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 insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results 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 located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this 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 metal-binding protein.
Hensbergen
P J
PJ
Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1087, 1081 HV, Amsterdam, Netherlands.
Donker
M H
MH
Hunziker
P E
PE
van der Schors
R C
RC
van Straalen
N M
NM
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000378:metabolism*; D015870:Gene Expression; D007313:Insecta / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism
D008670:Metals; D010455:Peptides; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase