Mapping the heparin‐binding site of the osteoinductive protein NELL1 by site‐directed mutagenesis

Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D.; Kuroda, Shinya; Niimi, Tomoaki

doi:10.1016/j.febslet.2015.11.032

Cited by 10 publications

(5 citation statements)

References 28 publications

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“…More recently, Nell1 was shown to activate canonical Wnt signaling [101]. Nonetheless, the cognate receptor(s) for Nell proteins have yet to be identified although Nell1 was shown to form oligomers and interact with heparin and integrins [105,[112][113][114][115]. Thus, the molecular mechanisms underlying NELL1-induced osteogenic differentiation remain to be fully understood.…”

Section: Discussionmentioning

confidence: 99%

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

Wang¹,

Liao²,

Zhang³

et al. 2017

Cell Physiol Biochem

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Background: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. Methods: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. Results: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. Conclusion: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.

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Section: Discussionmentioning

confidence: 99%

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

Wang¹,

Liao²,

Zhang³

et al. 2017

Cell Physiol Biochem

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“…Nonetheless, due to the fact that the regions responsible for heparin binding sites are composed of several positively charged residues that contribute to heparin binding. Hence, it is difficult to mutate certain binding sites and thus completely inhibit the binding of heparin [39, 40].…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding

Somsoros

Sangawa

Takebe

et al. 2021

Journal of General Virology

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White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

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“…It was later reported that Nell-1 can promote osteoblastic cell adhesion and differentiation through binding to integrin α3β1 with use of the last 2 von Willebrand factor C domains on its C-terminal (Nakamura et al 2014). In addition, 2 positively charged patches in the TSPN domain of Nell-1 have moderate heparin-binding activity, and the interaction of Nell-1 with heparan sulfate proteoglycans on the cell surface may assist in Nell-1-integrin binding (Takahashi et al 2015). Nell-1 was recently found to bind to roundabout 2 (Robo2) through its second and third EGF domains (Yamamoto et al 2019).…”

Section: Further Understanding Of Nell-1 Protein Structure and Functimentioning

confidence: 99%

Nell-1 Is a Key Functional Modulator in Osteochondrogenesis and Beyond

Zhang

Zheng

et al. 2019

J Dent Res

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Neural EGFL-like 1 (Nell-1) is a well-studied osteogenic factor that has comparable osteogenic potency with the Food and Drug Administration–approved bone morphogenic protein 2 (BMP-2). In this review, which aims to summarize the advanced Nell-1 research in the past 10 y, we start with the correlation of structural and functional relevance of the Nell-1 protein with the identification of a specific receptor of Nell-1, contactin-associated protein-like 4 (Cntnap4), for osteogenesis. The indispensable role of Nell-1 in normal craniofacial and appendicular skeletal development and growth was also defined by using the newly developed tissue-specific Nell-1 knockout mouse lines in addition to the existing transgenic mouse models. With the achievements on Nell-1’s osteogenic therapeutic evaluations from multiple preclinical animal models for local and systemic bone regeneration, the synergistic effect of Nell-1 with BMP-2 on osteogenesis, as well as the advantages of Nell-1 as an osteogenic protein with antiadipogenic, anti-inflammatory, and provascularized characteristics over BMP-2 in bone tissue engineering, is highlighted, which lays the groundwork for the clinical trial approval of Nell-1. At the molecular level, besides the mitogen-activated protein kinase (MAPK) signaling pathway, we emphasize the significant involvement of the Wnt/β-catenin pathway as well as the key regulatory molecules Runt-related transcription factor 2 (Runx2) in Nell-1-induced osteogenesis. In addition, the involvement of Nell-1 in chondrogenesis and its relevant pathologies have been revealed with the participation of the nuclear factor of activated T cells 1 (Nfatc1), Runx3, and Indian hedgehog (Ihh) signaling pathways, although the mechanistic insights of Nell-1’s osteochondrogenic property will be continuously evolving. With this perspective, we elucidate some emerging and novel functional properties of Nell-1 in oral-dental and neural tissues that will be the frontiers of future Nell-1 studies beyond the context of bone and cartilage. As such, the therapeutic potential of Nell-1 continues to evolve and grow with continuous pursuit.

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Mapping the heparin‐binding site of the osteoinductive protein NELL1 by site‐directed mutagenesis

Cited by 10 publications

References 28 publications

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding

Nell-1 Is a Key Functional Modulator in Osteochondrogenesis and Beyond

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