligand specificity, which may explain the observed sequence heterogeneity in mammalian Notch paralogues, and should allow the development of paralogue-specific ligand-blocking antibodies. As a proof of principle, we have generated a Notch-1-specific monoclonal antibody that blocks binding, thus paving the way for antibody tools for research and therapeutic applications.

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Fibrillin-1 is a 350 kDa calcium-binding protein which assembles to form 10-12 nm microfibrils in the extracellular matrix (ECM). The structure of fibrillin-1 is dominated by two types of disulfide-rich motifs, the calcium- binding epidermal growth factor-like (cbEGF) and transforming growth factor beta binding protein-like (TB) domains. Disruption of fibrillin-1 domain structure and function contributes to the pathogenic mechanisms underlying two inherited diseases with very different etiologies: Marfan syndrome (MFS) and homocystinuria (HC). MFS is a connective tissue disease caused by mutations in the fibrillin-1 gene FBN1. Many missense mutations cause variable degrees of fibrillin-1 domain misfolding, which may affect the delivery of fibrillin-1 to the ECM and/or its assembly into microfibrils. HC is a metabolic disorder which affects methionine metabolism and results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Patients with HC often exhibit ocular and skeletal defects resembling the MFS phenotype, suggesting that elevated homocysteine levels may lead to chemical reduction of disulfide bonds within fibrillin-1 domains resulting in the loss of native structure. Protein misfolding therefore is implicated in pathogenic mechanisms underlying MFS and HC.

Prokaryotic expression of limited fragments of the Notch receptor and its ligands followed by in vitro refolding has been used for the production of the significant amounts of protein required for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy. As an illustration of the protocol for the production of these EGF-containing constructs we have focused on a limited fragment of human Notch 1 that contains three calcium-binding EGF domains, hNotch-111-13. Following characterization by the methods described here, this construct has been shown to be functionally competent in a range of assays and the structure has been solved by X-ray crystallography and NMR.

Prokaryotic expression of limited fragments of the Notch receptor and its ligands followed by in vitro refolding has been used for the production of the significant amounts of protein required for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy. As an illustration of the protocol for the production of these EGF-containing constructs we have focused on a limited fragment of human Notch 1 that contains three calcium-binding EGF domains, hNotch-111-13. Following characterization by the methods described here, this construct has been shown to be functionally competent in a range of assays and the structure has been solved by X-ray crystallography and NMR.

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate T H 1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients. © 2012 Nature America, Inc. All rights reserved.

Background: The Notch pathway is an evolutionarily conserved cell-signaling system that plays an important role in both physiologic and pathologic conditions such as embryonic development and cancer. In the latter, Notch signaling has been reported to promote tumor growth by regulating different aspects of tumor biology such as cell survival, proliferation, angiogenesis, and stemness. Because of its importance, targeting of this pathway has been attempted via both small molecules (γ-secretase inhibitors; GSIs) and neutralizing antibodies (against individual Notch receptors or DLL4 ligand) but limitations still hamper the clinical use of such therapeutics, generally due to pathway complexity and toxicities caused by complete pathway inhibition (GSIs). Targeting Notch ligands such as Jagged1 (JAG1), offers the opportunity to selectively block specific elements of the pathway important in tumor biology thus avoiding normal tissue toxicities. Methods: Structural studies defined a region of JAG1 (DSL + EGF1-3) that bound Notch1, which was used as an immunogen. Using classical hybridoma technology we have generated and subsequently characterised a panel of monoclonal antibodies (mAbs) against the JAG1 ligand, both in vitro (cell signaling and cell biology assays) and in vivo (tumor xenografts in both mice and rats). Results: Four functional blocking mAbs recognised a unique JAG1 epitope within its DSL domain binding interface with Notch1, effectively blocking ligand-receptor interaction in vitro. Importantly, our mAbs were cross-reactive with rat Jag1 but not the murine orthologue. These were able to inhibit endogenous JAG1-induced signaling in tumor and stromal cells (eg. vascular smooth muscle cells). JAG1 mAb treatment reduced in vitro breast cancer 3D growth, exhibiting reduced expression of important genes such as HES1, IL6 and decreased numbers of cancer stem cells. Treatment did not affect JAG2-induced growth, or signaling mediated by other Notch ligands (eg. DLL4), confirming mAb specificity. Importantly, JAG1 mAb treatment in vivo inhibited Notch signaling and tumor growth in cancer xenograft models in two different host animals. Host body weight, blood tests and histological analysis detected no toxicity. Conclusions: We generated neutralizing mAbs able to inhibit JAG1-induced signaling both in vitro and in vivo. The ability of JAG1 blockade to impair tumor growth without toxicity indicates it has the potential to make a contribution to the current arsenal of cancer therapeutics. This mAb treatment will be subjected to further in vivo testing, both alone and in combination with other therapeutic approaches, to further expand our knowledge of its clinical potential and mechanism of action. Funding and conflicts of interest This work is supported by Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We have filed a patent application, WO/2014/111704, claiming therapeutic use of the JAG1 antibodies. Citation Format: Massimo Masiero, Demin Li, Pat Whiteman, Carol Bentley, Jenny Greig, Tasneem Hassanali, Sarah Watts, Stephen Stribbling, Jenna Yates, Ji-Liang Li, Susan Mary Lea, Penny Ann Handford, Adrian Llewellyn Harris, Alison Hilary Banham. Development of therapeutic anti-Jagged1 monoclonal antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C128.

FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments. Limited proteolysis, NMR spectroscopy and chromophoric calcium chelation experiments showed that the G267S and S227P substitutions cause long-range structural effects consistent with protein misfolding. Cellular studies using fibroblast cells further demonstrated that these recombinant forms of mutant fibulin-5 were not present in the extracellular medium, consistent with retention. In contrast, no significant effects of I169T and G202R substitutions on protein fold and secretion were identified. These data establish protein misfolding as a causative basis for the effects of G267S and S227P substitutions in AMD and CL, respectively, and raise the possibility that the I169T and G202R substitutions may be polymorphisms or may increase susceptibility to disease.

Fibrillin-1 is a modular glycoprotein and a major component of the 10-12 nm microfibrils of the extracellular matrix. Mutations in the fibrillin-1 (FBN 1) gene result in the connective tissue disease the Marfan syndrome (MFS) and related disorders. The calcium binding EGF-like (cbEGF) domain is the predominant structural motif of the protein and >70% of mutations leading to MFS disrupt this domain. A missense mutation which changes a proline to alanine (P1148A) in cbEGF domain 13 has been associated with a number of fibrillin disorders including MFS and Shprintzen-Goldberg syndrome. However, it has also been described as a polymorphism. In this study comparative NMR analyses on wild-type and mutant forms of covalently-linked fibrillin cbEGF domain pairs have been performed to investigate the structural consequences of this substitution. A comparison of the two-dimensional NOESY spectra of the wild-type and mutant forms of cbEGF domains 12 & 13 and cbEGF domains 13 & 14 indicated that the proline to alanine amino acid change does not introduce a significant structural defect into cbEGF domain 13 or the adjacent domains and most likely represents a polymorphism. These results demonstrate how, in the case of a protein with a well defined domain organisation such as fibrillin-1, comparative NMR analyses can be used to substantiate genetic evidence for the polymorphic status of an amino acid.

Human fibrillin-1 is an extra-cellular matrix glycoprotein with a modular organisation that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats interspersed with transforming growth factor beta binding protein-like (TB) domains. We have studied Marfan syndrome-causing mutations which affect calcium binding to cbEGF13, and demonstrate that in human fibroblast cells they cause unexpected endoplasmic reticulum retention, indicative of a folding defect. Biochemical and biophysical studies of in vitro refolded fragments from the TB3-cbEGF14 region indicate long-range and unidirectional effects of these substitutions on the adjacent N-terminal domain cbEGF12. In contrast, only short-range effects of a pathogenic mutation affecting calcium binding to cbEGF19 are observed, and secretion of this mutant protein occurs. Further NMR studies on wild-type cbEGF12-13 and cbEGF12-14 identify a co-operative dependence of domain folding where calcium binding to cbEGF13 is required before cbEGF12 can adopt a native Ca(2+)-dependent fold. These data demonstrate that during biosynthesis of fibrillin-1, multiple tandem repeats of cbEGF domains may not necessarily fold independently and therefore missense mutations resulting in identical substitutions may have different effects on the fate of the mutant protein. Complex folding of modular proteins should therefore be considered when interpreting the molecular pathology of single-gene disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29-31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29-30, but not cbEGF30-31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.

Fibrillin-1 is a large modular glycoprotein that assembles to form 10-12 nm microfibrils in the extracellular matrix. Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome and related connective tissue disorders (fibrillinopathies) that show autosomal dominant inheritance. The pathogenic mechanism is thought to be a dominant negative effect of a mutant protein on microfibril assembly, although direct evidence is lacking. A significant group of disease-causing FBN1 mutations are cysteine substitutions within EGF domains that are predicted to cause misfolding by removal of disulphide bonds that stabilize the native domain fold. We have studied three missense mutations (C1117Y, C1129Y and G1127S) to investigate the effect of misfolding on the trafficking of fibrillin-1 from fibroblast cells. We demonstrate that both C1117Y and C1129Y, expressed as recombinant fragments of fibrillin-1, are retained and accumulate within the cell. Both undergo core glycosylation but lack the complex glycosylation observed in the secreted wild-type fragment, suggesting retention in the endoplasmic reticulum (ER). In addition, co-immunoprecipitation experiments show association with the ER chaperone calreticulin, but not calnexin, 78 kDa glucose-regulated protein (Grp78/BiP) or protein disulfide isomerase. In contrast, G1127S, which causes a moderate change in the EGF domain fold, shows a pattern of glycosylation and trafficking profile indistinguishable from the wild-type fragment. Since expression of the recombinant fragments does not disrupt the secretion of endogenous fibrillin-1 by the cell, we propose that G1127S causes disease via an extracellular dominant negative effect. In contrast, the observed ER retention of C1117Y and C1129Y suggests that disease associated with these missense mutations is caused either by an intracellular dominant negative effect or haploinsufficiency.

Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal growth factor-like (cbEGF) domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these domains are critical for fibrillin-1 function. The cbEGF12-13 domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca(2+)-loaded cbEGF12-13 exhibits a near-linear, rod-like arrangement of domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interdomain linker residue, possess this rod-like structure. The domain arrangement of cbEGF12-13 is stabilized by additional interdomain packing interactions to those observed for cbEGF32-33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11-15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12-13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refoldedin vitro adopt the native fold. In contrast, the Gly → Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.