Deutsch
Department of Botany I - Plant-Physiology and Biophysics

Sclerostin - a negative regulator of bone growth

The SOST gene was identified in 2003 in a genome-wide screening to find the molecular cause of the two hyperostosis diseases sclerosteosis and the van Buchem syndrome. Both rare autosomal recessive diseases are characterized by massive bone overgrowth frequently affecting also the cranium. Untreated the craniofacial malformation will lead to facial palsy and deafness due to progressive compression of cranial nerves. Currently these consequences are avoided only by surgery, which removes excessive bone material. As both diseases are due to loss of function mutations in the SOST gene product, the secreted glycoprotein sclerostin therefore constitutes a negative regulator of bone growth. Contrary to initial suggestions sclerostin does not regulate bone homeostasis by directly modulating the BMP signaling pathway but rather affects the Wnt cascade.

Biophysical and biochemical analyses also from our group have revealed the molecular mechanism by which sclerostin functions as Wnt antagonist (1-3). Binding of sclerostin to the Wnt co-receptors of the LRP5/6 family blocks binding of Wnt factors thereby impeding receptor activation, which is accomplished by Wnt-mediated assembly of receptors of the frizzled family and members of the LRP5/6 co-receptor group. Determining its structure by NMR together with an extensive mutagenesis study showed that a short stretch in the second loop of the cystine-knot protein sclerostin constitutes the binding site to LRP5/6 and is essential for sclerostin’s negative regulatory function on bone growth (3,4).

Neutralizing antibodies against sclerostin have gained special interest, as these can abrogate sclerostin’s negative function on bone growth. Such antibodies can restore bone density in diseases, where bone growth has ceased or is limited. This osteoanabolic concept might be a highly valuable and superior treatment option for osteoporosis, particularly with regard to the current therapies, which attempt to mainly stop further bone loss. In collaboration with Bio-Rad AbD Serotec our lab has also developed two sclerostin-neutralizing antibodies (5,6). Determining their structures by X-ray diffraction revealed new insights into the epitopes of sclerostin that must be ideally blocked for muting sclerostin function (7,8). These data can now be used for the design of novel non-protein based inhibitors of sclerostin for future osteoporosis therapies.

  1. Krause C, Korchynskyi O, de Rooij K, Weidauer SE, de Gorter DJ, van Bezooijen RL, Hatsell S, Economides AN, Mueller TD, Lowik CW et al: Distinct modes of inhibition by sclerostin on bone morphogenetic protein and Wnt signaling pathways. J Biol Chem 2010, 285(53):41614-41626.
  2. Piters E, Culha C, Moester M, Van Bezooijen R, Adriaensen D, Mueller T, Weidauer S, Jennes K, de Freitas F, Lowik C et al: First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function. Hum Mutat 2010, 31(7):E1526-1543.
  3. Boschert V, van Dinther M, Weidauer S, van Pee K, Muth EM, Ten Dijke P, Mueller TD: Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for wnt-signaling inhibition. PLoS One 2013, 8(11):e81710.
  4. Weidauer SE, Schmieder P, Beerbaum M, Schmitz W, Oschkinat H, Mueller TD: NMR structure of the Wnt modulator protein Sclerostin. Biochem Biophys Res Commun 2009, 380(1):160-165.
  5. Back JW, Frisch C, Van Pee K, Boschert V, van Vught R, Puijk W, Mueller TD, Knappik A, Timmerman P: Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin. Protein Eng Des Sel 2012, 25(5):251-259.
  6. Boschert V, Muth EM, Knappik A, Frisch C, Mueller TD: Crystallization and preliminary X-ray crystallographic analysis of the sclerostin-neutralizing Fab AbD09097. Acta Crystallogr F Struct Biol Commun 2015, 71(Pt 4):388-392.
  7. van Dinther M, Zhang J, Weidauer SE, Boschert V, Muth EM, Knappik A, de Gorter DJ, van Kasteren PB, Frisch C, Mueller TD et al: Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling. PLoS One 2013, 8(4):e62295.
  8. Boschert V, Frisch C, Back JW, van Pee K, Weidauer SE, Muth EM, Schmieder P, Beerbaum M, Knappik A, Timmerman P et al: The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6. Open Biol 2016, 6(8).
The hyperostosis sclerosteosis and van Buchem syndrome are characterised in a massive bone overgrowth. The dramatic increase in bone mass is illustrated in this figure by comparing the mass of a skull of a van Buchem’s patient with that of healthy individuals. The former has about a four-fold higher mass.
Fig. 1: The picture illustrates the massive increase in bone mass of the skull of a Van Buchem’s disease (VBD) patient. The loss of a 52 kilobase long sequence at the untranslated 3’ end of the SOST gene, which likely contains a transcriptional enhancer element, results in an attenuated expression of sclerostin, a negative regulator of bone growth and an antagonist of the Wnt pathway. The enhanced bone growth in VBD patients leads to an about 4fold increased bone mass compared to healthy individuals. (Graphic: Oxford University Press)
Structural alignment of 15 NMR structures of murine sclerostin. In the right panel a secondary structure cartoon is shown highlighting the characteristic cystine-knot motif. The latter is formed by two disulfide bonds forming an outer ring which is penetr
Fig. 2: (left panel) NMR structure ensemble of murine sclerostin, for clarity only the main chain atoms O, C, C and N are shown. A structural alignment of 15 individual NMR structures is shown, which were calculated from NMR data of (4) and do no violate any of the NMR restraints. Sclerostin exhibits an extended architecture; the two loops emanating from the central cystine knot into the same direction form two 2-stranded -sheets (loops 1 and 3), while the second loop running into the opposite direction is unstructured and displays high flexibility and dynamics. (right panel) Cartoon ribbon plot of a representative NMR structure of murine sclerostin highlighting its secondary structure elements. Sclerostin harbours a cystine knot motif; here six cysteine residues form three disulfide bonds, of which two build an outer ring through which is penetrated by the third disulfide bond thereby tying the knot.
Grafik
(Grafik: T. Müller, Universität Würzburg 2017)
Contact

Chair of Botany I - Plant Physiology and Biophysics
Julius-von-Sachs-Platz 2
97082 Würzburg

Phone: +49 931 31-86101
Fax: +49 931 31-86157
Email

Find Contact

Julius-von-Sachs-Platz 2 Hubland Süd Hubland Nord Fabrikschleichach Hubland Süd, Geb. B2 Hubland Süd, Geb. B3