Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.
Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.
Dublet
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B
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS, Unité Propre de Recherche 9015), 41, rue Jules Horowitz, F-38027 Grenoble Cedex 01, France. dublet@ibs.fr
Vernet
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T
van der Rest
M
M
D018834:Chaperonin 60 / Q000378:metabolism; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D010009:Osteochondrodysplasias / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D018834:Chaperonin 60; D003094:Collagen; D014357:Trypsin