Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).
We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP.
The Center for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany.
Luitgard
L
Spitznagel
D Patric
DP
Nitsche
Mats
M
Paulsson
Patrik
P
Maurer
Frank
F
Zaucke
D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D064236:Cartilage Oligomeric Matrix Protein; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006801:Humans; D064235:Matrilin Proteins; D009154:Mutation; D010009:Osteochondrodysplasias / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D064236:Cartilage Oligomeric Matrix Protein; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D064235:Matrilin Proteins; C577553:TSP5 protein, human; D006639:Histidine; D003094:Collagen; D001120:Arginine