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

Abstract

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.

Figure 1. cDNA sequence and deduced amino acid sequence of G. mellonella IMPI

Nucleotides are numbered from the first base at the 5′-end. Amino acids are numbered from the initiating methionine. The putative signal peptide is shown in italics, and the putative IMPI protein is underlined. The amino acid sequence in parentheses was determined by peptide sequencing. The SPC cleavage site (R-R) is given in pale grey lettering.

Figure 2. Relative quantification of IMPI cDNA in comparison with actin cDNA by real-time PCR

(A) IMPI mRNA is up-regulated upon LPS stimulation, as demonstrated by the lower number of cycles needed to amplify IMPI. (B) PCR with β-actin-specific primers using the same cDNA preparations as in (A) indicates no difference in the total cDNA amount between the different probes.

Figure 3. MALDI MS of rIMPI-1 (A), rIMPI-1* (B) and rIMPI-2 (C)

The rIMPI* was purified by affinity chromatography on a thermolysin–Sepharose column to remove the vector part from rIMPI-1 (see Figure 5). rIMPI-1 and rIMPI-2 were isolated from the supernatant of the Drosophila Schneider 2 cell culture medium by affinity chromatography using a chelating Sepharose matrix. For details, see the Materials and methods section.

Figure 4. Western blot analysis of rIMPIs

Samples of 25 ng of rIMPI proteins were loaded on a 12% (w/v) polyacrylamide Bis-Tris gel in reducing loading buffer. After incubation with anti-V5 as primary antibody and goat anti-mouse Ig conjugated to horseradish peroxidase as secondary antibody, rIMPI-1* revealed no signal, in contrast with rIMPI-1 and rIMPI-2.

Figure 5. Possible cleavage sites for thermolysin along the vector part of rIMPI-1*

The part of the sequence contributed by IMPI is in bold. Possible sites of cleavage by thermolysin are indicated by | (N-terminal sides of Ile, Leu, Val, Ala, Phe and Met). MS analysis of rIMPI-1* matches best with the cleavage indicated by ↓.

Figure 6. Inhibitory activity of native IMPI, rIMPI-1, rIMPI-1*, rIMPI-2 and phosphoramidon against bacterial metalloproteinases belonging to the gluzincin family

For each assay, positive controls were performed and the proteinases were added at concentrations that provided similar proteolytic activity (Thl, thermolysin; Bal, bacillolysin; Psl, pseudolysin). The resulting proteinase activity was defined as 100%. The IC₅₀ values for tested inhibitors (I-IC₅₀ for IMPI and P-IC₅₀ for phosphoramidon) were determined in the presence of inhibitors, which were serially diluted 3-fold and preincubated for 10 min with the proteinase before initiating the reaction by substrate addition. (A) Thermolysin activity was monitored in the presence of rIMPI-1 or rIMPI-2 using the MMP-1/MMP-9 substrate assay (assay I). (B) Comparison of the IC₅₀ values for native IMPI and rIMPI-1* (both isolated on thermolysin–Sepharose columns), and comparison of IC₅₀ values for rIMPI-1 (isolated on Ni²⁺-chelating Sepharose column) and rIMPI-1* (purified additionally on a thermolysin–Sepharose column) for thermolysin activity using the MMP-1/MMP-9 substrate assay (assay II). (C) Thermolysin, bacillolysin and pseudolysin activities were analysed in the presence of rIMPI-1 and phosphoramidon using the Azocoll assay.