MIG-17, a glycoprotein belonging to the ADAM family, is secreted from the body wall muscle cells and localizes to the gonadal basement membrane to control the migration of gonadal distal tip cells (DTCs). MIG-17 has four domains following the signal peptide; pro-, metalloprotease (MP), disintegrin-like (DI) and cysteine-rich (CR) domains. MIG-17 has nine potential N -glycosylation sites (i.e. NXS/T): six in the prodomain (Asn 52, Asn 65, Asn 123, Asn 172, Asn 183, Asn 189), and three in the metalloproteinase domain (Asn 218, Asn 219, Asn 350). To understand the significance of glycosylation in MIG-17, we generated MIG-17 lacking glycosylation either from pro- or MP domain. The asparagine residues in these sites were changed to glutamines in various combinations to eliminate glycosylation in this study. The MIG-17 protein failed to localize to the gonad and cannot rescue the DTC migration defects of
mig-17 mutants in the total absence N-glycans. The analysis of MIG-17 lacking N-glycans either from the pro- or metalloproteinase domain clearly revealed that the N-glycosylation of the prodomain, but not the metalloproteinase domain, is required for localization and function of MIG-17, suggesting a critical role of the prodomain N-glycosylation. The analysis of MIG-17 lacking individual N-glycosylation sites revealed that the substitution of Asn52, Asn65, Asn172 and Asn189 in the prodomain cannot efficiently rescue the cell migration defects of
mig-17 mutants. Surprisingly, however, the MIG-17 lacking prodomain glycosylation successfully rescued
mig-17 mutants when expressed in the DTCs, suggesting that the prodomain glycosylation is important for targeting MIG-17 to the gonad but not for its activity. The prodomain is proteolytically removed when the enzyme becomes an active matured form. Therefore, it is possible that MIG-17 first localizes to the gonad in a prodomain-dependent manner and then the prodomain is processed. The mutant protein with amino-acid changes from KK to LL at the border of pro- and MP domains failed to be processed and could not rescue
mig-17 mutants, but it strongly localized to the gonad, indicating that processing of the prodomain is not required for localization. The prodomains of ADAM proteases have been thought to be required for keeping catalytic latency or for proper folding of polypeptides. Our findings shed light on the function of prodomains whose glycosylation is essential for targeting secreted ADAM proteins to specific tissues or cells.