Cuticular collagen synthesis by Ascaris suum during development from the third to fourth larval stage: Identification of a potential chemotherapeuticagent with a novel mechanism of action

The dominant proteins released by Ascaris suum during development in vitro from the L3 to L4 stage were identified as collagenous cuticular proteins b y sequence analysis and susceptibility to digestion by collagenase. Under r educing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ), the colla.-en proteins separated into 3 groups with molecular weights es timated at 32 kDa, 54-60 kDa, and 71-91 kDa. The 32-kDa protein represents monomeric collagen; the 54-60- and 71-91-kDa components represent dimeric a nd trimeric forms, respectively, polymerized by nonreducible cross-links. F urthermore, the release of these forms of colla.-en was developmentally reg ulated, as exemplified by a sequential temporal progression from monomeric to dimeric to trimeric forms in association with the in vitro transition fr om L3 to L4. The data suggest that collagen released in vitro during develo pment of A. suum L3 to L4 reflects the increased translation of collagen ge ne products and their initial assembly into higher molecular weight molecul es associated with the synthesis of the L4 cuticle. A biotinylated dipeptid yl fluoromethylketone cysteine protease inhibitor (Bio-phe-ala-FMK) bound s pecifically to the 32-kDa collagen Ind, to a lesser extent, to a 30-kDa pro tein; binding was dependent on the presence of dithiothreitol (DTT) and was prevented by iodoacetamide. Because cysteine residues play an essential ro le in the initial assembly of the collagen monomers into the higher molecul ar weight oligomers present in the mature nematode cuticle, inhibition of m olting of A. suum L3 to L4 by the cysteine protease inhibitor Z-phe-ala-FMK might be due to its binding to thiol groups of collagen monomers during a critical phase of collagen assembly. Prevention of cystine cross-links duri ng this critical period of cuticle assembly by peptide-FMK inhibitors may r epresent a potential control mechanism having a novel mechanism of action.