C. elegans has eight genes coding for proteins homologous to the members of the serine protease family S9, which are subdivided into the subfamilies S9B and S9C. S9B includes dipeptidyl peptidase IV (DPPIV)/CD26 and seprase/fibroblast activation protein (FAP), and S9C includes acylaminoacyl peptide hydrolase. Recently, DPPIV/CD26 and seprase/FAP have been reported to participate in cell migration in mammals, but the details of their functions are not well known. In this study, we performed RNAi analysis on six of the family S9 genes including C27C12.7, T23F1.7 and K02F2.1 in S9B, and F01F1.5, R11E3.8 and F44B9.1 in S9C. When each gene was individually interfered, no abnormality was observed. Simultaneous interference of two genes, however, led to abnormal migration of distal tip cells (DTCs) in every combination of the genes. The effect of RNAi was most prominent when C27C12.7 and T23F1.7 were inactivated. We isolated a deletion allele,
tm619, of the F01F1.5 gene. Although this mutant showed a normal phenotype, abnormality in gonadal shape was observed when any one of the other S9 genes was knocked down by RNAi in this genetic background. By in situ hybridization and immunostaining of the family S9 gene products, the mRNA and proteins were all detected in the DTCs at the L1 and L2 stages. The proteins were not detected in the RNAi-treated worms.Although all of the gene products belong to the family S9, their identity in amino acid sequence was less than 25% to one another. In order to enzymatically characterize them, the proteins were expressed in insect cells and purified. They were assayed using synthetic substrates Gly-Pro-MCA (a substrate for DPPIV/CD26), Ac-Ala-MCA (a substrate for acylaminoacyl peptide hydrolase), and Ala-MCA (a substrate for aminopeptidase). All of the proteins were found to be active toward Gly-Pro-MCA. The activities toward Ac-Ala-MCA and Ala-MCA, however, were hardly detected. The specific activities toward Gly-Pro-MCA of the enzymes were as follows: C27C12.7