Matrix metalloproteinases (MMPs) are a class of endopeptidases belonging to the superfamily of metalloproteinases, metzincins (Bode et al., 1993). A common feature of the metzincins is the presence of the zinc-binding domain, HEXXHXXGXXH (Bode et al., 1993). The metzincins are sub-divided based on the presence of other domains into ADAMs/adamalysins, astacins, serralysins and the MMPs. MMPs can be identified based on their primary amino acid sequence, tertiary protein structure, substrate specificity, and inhibition by MMP-specific inhibitors. MMPs are named, in part, because of their ability to cleave extra-cellular matrix proteins, but their substrates include a wide range of other molecules, such as growth factors, cell adhesion molecules, chemokines, and cytokines (Sternlicht and Werb, 2001; Greenlee et al., 2007).
MMPs are thought to have evolved from a much simpler enzyme that appeared even before the divergence of vertebrates from invertebrates and are highly conserved throughout these groups (Massova et al., 1998). The first MMP discovered was a collagenase, found in 1962 in the tail of a metamorphosing tadpole larva (Gross and Lapiere, 1962). Since then, MMPs have been found in various organisms ranging from bacteria to plants to mammals, including 25 in mice and 24 in humans (Brinckerhoff and Matrisian, 2002; Mott and Werb, 2004). Activity of MMPs is often associated with tissue degradation, as seen in cancer metastasis, arthritis, or asthma. Because of this, research on therapeutic agents has focused on the inhibition of MMPs (Overall and Kleifeld, 2006). Knockout mice lacking one or more MMPs often have deleterious phenotypes when subjected to disease models (Parks et al., 2004; Greenlee et al., 2007). In contrast, even though expression of several MMPs is highly correlated with development, there is little evidence for altered developmental phenotypes in mice lacking MMP2, MMP9, and MMP14 (Vu and Werb, 2000; Kheradmand et al., 2002; Oblander et al., 2005). These various phenotypic observations are most likely due to the fact that many MMPs have been shown to have overlapping and compensatory functions. Thus, the complex milieu of MMPs in vertebrates makes understanding their function in these organisms very difficult.
A simpler system for studying MMP function can be found in insects, which have at most three MMPs per species (Knorr et al., 2009). Two MMPs have been identified in the fruit fly, Drosophila melanogaster (Dm1-MMP and Dm2-MMP; Llano et al., 2000; Llano et al., 2002); one in the waxmoth, Galleria mellonella (Gm1-MMP; Altincicek and Vilcinskas, 2008); and three in the flour beetle, Tribolium castaneum (Knorr et al., 2009). Both Dm-MMPs are critical for larval tracheal growth, metamorphosis, and tissue remodeling (Page-McCaw et al., 2003; Glasheen et al., 2010). Gm1-MMP and Tribolium MMP-1 are closely related to Dm1-MMP both in structure and function (Altincicek and Vilcinskas, 2008; Knorr et al., 2009). Inhibition of Tribolium MMP-1 using RNAi resulted in defects in antennae, eyes, appendages, and head. In addition, these insects were unable to complete pupation and died during metamorphosis. While both Dm1-MMP mutant larvae and MMP knockdown beetle larvae exhibited broken tracheal system trunks during juvenile development, these defects were only lethal in Drosophila (Altincicek and Vilcinskas, 2008; Glasheen et al., 2010).
Because of our interest in MMPs due to their importance in disease processes and development, and our specific interest in growth during juvenile development, we sought to identify a putative MMP in the model organism, Manduca sexta, the tobacco hornworm. In this study, we correlate proteolytic activity and gene and protein expression in the tracheae with developmental stage. To directly test the hypothesis that Ms-MMP plays a critical role in juvenile development of M. sexta, we injected a broad-spectrum MMP inhibitor and recorded its effects on growth and development.
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- MATERIALS AND METHODS
- LITERATURE CITED
In this study, we identify a novel MMP in the caterpillar, M. sexta, that is involved in juvenile development, slowing 5th instar growth and delaying the larval–pupal transition. Ms-MMP gene and protein expression both increase at the time of molting and decrease throughout the instar, suggesting that it may be important for molting itself or for tissue remodeling that occurs immediately after molting. In vivo inhibition of Ms-MMP resulted in decreased growth rates and delayed molting, suggesting that there may be dual roles for this protease.
To verify that the protein we identified was a MMP, we used phylogenetic analyses. Because the zinc-binding motif is also present in other zinc-dependent endopeptidases, including the adamalysins, the astacins, and the serralysins (Bode et al., 1993), these proteases were included in the phylogenetic analysis. Ms-MMP grouped with the other insect MMP1s, providing additional support to the hypothesis that the protease we identified is a MMP. Together, the sequence alignment and the phylogenetic analysis provide strong evidence that the protease we identified is a MMP (Figs. 1 and 2).
While our search only identified one MMP, most insects studied have two or three MMPs (Llano et al., 2002; Knorr et al., 2009). A BLAST search on NCBI turns up an unrelated zinc-dependent protease, but no other matches in M. sexta. However, using the conserved catalytic domain of Ms-MMP, a BLAST search of the current draft of the M. sexta genome (Agricultural Pest Genomics Resource Database: www.agripestbase.org) identified three possible similar sequences, supporting the hypothesis that other MMPs exist. Since Ms-MMP clusters with MMP1 from other insects (Fig. 2C), it is likely that other MMPs will be homologous with insect MMP2 or MMP3.
MMPs are expressed as zymogens (Woessner and Nagase, 2000), and both the pro- and active forms can be detected using Western blotting or zymography. The presence of multiple bands in our Western blots (Figs. 3B and 4B) indicates that this is likely also the case for Ms-MMP. We are confident that the polyclonal antibodies detected Ms-MMP proteins because pre-incubation of our custom synthesized antibodies with either the peptide-antigen (Fig. 3C) or mouse-MMP2 (data not shown) resulted in the disappearance of most bands. The size of the full length Ms-MMP protein is comparable to that found in D. melanogaster, 60.3 kDa (Llano et al., 2000). As in D. melanogaster, we detected multiple sizes of Ms-MMP (Figs. 3B and 4B). For example, active, recombinant Dm1-MMP expressed in E. coli was identified on Western blot as a 19 kDa band, while larval extracts exhibit two bands of Dm1-MMP (61 and 49 kDa). From the deduced amino acid sequence, one can predict the size of the active band by calculating the MW of the sequences downstream of the cysteine switch (starting at residue 40, Fig. 1). By this method, activated Ms-MMP is predicted to be 49.8 kDa. However, post-translational processing, including phosphorylation and proteolytic cleavage, alternative splicing, and the formation of dimers are common among MMPs and may result in multiple bands (Woessner and Nagase, 2000; Greenlee et al., 2007; Cauwe and Opdenakker, 2010). Alternatively, the extra bands detected in Figures 3B and 4C could be other MMPs, since the peptide used to generate the antibody is from the highly conserved region of the protein. Clearly, more work needs to be done to understand the functional significance of the tissue-specific Ms-MMP expression patterns.
To date, the role of MMPs in insect development has been investigated in only a few studies, including the present study (Page-McCaw et al., 2003; Knorr et al., 2009; Glasheen et al., 2010). Two studies showed that lack of MMP1, either by mutation (D. melanogaster) or RNAi (T. castaneum), caused defects in tracheal respiratory structures, which were lethal in D. melanogaster. Dm1-MMP is required for elongation and growth of tracheae, as well as degradation of cuticle for proper ecdysis (Glasheen et al., 2010). In addition, T. castaneum and D. melanogaster were unable to successfully pupate, suggesting that there are multiple roles for MMPs in insect development (Knorr et al., 2009; Glasheen et al., 2010). The mechanism of MMP action in insects is largely unknown, but MMPs in mammals have been shown to cleave chemotactic factors (Greenlee et al., 2006) and cytokines (reviewed in Greenlee et al., 2007), as well as serine proteases (Brown et al., 1995) and their inhibitors (Liu et al., 2000), all of which may be important for tissue remodeling or degradation. Indeed, serine proteases are a major component of molting fluid (Katzenellenbogen and Kafatos, 1970, 1971; Bade and Shoukima, 1974; Bade and Stinson, 1978; Samuels et al., 1993a, 1993b; Samuels and Reynolds, 2000).
Inhibition of Ms-MMP in our system resulted in decreased growth and delayed appearance of the dorsal vein. While the underlying cause of the delay has not yet been investigated, it could have resulted from tracheal defects, as was found in D. melanogaster (Page-McCaw et al., 2003). Tracheal defects could result in decreased oxygen availability and cellular hypoxia, which has been shown to slow development in other insects (Loudon, 1988; Greenberg and Ar, 1996; Klok et al., 2009). Alternatively, the decrease in growth could be due to dysfunction or dysregulation of metabolic enzymes that are substrates of MMPs. MMPs may cleave many intracellular proteins, including enzymes required for metabolism and oxidation of reactive oxygen species (reviewed in Cauwe and Opdenakker, 2010). Inhibition of Ms-MMP at the transcriptional level would yield more clear results, since ensuring complete inhibition of enzymatic activity is difficult with injection of chemical inhibitors.
The increased expression of Ms-MMP near the 4th to 5th instar molt hints at its role in the process of molting. Previous use of the MMP inhibitor, 1,10-phenanthroline in M. sexta resulted in an 83% reduction of proteolytic activity of the molting fluid from 5th instar larvae and 98% of proteolytic activity from molting fluid of pupae that were nearing adult emergence (Samuels et al., 1993a), suggesting that one or more MMPs may be involved in molting, either directly or indirectly. The process of molting includes both the separation and shedding of the old cuticle, apolysis and ecdysis, respectively (Chapman, 1998). These processes require several enzymes, many of which have been identified from the fluid filling the space between the old and new cuticle, called molting fluid. These enzymes include chitinases, phosphatases, and phenoloxidases (Reynolds and Samuels, 1996), which could be activated or inactivated by MMPs. Furthermore, two trypsin-like serine proteases have been identified from the molting fluid of B. mori (Katzenellenbogen and Kafatos, 1971), and serine proteases are commonly substrates of MMPs (Overall and Blobel, 2007).
Despite the strong evidence for a potential role for Ms-MMP in larval–larval molting, our in vivo inhibition experiment did not prevent the 4th to 5th instar molt. Inhibitor injections began on the third day of the 4th instar with the goal of inhibiting the molt to the 5th instar. However, caterpillars molted synchronously. Because the 4th instar is short (the 4th instar larvae start to molt on the 4th day), injections from the first day of the 4th instar may help to elicit a stronger effect of the inhibitor on molting.
Although we were unable to prevent larval–larval molting, in vivo inhibition of Ms-MMP resulted in a decrease in growth (Fig. 5A). The decreased growth rates of inhibitor-treated caterpillars manifested as a delay in the events leading up to pupation, in which the dorsal heart appeared a day and a half after those of control caterpillars (Fig. 5B). Similar results were obtained in the flour beetle T. castaneum, where the larval–pupal transition was inhibited when MMP-1 was knocked down using RNAi (Knorr et al., 2009) and in M. sexta fed 1,10-phenanthroline, which had delayed and arrested development (Bade and Shoukima, 1974). These results yield several hypotheses about the role of MMP in developing insects. One hypothesis is that Ms-MMP may regulate PTTH, which initiates gut purging along with the appearance of dorsal heart (Nijhout and Williams, 1974). Alternatively, Ms-MMP may regulate one or more proteases involved in tissue degradation, including clearance of tissue surrounding the dorsal blood vessel (Nijhout and Williams, 1974). Since we observed a general decrease in growth rate, but inhibited caterpillars eventually reached the same mass at pupation, Ms-MMP may activate or inactivate intracellular enzymes, such as those involved in carbohydrate metabolism or protein synthesis (Cauwe and Opdenakker, 2010). Lastly, MMPs are known to be important in the innate immune response in mammals (Li et al., 2004; Hong et al., 2011) and in insects (Altincicek and Vilcinskas, 2008; Knorr et al., 2009), and it is possible that the decreased growth occurred because the inhibitor-treated caterpillars had sub-lethal infections and exhibited a trade-off between immunity and growth.
While MMP research in mammals is extensive, research on MMPs in invertebrates is just beginning. MMPs are important proteases, functioning in processes as varied as development, wound healing, and immunity, and yet many of their functions and substrates are still unknown. Our data along with the handful of other studies on insect MMPs show an intriguing pattern of MMP involvement in insect development and immunity. Understanding how the functions of similar MMPs vary with taxa from flies to beetles to butterflies may yield important information about how vertebrate MMPs evolved. In addition, the similarity between insect MMPs and vertebrate MMPs makes these a desirable model for testing hypotheses about MMP function. For example, if MMPs are involved in carbohydrate metabolism, at the cellular level, vertebrate functions may also be similar. Insects can also serve as useful models for innate immunity, since hemocytes are very similar to mammalian immune cells (Lemaitre and Hoffmann, 2007; Strand, 2008) and they both express MMPs (Johansson et al., 2005; Knorr et al., 2009). Finally, elucidating functions for MMPs in lower taxa may yield critical information about the evolution of the numerous MMPs found in vertebrates.