Recombinant and tissue-derived mouse BM-40 bind to several collagen types and have increased affinities after proteolytic activation.
The calcium-binding extracellular matrix protein BM-40 was obtained as a mouse cDNA product from a stably transfected kidney cell clone. Electrophoresis and N-terminal sequence analysis demonstrated absence of the proteolytic processing previously observed for a mouse tumour-derived BM-40. Yet the two forms of BM-40 were very similar in their CD spectra, their calcium-dependent change in alpha helix content and their immunological epitopes. In surface plasmon resonance assays, recombinant mouse BM-40 showed distinct binding to the triple-helical domains of collagens I, II, III, IV and V with Kd = 1-4 microM but no binding to collagen VI. These interactions were abolished in the presence of EDTA. Tissue-derived mouse BM-40, however, bound collagens I and IV with Kd = 0.1-0.2 microM. Activation of collagen binding to give a similar Kd could be achieved for recombinant mouse BM-40 by treatment with the matrix metalloproteinase collagenase-3. The major cleavage site was located in helix C of the extracellular calcium-binding module of BM-40 and other less prominent cleavages occurred close to the N-terminus. The sensitive helix C site was just one residue away from that sensitive to endogenous tissue proteolysis, suggesting that cleavage could be a physiological mechanism to modulate collagen binding.
Maurer
P
P
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Göhring
W
W
Sasaki
T
T
Mann
K
K
Timpl
R
R
Nischt
R
R
D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism; D006801:Humans; D053509:Matrix Metalloproteinase 13; D051379:Mice; D015676:Osteonectin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015676:Osteonectin; D011993:Recombinant Fusion Proteins; D003094:Collagen; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C507081:Mmp13 protein, mouse