R. Ruppert et al., HUMAN BONE MORPHOGENETIC PROTEIN-2 CONTAINS A HEPARIN-BINDING SITE WHICH MODIFIES ITS BIOLOGICAL-ACTIVITY, European journal of biochemistry, 237(1), 1996, pp. 295-302
Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone
regeneration and repair as well as during various stages of embryonal
development. A cDNA encoding mature human BMP-2 could be efficiently
expressed in Escherichia coli, and after renaturation a dimeric BMP-2
protein of M(r) 26 000 was prepared with a purity greater 98%. The rec
ombinant BMP-2 was functionally active as demonstrated by the inductio
n of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell li
ne (EC(50) of 70 nM) and proteoglycan synthesis in embryonic chicken l
imb bud cells (EC(50) of 15-20 nM). A peptide 1-17 representing the N-
terminal basic part of BMP-2 as well as heparin increased the specific
activity of the protein about fivefold in the limb bud assay. These o
bservations suggested that the N-termini reduce the specific activity
of BMP-2, probably by interacting with heparinic sites in the extracel
lular matrix. This conclusion was supported by a variant EHBMP-2, wher
e the N-terminal residues 1-12 of BMP-2 had been substituted by a dumm
y sequence of equal length and which showed an EC(50) value of around
1 nM which was affected neither by heparin nor by peptide 1-17. A phys
ical interaction between BMP-2 and heparin could be seen in biosensor
experiments, where BMP-2 bound to immobilized heparin with a dissociat
ion constant, K-d, of approximately 20 nM, whereas the heparin-binding
of variant EHBMP-2 was negligible. These results identify the basic N
-terminal domains of dimeric BMP-2 as heparin-binding sites that are n
ot obligatory for receptor activation but modulate its biological acti
vity.