Tài liệu Báo cáo khoa học: The pro-form of BMP-2 interferes with BMP-2 signalling by competing with

The pro-form of BMP-2 interferes with BMP-2 signalling by

competing with BMP-2 for IA receptor binding

Anja Hauburger1

, Sabrina von Einem1

, Gerburg K. Schwaerzer2

, Anja Buttstedt1

, Matthias Zebisch3

,

Michael Schra¨ml4,

, Peter Hortschansky5

, Petra Knaus2 and Elisabeth Schwarz1

1 Institut fu¨r Biochemie und Biotechnologie, Martin-Luther-Universita¨t Halle-Wittenberg, Germany

2 Institut fu¨r Chemie ⁄ Biochemie, Freie Universita¨t Berlin, Germany

3 Biotechnologisch-Biomedizinisches Zentrum, Universita¨t Leipzig, Germany

4 Scil Proteins GmbH, Halle, Germany

5 Leibniz-Institut fu¨r Naturstoffforschung und Infektionsbiologie, Hans-Kno¨ll-Institut (HKI), Jena, Germany

Keywords

alkaline phosphatase; BMPR-IA; BMPR-II;

bone morphogenetic protein-2; pro-domain

Correspondence

E. Schwarz, Institut fu¨r Biochemie and

Biotechnologie, Martin-Luther-Universita¨t

Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120

Halle, Germany

Fax: +49 345 55 27 013

Tel: +49 345 55 24 856

E-mail: elisabeth.schwarz@biochemtech.

uni-halle.de

Present address

Roche Diagnostics GmbH, Nonnenwald 2,

82372 Penzberg, Germany

(Received 2 April 2009, revised 18 August

2009, accepted 3 September 2009)

doi:10.1111/j.1742-4658.2009.07361.x

Pro-forms of growth factors have received increasing attention since it was

shown that they can affect both the maturation and functions of mature

growth factors. Here, we assessed the biological function of the pro-form of

bone morphogenetic protein-2 (BMP-2), a member of the transforming

growth factor b (TGFb)⁄BLP superfamily. The role of the 263 amino acids

of the pro-peptide is currently unclear. In order to obtain an insight into the

function of the pro-form (proBMP-2), the ability of proBMP-2 to induce

alkaline phosphatase (AP), a marker enzyme for cells differentiating into os￾teoblasts, was tested. Interestingly, in contrast to mature BMP-2, proBMP-2

did not lead to induction of AP. Instead, proBMP-2 inhibited the induction

of AP by BMP-2. This result raised the question of whether proBMP-2 may

compete with mature BMP-2 for receptor binding. ProBMP-2 was found to

bind to the purified extracellular ligand binding domain (ECD) of BMPR￾IA, a high-affinity receptor for mature BMP-2, with a similar affinity as

mature BMP-2. Binding of proBMP-2 to BMPR-IA was confirmed in cell

culture by cross-linking proBMP-2 to BMPR-IA presented on the cell sur￾face. In contrast to this finding, proBMP-2 did not bind to the ECD

of BMPR-II. ProBMP-2 also differed from BMP-2 in its capacity to induce

p38 and Smad phosphorylation. The data presented here suggest that the

pro-domain of BMP-2 can alter the signalling properties of the growth factor

by modulating the ability of the mature part to interact with the receptors.

Structured digital abstract

l MINT-7261817:BMPR-IA (uniprotkb:P36894) and proBMP2 (uniprotkb:P12643) physically

interact (MI:0915) by cross-linking studies (MI:0030)

l MINT-7261681, MINT-7261693: BMP2 (uniprotkb:P12643) binds (MI:0407) to BMPR-IA

(uniprotkb:P36894) by enzyme linked immunosorbent assay (MI:0411)

l MINT-7261751, MINT-7261794: proBMP2 (uniprotkb:P12643) binds (MI:0407) to BMPR-IA

(uniprotkb:P36894) by competition binding (MI:0405)

l MINT-7261806, MINT-7261846: BMPR-IA (uniprotkb:P36894) physically interacts

(MI:0915) with BMP2 (uniprotkb:P12643) by anti bait coimmunoprecipitation (MI:0006)

l MINT-7261628, MINT-7261642: noggin (uniprotkb:Q13253) binds (MI:0407) to proBMP2

(uniprotkb:P12643) by surface plasmon resonance (MI:0107)

Abbreviations

AP,alkaline phosphatase; BMP-2, bonemorphogenetic protein-2; ECD, extracellular domain;GDF-8, growthand differentiation factor-8; HA,

haemagglutinin;MBP, maltosebinding protein;PFC, pre-formed receptorcomplex; Smad, small mothersagainstdecapentaplegic;

TGFb,transforming growthfactor b.

6386 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS