Identification of a novel metalloproteinase and its role in juvenile development of the tobacco hornworm, Manduca sexta (Linnaeus)

Abstract

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases that are highly conserved across numerous taxa, from bacteria to humans. Recently, MMPs have been identified in several insect species and are hypothesized to function in immunity and development. In this study, we identify a putative MMP and correlate its proteolytic activity and gene and protein expression in the tracheae with developmental stage. Ms-MMP gene expression increases 10-fold during molting, which is accompanied by an increase in both protein expression and gelatinolytic activity. To directly test the hypothesis that Ms-MMP plays a critical role in juvenile development of Manduca sexta, we injected a broad-spectrum MMP inhibitor and recorded its effects on growth and development. Inhibition of MMPs caused a delay in juvenile development and decreased growth rates. Understanding the function of MMPs will help us better understand molting and control of body size in insects. Furthermore, elucidating functions for MMPs in lower taxa may yield critical information about the evolution of the numerous MMPs found in vertebrates.

Figure 1

A: Nucleotide sequence and the deduced amino acid sequence of Ms-MMP. The underlined M residue indicates the putative translation start site. The amino acid sequence highlighted in yellow indicates the cysteine switch, while the orange highlighting indicates the conserved catalytic domain. The amino acid sequence underlined with a dotted line indicates the peptide used for antibody synthesis. B: Structural analysis of Ms-MMP shows the signal peptide (red; position 1–39) overlapping the transmembrane domain (position 21–43), a peptidoglycan binding domain (Pfam; position 47–104), the catalytic domain (ZnMc; position 128–285), and four hemopexin repeats (HX; positions 326–370, 372–416, 418–465, and 467–514).

Figure 2

A: Amino acid sequence alignment used to generate the phylogram. Shaded box denotes the conserved zinc-binding catalytic domain. B: Phylogenetic relationships between metzincin superfamily members, including several MMPs, astacins, serralysin, and adamalysin. Numbers above branches indicate substitutions per site, while numbers to the right of nodes indicate the consensus percent. C: Phylogenetic relationships between insect MMPs.

Figure 3

Tissue distribution of Ms-MMP in 5th instar larvae. A: Ms-MMP was amplified from cDNA from hemocytes (HC), fat body (FB) and tracheae (TR). B: Anti-Ms-MMP detected Ms-MMP in TR, FB, hemolymph (HL), and HC. C: Pre-incubation of the antibody with the Ms-MMP peptide abolished most signal from the membrane.

Figure 4

Expression of Ms-MMP varies throughout development. A: Ms-MMP gene expression in tracheae peaked on the first day of the 5th instar (n = 4 caterpillars per day). Asterisk indicates that day 0 is significantly higher than other days (P < 0.02). B: Western blot of tracheae using rabbit anti-Ms-MMP (numbers to left are the molecular masses in kDa). Each lane represents tracheae from one caterpillar. C: Zymograms of tracheae collected daily during the 5th instar show gelatinolytic activity early in the instar (clear bands). Each panel shows tracheal samples from six individual caterpillars. Numbers to left are the molecular masses in kDa. Experiments were repeated at least three times.

Figure 5

Effect of MMP inhibition on the growth of caterpillars. A: Caterpillars injected with GM6001 (triangles) weighed significantly less than control caterpillars (circles, n = 5 per group) by day 4 of the 5th instar. B: Juvenile development was significantly delayed, as indicated by the delayed appearance of the dorsal heart in the MMP-inhibited larvae. Asterisk indicates significant differences (P < 0.01).