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 examin ed by analyzing site-directed mutations of the sqt-1 and rol-6 collage n genes in transgenic animals. Altered collagen genes on transgenic ar rays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 gene rally produced the same phenotypes, indicating that conserved amino ac ids in these two collagens have similar functions. Serine substitution s 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 f or normal function but not required for assembly. Arg-1 or Arg-4 to Cy s mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the ami no non-Gly-X-Y domain) caused RRol phenotypes, while the same alterati on at Arg-3 had no effect, indicating that Arg-3 is functionally diffe rent from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Gl u also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions f or Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefo re, strong positively charged residues, Arg or Lys, are required at po sitions 1 and 4 for normal function. The conserved pattern of arginine s in HBA matches the cleavage sites of the subtilisin-like endoprotein ases. HBA may be a cleavage site for a subtilisin-like protease, and c leavage may be important for cuticle collagen processing.