title: Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella.
abstract: In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor.
authors.affiliation: Institute of Zoology, Free University of Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Wedde
authors.forename: C
authors.initials: C
authors.lastname: Weise
authors.forename: P
authors.initials: P
authors.lastname: Kopacek
authors.forename: P
authors.initials: P
authors.lastname: Franke
authors.forename: A
authors.initials: A
authors.lastname: Vilcinskas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D006458:Hemolymph / Q000737:chemistry*; D007814:Larva / Q000737:chemistry; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D013820:Thermolysin / Q000633:toxicity
chemical_list: D011480:Protease Inhibitors; D008666:Metalloendopeptidases; D013820:Thermolysin