Haemonchus contortus: evidence that the 3A3 collagen gene is a member of an evolutionarily conserved family of nematode cuticle collagens

Rabbit antisera were raised against an 18 amino acid-long peptide that corresponds to the predicted sequence of the carboxy-terminal, nontriple helical region of the Haemonchus contortus 3A3 collagen gene. This sequence is highly conserved and diagnostic for members of the col-l collagen family, which includes the 3A3 gene. We find that these antisera react predominantly with multiple, high molecular weight (greater than 68 kDa) proteins on Western blots of whole worm extracts. The number and molecular weights of the reacting proteins vary depending upon the developmental stage of the worms analyzed. All of the reacting proteins are collagenase sensitive. The reacting collagens copurify with cuticles and are released from cuticles by reducing agents. In indirect immunofluorescence assays the antisera react only with the broken edges of isolated cuticles, suggesting that the antisera are reacting with an internal cuticle layer. This layer appears to be circular and to extend throughout the length of the worm. The antisera react on Western blots with multiple, high molecular weight collagens of eight other nematodes examined, representing two classes and several orders. These data provide additional support for the notion that the 3A3 collagen gene, and other members of the col-l collagen family, encode cuticle collagens. Collagens with this peptide sequence, presumably other members of the col-l collagen family, appear to be widely distributed in the phylum Nematoda.