Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insec ts, primary structures of MTs are known only for Drosophila and the collemb olan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstra ted the presence of two smaller metal-binding peptides, whose amino acid se quences correspond to parts of the cDNA, and which apparently result from c leavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein is olation techniques in combination with mass spectrometry and N-terminal seq uence analysis. Further, recombinant expression allowed us to study the int rinsic stability of the MT and to perform in vitro cleavage studies. The re sults show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is loc ated in the N-terminal region and the other in the linker region between tw o putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, thi s protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain meta l-binding protein. (C) 2001 Elsevier Science Ltd. All rights reserved.