Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity

• Published in: Biochemical journal 2004-08, Vol.382 (Pt 1), p.315-322
• Main Authors: Clermont, Anja, Wedde, Marianne, Seitz, Volkhard, Podsiadlowski, Lars, Lenze, Dido, Hummel, Michael, Vilcinskas, Andreas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins - biosynthesis; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - physiology; Cloning, Molecular - methods; Hemolymph - chemistry; Humans; Immunity, Innate - physiology; Insect Proteins - biosynthesis; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - physiology; Insecta - chemistry; Insecta - immunology; Larva - immunology; Mass Spectrometry - methods; Metalloendopeptidases - antagonists & inhibitors; Metalloproteases - antagonists & inhibitors; Molecular Sequence Data; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Analysis, Protein - methods; Up-Regulation - genetics
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20031923

The first IMPI (inhibitor of metalloproteinases from insects) was identified in the greater wax moth, Galleria mellonella [Wedde, Weise, Kopacek, Franke and Vilcinskas (1998) Eur. J. Biochem. 255, 535-543]. Here we report cloning and expression of a cDNA coding for this IMPI. The IMPI mRNA was identified among the induced transcripts from a subtractive and suppressive PCR analysis after bacterial challenge of G. mellonella larvae. Induced expression of the IMPI during a humoral immune response was confirmed by real-time PCR, which documented up to 500 times higher amounts of IMPI mRNA in immunized larvae in comparison with untreated ones. The IMPI sequence shares no similarity with those of tissue inhibitors of metalloproteinases or other natural inhibitors of metalloproteinases, and the recombinant IMPI specifically inhibits thermolysin-like metalloproteinases, but not matrix metalloproteinases. These results support the hypothesis that the IMPI represents a novel type of immune-related protein which is induced and processed during the G. mellonella humoral immune response to inactivate pathogen-associated thermolysin-like metalloproteinases.