In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.
The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.
Yang
J
J
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Kramer
J M
JM
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000378:metabolism*; D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D004220:Disulfides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D003094:Collagen; D013381:Subtilisins