The role of coiled-coil alpha-helices and disulfide bonds in the assembly and stabilization of cartilage matrix protein subunits. A mutational analysis.
Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.
Haudenschild
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DR
Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, USA.
Tondravi
M M
MM
Hofer
U
U
Chen
Q
Q
Goetinck
P F
PF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002356:Cartilage; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D003545:Cysteine; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004220:Disulfides; D004355:Drug Stability; D004815:Epidermal Growth Factor / Q000737:chemistry; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D046911:Macromolecular Substances; D064235:Matrilin Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D017931:DNA Primers; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D064235:Matrilin Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D003545:Cysteine