Interaction properties of the procollagen C-proteinase enhancer protein shed light on the mechanism of stimulation of BMP-1.
Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.
Ricard-Blum
Sylvie
S
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale, CNRS UMR 5075, 38027 Grenoble cedex 1, France.
Bernocco
Simonetta
S
Font
Bernard
B
Moali
Catherine
C
Eichenberger
Denise
D
Farjanel
Jean
J
Burchardt
Elmar R
ER
van der Rest
Michel
M
Kessler
Efrat
E
Hulmes
David J S
DJ
D001665:Binding Sites; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000502:physiology*; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism / Q000502:physiology*; D011347:Procollagen / Q000378:metabolism*
D019485:Bone Morphogenetic Proteins; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C491555:PCOLCE protein, human; D011347:Procollagen; D008666:Metalloendopeptidases; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1; D002118:Calcium