Introduction

Included in the five classes of proteinases catalyzing hydrolysis of peptide bonds, serine endoproteinases are divided into two superfamilies that evolved similar catalytic mechanisms independently. The trypsin superfamily includes trypsins and chymotrypsins, which are ubiquitous in animals, while the subtilysine superfamily is present in most bacteria and fungi studied to date (Hu and Leger 2004). Regardless of origin, trypsins are classified in clan SA, family S1 (Webb 1993; Barrett et al. 1998). Trypsins possess a catalytic triad that characterize all serine proteinases, consisting of His, Asp, and Ser amino acid residues and typically show two characteristics that distinguish them from other serine proteinases: (1) specificity for the peptide bond formed by the carboxylic-side of Arg or Lys residues and (2) ability to activate other pancreatic zymogens (de Albuquerque et al. 2001).

The ability to digest protein must have emerged early in evolution, as shown by the finding of trypsin coding genes in eubacterial genomes (Rypniewski et al. 1994). Primitive organisms selectively fixed the ability to use foreign protein as a source of amino acids by synthesizing enzymes, now termed proteinases, which were capable of recognizing and hydrolyzing peptide bonds in a specific region of a polypeptide chain, based on specificity for the side chains of amino acid residues in the carboxylic side of the site of cleavage.

Invertebrate proteolytic enzymes have been widely studied and special attention has been directed to the trypsin group (EC 3.4.21.4) because they take part in a number of physiological processes, playing a major and better-understood key role in hydrolyzing food protein (Díaz-Mendoza et al. 2005; Delcroix et al. 2006). Among invertebrates, crustaceans and insects have been the traditional model, but an increasing list of species belonging to other taxa have contributed to understanding and characterizing physiological and biochemical processes in which these enzymes are involved (Noriega and Wells 1999; Maeda-Martínez et al. 2000; de Albuquerque et al. 2002). The first studies of proteinases, mainly trypsin and chymotrypsin in vertebrate species, started during the first part of the twentieth century, describing kinetic properties, isolation, some molecular characteristics, and evolution of trypsin and trypsinogen. The first reports of invertebrate trypsins were published in the 1960s, when secretion of digestive enzymes responsible for hydrolysis of polymeric nutrients in food were described in insects and crustaceans (Davie and Neurath 1955; Keil et al. 1968; Arnon and Neurath 1969; Pfleiderer et al. 1970; Yang and Davies 1971; Gooding 1975; Gibson and Barker 1979), showing that most terrestrial and aquatic invertebrates synthesize and secrete trypsin and other related enzymes. It is now well documented that invertebrates synthesize paralogue (homologue enzymes in the same organisms) and orthologue (homologue enzymes within a taxa) trypsins (Zhu et al. 2000; Heger and Ponting 2008). In 1876, Wilhelm Friederich Kuehne discovered a protein substance, in pancreatic juice of pig that degraded other protein substances and named it trypsin. Kuehne (1876) soon learnt that this pancreatic proteinase was initially in an inactive state, but spontaneously converts to the active form and is inactivated (denatured) by heat. One of the first proteinases isolated with sufficient purity and enough quantity for accurate biochemical analysis was trypsin from bovine pancreas (Northrup et al. 1948). Bovine trypsin was purified by crystallization in the early 1930s (Northrup and Kunitz 1931) and almost the entire amino acid sequence of the zymogen was known by protein sequencing in the 1960s (Walsh et al. 1964; Walsh and Wilcox 1970). With the advent of X-ray crystallography, the three-dimensional structure of bovine trypsin was resolved in the 1970s (Huber et al. 1974; Sweet et al. 1974; Kossiakoff et al. 1977; Rypniewski et al. 1993). Thus, over several decades, details of the amino acid sequence, structure, and mechanism of action of trypsin were revealed in a large number species, including invertebrates (Male et al. 1995; Noriega and Wells 1999; Heheman et al. 2007).

Given the importance of trypsin’s functions and ubiquity, its evolution has been intensely studied. Using Fourier transformation of biological sequences, Baptista et al. (1998) found strong evidence for tandem gene duplication. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which contemporary trypsins have developed. Since invertebrates have a body temperature close to that of the environment, adaptations to temperature should have evolved. Comparative studies of structures from several trypsins revealed that several characteristics are unique to cold-adapted trypsins. An example of such characteristics is the increase in substrate affinity from a lower electrostatic potential of the S1-binding pocket provided by Glu221B and the absence of five hydrogen bonds adjacent to the catalytic triad, compared to mesophilic counterparts (Schrøder-Leiros et al. 2000).

In this review, we summarize the knowledge concerning invertebrate trypsins and make comparisons with vertebrates when a topic is poorly understood in invertebrates. The review encompasses molecular and kinetic characteristics, mechanisms regulating measurable activity in digestive organs, mechanisms dealing with unwanted autolysis of cell and tissue proteins, genetics, evolution, and function.

Characteristics of invertebrate trypsin

Trypsins show species-specific characteristics since there are significant differences in feeding habits, ingested food composition, and differences in the protein digestion process, according to the requirements of the species. These differences are adaptive responses to different life styles, environments, and mostly, different survival abilities among species. However, trypsins share some general characteristics, as observed in Tables 1 and 2, mainly related to their catalytic features.

Table 1 General characteristics of various trypsins
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Table 2 Trypsin genes from invertebrate species and translated/ isolated protein characteristics
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Ca2+ dependence

To date, it is not conclusive whether invertebrate trypsins depend on a moderate concentration of Ca+2 for maximum activity and stability, as observed in mammalian trypsins. Trypsin from starfish Asterias amurensis and Lysastrosoma anthosticta and insects Diatraea saccharalis, Periplaneta americana, Tenebrio mollitor, and Musca domestica are not dependent on Ca+2 for activation or stability. In fact, the calcium-binding motif that is commonly observed in mammalian trypsins does not occur in most of these insect trypsins (Dionysius et al. 1993; Kishimura and Hayashi 2003; Lopes et al. 2006). However, trypsin structure from the crayfish Astacus leptodactylus has a high-affinity, calcium-binding site formed by the Glu70–Glu80 loop (Fodor et al. 2005). Some authors report activating and stabilizing effects of calcium on serine proteinases from invertebrates, such as the crab Cancer pagurus (Saborowski et al. 2004) and Australian redclaw crayfish Cherax quadricarinatus (Figueiredo et al. 2001).

Structure

The three-dimensional structure of enzymes in the S1 family is highly conserved, even though primary structures vary to a large extent. To date, the crystal structure of trypsin enzymes has been studied for only two crustaceans, the freshwater crayfish A. leptodactylus (Fodor et al. 2005) and the marine crab C. pagurus (Heheman et al. 2007). Active trypsin consists of a single polypeptide chain; the catalytic amino acid residues lie between two-stranded, β-barrel domains packed against each other. Ser195, in the numbering system of bovine chymotrypsin (Rypniewski et al. 1994), acts as a nucleophile in hydrolysis and produces an acyl-enzyme intermediate with the substrate, while His57 acts as a general base and Asp102 stabilizes the correct tautomer of His57 and provides compensation for developing of a positive charge during the catalytic reaction. The substrate forms an anti-parallel β sheet with the protein-binding site and specificity is related to the Asp189 side chain that is located at the bottom of the binding pocket, which provides a negative charge for attracting the positively charged Lys or Arg amino acid residue in the substrate. Fodor et al. (2005) reported some important differences between crayfish trypsin structure and that of vertebrates. They found four loops (37, 60, 145, and 202) in crayfish trypsin that are different from vertebrates. Also, an extended hydrophobic region in crayfish trypsin includes a five-residue insertion at position 37, which is suspected to be involved with inhibitor interaction and a seven-residue insertion at position 60. These insertions affect the molecular mass (kDa) and pI values, yielding both cationic and anionic enzymes (Table 1; Barrett et al. 1998). Most relatives of invertebrate trypsins have an N-terminal signal peptide and are synthesized as precursors with an N-terminal extension acting as proenzymes that require proteolytic cleavage to yield the active enzyme (Amino et al. 2001; Sainz et al. 2004b).

Evolution

The two central elements of neo-Darwinian evolution are small random variations and natural selection. Comprehension of evolution requires the understanding of the details of random variation of DNA and the natural selection acting on gene products. The comparison of two or more sequences is the basis for evolutionary analysis, and it may reveal similarities that arise through random chance, sharing a common ancestor, or sharing a common selective pressure. In general, trypsin sequences in invertebrate species are still a small number, which provides an incomplete description of the long evolutionary process that these enzymes might have undergone.

In the late 1970s, immunological methods were used in studies on the evolution of peptidases. Studies of trypsins of vertebrates suggested an evolutionary relationship among these enzymes, finding that mutations affecting the structure of enzymes were extensive, although catalytic activity and specificity were barely affected (Pfleiderer et al. 1967; Arnon and Neurath 1969; Pfleiderer et al. 1970). The high degree of cross-reactivity among different trypsins, including those in invertebrates, indicated that several features on the surface of the molecule are common in these proteins. These data, in turn, suggested a high-degree of similarity in the three-dimensional structure of vertebrate and invertebrate trypsins, thus establishing that trypsins appeared before invertebrates and vertebrates diverged (Arnon and Neurath 1969).

Studies of the phylogeny of trypsins and trypsinogens of vertebrates and invertebrates provided some clues to the evolution of these protein families (Roach et al. 1997). Trypsin of the midgut gland of the crayfish Astacus fluviatilis, a species on the evolutionary pathway when decapods and vertebrates diverged 700 mya, shows that some essential structural properties are more closely shared with bacteria trypsin than with bovine trypsin, suggesting that crayfish trypsins should be placed closer to bacteria trypsins than to vertebrate trypsins. Vertebrate trypsins evolved new characteristics, such as a larger number of disulfide bridges (Zwilling and Neurath 1981; Titani et al. 1983; Neurath 1984). However, the main and more conserved sequence characteristics of vertebrate trypsins have also been observed in invertebrate trypsins, like the key amino acid residues in the specificity pocket Asp, Gly, Gly (Roach et al. 1997) and the three amino acid residues at the active site, His, Asp, and Ser, that are involved in the catalytic mechanism of serine proteinases (Neurath 1984; Pancer et al. 1996). Also, in the invertebrate trypsinogens, the presence of a signal peptide with a typical cleavage site, Arg or Lys/Ile, in vertebrates is observed (Table 2; De Haën et al. 1975; Klein et al. 1996; Lehane et al. 1998). However, important differences between vertebrate and invertebrate trypsinogens have been found. The shrimp and crayfish signal peptides in trypsinogens appear to be longer than those of vertebrates and other invertebrates, according to Peterson et al. (1994), but it seems that such results are not in agreement with new information (Fig. 1); however, we suggest that crustacean trypsins are more similar in size than those in insects.

Fig. 1
figure 1

Amino acid sequences of purified proteins or deduced trypsinogen proteins from invertebrates. IVGG indicates the N-terminal residues of the active enzymes starting at Ile 16 (as numbered in vertebrate trypsinogens). Six conserved cysteines are marked with circles; two additional cysteines are marked in Diaprepes abbreviatus and Penaeus vannamei sequences. Active-site, triad-position amino acids are indicated with an asterisk on the top, and amino acid residues in the specific binding pocket are indicated with a black square on the top

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Other important difference found between vertebrate and invertebrate trypsins is the number of Cys residues and, as a consequence, in the number and location of disulfide bridges. Vertebrate species commonly have six disulfide bridges, and some insect and crustacean trypsins have only three at conserved positions, which seems to be conserved bonds because their position is closer to the active site of the enzyme, as seen in the crayfish Pacifastacus leniusculus and A. leptodactylus and insects Anopheles gambiae and Pediculus humanus (Table 2; Fig. 1; Fletcher et al. 1987; Müller et al. 1993; Hernandez-Cortés et al. 1999a; Kollien et al. 2004; Fodor et al. 2005). In the shrimp P. vannamei (Klein et al. 1996) and the citrus weevil Diaprepes abbreviatus, eight Cys residues and four disulfide bonds are present. Differences in the number of Cys residues have been linked to the early evolutionary separation of vertebrates and invertebrates (Titani et al. 1983). De Haën et al. (1975) suggested that their common ancestor had the same number of disulfide bonds at the same positions as all vertebrate trypsins.

Rypniewsky et al. (1994) described a phylogenetic tree with a continuous evolution of trypsins from a single ancestral gene. A few years later, Roach et al. (1997) suggested that vertebrate trypsinogen evolution was dynamic and multi-modal, including gross duplication of a whole gene or part of the trypsinogen gene locus after elasmobranches diverged, giving rise to two groups of trypsinogen genes: anionic and cationic trypsinogens. This idea supports the hypothesis that a single, gene-duplication occurred prior to the divergence of mammals (Rypniewsky et al. 1994). The time needed for the divergence of trypsin into cationic and anionic forms has been subject to speculation. Fletcher et al. (1987) stated that, because of the high homology among the amino acid sequences of cationic trypsinogens in different species, divergence of a primordial trypsinogen gene should occur before the divergence of rodents and ungulates. The structure of anionic and cationic trypsinogens has been studied in vertebrates; however, the manner in which the sequences of these proteins dictate their structures is not understood (Pasternak et al. 1999).

Anionic and cationic trypsins have been observed in invertebrates. Ward (1975) reported the purification and properties of anionic trypsin in larvae of the webbing clothes moth Tineola bisselliella and Pancer et al. (1996) reported the sequences of two cDNAs coding for putative anionic trypsinogens from the colonial ascidian Botryllus scholosseri. In the insects T. mollitor and Locusta migratoria, two or three forms of anionic trypsinogen have been found in the midgut, together with a single cationic trypsinogen (Lam et al. 2000; Lopes and Terra 2003). Thus, besides differences in the overall net electric charge between cationic and anionic trypsinogens, the presence of trypsins, either cationic or anionic, is species-specific and the electric charge could be functionally important; however, little is known about the practical significance of different trypsins and its conservation during evolution (Fletcher et al. 1987; Pancer et al. 1996).

Mechanisms regulating trypsin activity

Trypsins are paramount effectors in the network of enzymatic functions, since its activation is the first step in a series of consecutive reactions, where the function of multiple proteinases of different classes is part of a coordinated physiological process. Digestion of food protein is a prime example (Neurath 1984; Ehrmann and Clausen 2004; Delcroix et al. 2006).

Because synthesis and storage of active proteinases impose a risk of tissue damage by hydrolysis of autologous protein, organisms must possess a mechanism controlling activation until needed for a precise function. This is achieved by a complex system that involves control of synthesis; transcription and translation, storage, secretion, and activation of zymogens. Several mechanisms of regulation of proteolytic enzyme activity and function are known in vertebrates, and recently some of those mechanisms have been found to share similarities to invertebrate models, such as mosquitoes, other hematophagous insects, and crustaceans (Carreira et al. 1996; Lehane et al. 1998; Sahin-Tóth 2000). Different mechanisms of regulation at transcriptional, post-transcriptional, and translational levels have been suggested to exist in invertebrates, either alone or combined, and that they can be organized spatially and/or sequentially (Xiong and Jacobs-Lorena 1995; Noriega and Wells 1999).

Trypsin genes and transcriptional regulation

Transcription of genes encoding trypsin in vertebrates and invertebrates has been suggested as a major regulatory mechanism. Even though a few invertebrate trypsinogen nucleotide sequences have been reported, trypsin genes appear to be commonly represented as families in many invertebrates (Table 2; Zwilling and Neurath 1981; Zhu et al. 2000; Diaz-Mendoza et al. 2005). In the mosquito A. gambiae, trypsin genes are arranged as a tightly clustered gene family consisting of seven related coding sequences (Müller et al. 1995). The sheep blowfly Lucilia cuprina has a four-member multi-gene family of trypsins (Casu et al. 1994). Wang et al. (1995) suggested that the ancestors of dipterans and lepidopterans had only one trypsinogen gene, and that extra copies were gained by gene duplication.

Kollien et al. (2004) found only one trypsin gene in the hematophagous louse P. humanus. Incubation of the translation product in the presence of chymotrypsin yielded bands at 33 and 26 kDa, suggesting a novel and uncommon mechanism of trypsin activation in insects. The significance of this finding remains to be investigated. Within a species, the way an individual regulates the presence and activity of a digestive enzyme is varied, and sometimes, opposing. Lehane et al. (1998) reported two trypsins produced as pre-proenzymes in the opaque zone of the midgut of the horsefly Stomoxys calcitrans, which are activated after a blood meal. In contrast, in the yellow fever mosquito Aedes aegypti, the absence of trypsin zymogens allows the organism to develop a particular regulation mechanism (Noriega and Wells 1999).

How trypsin multi-gene families provides adaptive value is not clear; however, Bown et al. (1997) and Zhu and Baker (1999) suggested that the complex provides an efficient mechanisms for protein degradation and an adaptive advantage for insects feeding on plants containing trypsin inhibitors (“Inhibition”). Various regulatory mechanisms of trypsins have been suggested for different invertebrates, especially during gene transcription in the cell nucleus and during translation or post-transcription. The female yellow fever mosquito A. aegypti is an example, possessing several regulatory mechanisms, since trypsin expression is a complex biphasic mechanism dependent on tissue and gender (Kalhok et al. 1993); transcription of early trypsin, preceded by juvenile hormone and ingestion of a blood meal, induces an increase of proteolytic activity by facilitating its translation. Early trypsin shows a negligible endopeptidase activity in the midgut. Late trypsin transcription reaches a maximum 18–24 h after the blood meal (Barrillas-Mury et al. 1991; Barrillas-Mury and Wells 1993; Lu et al. 2006). The presence and amount of each isoenzyme depends on a refined control mechanism after ingestion of blood; the amount of mRNA of early and late trypsins is also found to vary (Noriega et al. 1994). The mRNA for early trypsin is present in suitably small concentrations, affecting translation of late trypsin after ingestion of blood, then the concentration of the early trypsin mRNA drops. This sequence of events is repeated in the next gonadotropic cycle, when mRNA returns to its original level before the blood meal. The mature, early trypsin is synthesized as an active form without a trypsinogen stage (Noriega et al. 1996; Noriega and Wells 1999). It seems that this regulation allows the mosquito to assess the quality and amount of the blood protein by early trypsin proteolysis. For some species, trypsin genes have been studied to understand its regulatory mechanisms. Some of these mechanisms, as promoter sequences in malaria mosquito group A. gambiae, have been described and partially elucidated (Müller et al. 1993; Skavdis et al. 1996; Lehane et al. 1998).

Four genes, belonging to a clustered gene family coding for trypsin enzymes, were isolated from the Mediterranean fruit fly Drosophila melanogaster (Davis et al. 1985). The four genes in the family are transcribed in alternating orientations and the activation peptide sequence of the α gene is not similar to that of most trypsins reported, indicating a modified activation mechanism. In Drosophila, the promoter function was highly conserved during evolution and Drosophila transcription factors recognize promoter DNA sequences from other distantly related species, such as the black fly Simulium vittatum and the mosquito A. gambiae (Shen and Jacobs-Lorena 1998). In fact, some cis-acting DNA sequences, such as the TATA box, the initiator region TCAGT, and other upstream promoter elements, have been identified immediately upstream of non-Drosophila insect trypsin genes (Xiong and Jacobs-Lorena 1995; Harshman and James 1998). This means that transcriptional regulatory elements do exist in invertebrates, perhaps distributed in a ubiquitous manner, and that these elements, according to the authors’ suggestions, are evolutionarily conserved.

In contrast to the A. aegypti mosquito mechanism, where trypsin gene transcription seems to be inducible, in the midgut epithelium of the S. calcitrans fly, a large amount of trypsin mRNA is stored even in unfed individuals. This supports evidence that trypsin genes may be constitutively transcribed and that control over production of digestive trypsin can be regulated during post-transcription (Lehane et al. 1998). In other species, studies have demonstrated that a series of constitutive and blood meal-induced trypsin genes are present in the gut of both genders during the stages of the life cycle, as in the A. gambiae mosquitoes (Müller et al. 1995).

Studies of trypsins in marine invertebrates, especially crustaceans, have shown various trypsin genes dispersed in different multi-gene families, such as the whiteleg shrimp Penaeus vannamei, where the number of introns in the three trypsin genes varies from zero to three, showing relatively conserved positions and, commonly, shorter sequences than those in vertebrate species (Klein et al. 1998). Comparisons of DNA sequences and cDNAs show three exons and two introns, while vertebrates commonly have five exons separated by four introns (Craik et al. 1984). In P. vannamei, three isotrypsins, A, B, and C, found in the digestive gland were purified and characterized by molecular, biochemical, and kinetic parameters (Sainz et al. 2004a), confirming that digestive trypsin is a polymorphic enzyme, as anticipated by Klein et al. (1996) in a study of cDNA. P. vannamei isotrypsins are codified at two loci: Locus β, which is homozygous and yields isoenzyme C and Locus α, which is heterozygous, yielding isoenzymes A and B (Sainz et al. 2005). In shrimp species, Al-Mohanna et al. (1985) and Lehnert and Johnson (2002) proposed that fibrillar cells (F) of the midgut gland tubules are the only sites for the secretion of digestive enzymes, which must be synthesized as zymogen granules and stored in supranuclear vacuoles similar to the formation of zymogens in the pancreatic exocrine cells of vertebrates. Al-Mohanna and Nott (1989) and Icely and Nott (1992) found that digestive enzymes in the vacuoles are liberated periodically into the gut, depending on the species and nutritional conditions. To date, the published information concerning isolation of invertebrate trypsinogens is scarce. Hernández-Cortés et al. (1999b) reported a trypsinogen in the midgut gland of the crayfish P. leniusculus. Lehnert and Johnson (2002) and Sainz et al. (2004b) used immunological approaches to find digestive trypsins in the giant tiger prawn Penaeus monodon and the whiteleg shrimp P. vannamei, respectively, stored as zymogens, which is a common strategy of frequent-feeder species. This information is relevant because studies of the organization and sequence of genes alone may never anticipate information about post-transcriptional modifications of trypsinogens.

We hypothesize that animals having isotrypsins have adaptive advantages; for example, Atlantic salmon with isotrypsin TRP-2*92 are larger than salmon without it (Rungruangsak-Torrisen et al. 1999). These authors believe that the effect is a consequence of better food conversion and protein efficiency, at least under the conditions of the experiment. Some isoenzymes may be vestigial, which is not rare, according to Pils and Shultz (2004) who suggested, when analyzing complete metazoan genomes, that up to 15% of the members of all encoded enzymes families may have lost their catalytic activity. In pig, Barret et al. (1998) found that pepsin B has a weak, general proteolytic activity, about 4% of what occurs with pepsin A, and it does not contribute much to digestion.

Limited information concerning mechanisms of trypsin activity regulation in marine invertebrates has been obtained. It is generally accepted that proteinolytic digestive enzymes are transcriptionally regulated in mammals, with the quantity and quality of the protein in food being one of the major inducers (Lhoste et al. 1994). Studies of insect species also suggest the influence of food in trypsin activity as in the larvae of the red palm weevil Rhynchoporus ferrugineus (Alarcon et al. 2002) and in the mosquito A. aegypti (Noriega et al. 1994, 1996) indicating that one of the trypsin genes is mainly regulated for transcription by the quality and quantity of meal. Similar mechanisms have been described for some crustaceans. Le Moullac et al. (1994) suggested that food protein quality induces synthesis of digestive enzymes (trypsin, chymotrypsin, and amylase) in the larvae of whiteleg shrimp P. vannamei and some interesting observations of this species have been made in our lab. After starvation for 24 h, the amount of trypsin mRNA increased significantly, followed by a steep decline after feeding, compared to shrimp that had not been starved (Sánchez-Paz et al. 2003). Starvation affected midgut gland trypsin activity in a similar way (Muhlia-Almazán et al. 2002). Thus, a decrease of both trypsin activity and mRNA by starvation suggests a strategy to reduce enzyme synthesis, and consequently, to save energy required for protein synthesis, as well as preventing potential autolysis. Such results suggest a mechanism of regulation during transcription by external factors. Additionally, this could be an effective strategy for handling the effects of medium-to-long-term starvation and avoiding deleterious effects on fitness. This knowledge opens a door for management of the digestive system for greater efficiency, at least in pond-raised shrimp.

Zymogens

Vertebrates synthesize trypsin as a non-active precursor called trypsinogen, which is commonly stored in intracellular organelles until it is activated-secreted into the intestine lumen for food protein digestion. The active trypsin form is achieved by the hydrolytic post-translational removal of a short, highly charged peptide in the amino terminus of the zymogen, the trypsinogen activation peptide (TAP), by specific cleavage between lysine/arginine and isoleucine residues (Male et al. 1995). The transition from the inactive to an active state is achieved by reorientation of the so-called activation domain: the new N-terminus (Ile 16) folds into a pocket and makes a salt bridge with Asp 194, which results in an ordered, conformational change in the activation domain that creates the substrate binding site (Fig. 1; Pasternak et al. 1999; Ehrmann and Clausen 2004). Activation of trypsin zymogen and cleavage of signal peptides are two of various post-translational controls that digestive proteinases undergo. During the 1950s, bovine trypsinogen was shown to be activated through the cleavage of a peptide Val-Asp-Asp-Asp-Asp-Lys-Ile located on the N-terminus of the protein. The heptapetide was cleaved autocatalytically and by its physiological activator, enteropeptidase (Chen et al. 2003). The Asp4 sequence was found conserved in several mammal species, including human (Guy et al. 1978; Emi et al. 1986), cow (Davie and Neurath 1955; Le Huerou et al. 1990), goat (Bricteux-Gregoire et al. 1971), sheep (Schyns et al. 1969), pig (Louvard and Puigserver 1974), dog (Pinsky et al. 1985), cat (Steiner et al. 1997), and rat (MacDonald et al. 1982). However, the number of consecutive aspartyl residues seems to reach a maximum in TAPs of mammalian vertebrates, while in other vertebrates this number decreases. In this way, none of the TAP sequences found in bacteria (Kim et al. 1991), fungi (Rypniewsky et al. 1993), crustaceans (Hernández-Cortes et al. 1999b; Johnson et al. 2002) and insects (Müller et al. 1993; Wang et al. 1999; Kollien et al. 2004) possess an acidic residue immediately before the Lys or Arg prior to the conserved sequence Ile-Val-Gly-Gly of the amino terminus of mature trypsins (Zhu and Baker 1999). In the tunicates (Roach et al. 1997) and the ascidian Botryllus schlosseri (Pancer et al. 1996), TAP sequences have an Asp before the Lys23-Ile24 cleavage bond. Additional Asp residues were added during the course of vertebrate evolution: one in sea lamprey (Roach et al. 1997); two, three, or four in fish, amphibians, and birds (Shi and Brown 1990; Gudmundsdottir et al. 1993; Male et al. 1995; Wang et al. 1995; Douglas and Gallant 1998; Kurokawa et al. 2002); and four in mammals. This fact suggests two features: (1) progressive increase in selective pressure for such acidic residues during the course of vertebrate evolution (Chen et al. 2003), and (2) increasing number of Asp residues preceding the Lys23-Ile24, associated with a progressive decline in the tendency for auto-activation. Studies using small, synthetic peptides indicate that Asp4 significantly slowed hydrolysis of the Lys23-Ile24 bond by bovine trypsin, but enhanced the action of enteropeptidase. This suggests that, although Asp4 might play a protective role against spontaneous activation of the zymogen within the pancreas, it must constitute the signal for specific activation of trypsinogen by enteropeptidase in the duodenum (Chen et al. 2003). Peterson et al. (1994) found that activation peptides in the midgut gland of insects, such as Manduca sexta, lack the enteropeptidase consensus sequence (DDDK), which is specific for cleavage in most vertebrate trypsinogens. This observation was confirmed for the human body louse P. humanus (Kollien et al. 2004) and fruit fly D. melanogaster (Davis et al. 1985). Recent reports show evidence suggesting the existence of reversible zymogen activation in bacteria and vertebrates, this on/off switch of proteinase activity may be regulated by concentrated salts (Huang et al. 2001; Friedrich et al. 2003) or temperature (Spiess et al. 1999; Ehrmann and Clausen 2004). However, no reports concerning this activity have been published for invertebrate trypsins. Undoubtedly, the activation peptide is located at the amino acid end of the molecule, which, by selective pressure, is a useful feature because zymogen is synthesized beginning with the activation peptide for the emerging proteinase born inactive.

Endocrinology

One of the distinctive features in studies of insect gene regulation is the support provided by the rich tradition of insect endocrinology. Insect endocrinology is based on a well-established experimental logic that uses extirpation, ligation, transplantation, and administration of hormonally active compounds (Harshman and James 1998). Only a few studies about hormone control of genes expression or protein activation have been reported in invertebrates (Harshman and James 1998). Hence, endocrine control of trypsin secretion in invertebrates is far from being fully understood.

During the late 1970s, researchers found that median neurosecretory cells of the yellow fever mosquito A. aegypti contain factors that enhance the secretion of trypsin twofold (Briegel and Lea 1979), and that ecdysteroids secreted by the ovaries were capable of enhancing the secretion of proteases (Briegel and Lea 1977). Later, Graf et al. (1998) showed that trypsin synthesis was reduced to less than half of its normal output when mosquitoes were decapitated or ovariectomized, indicating that hormonal factors released from the brain and ovaries influenced the rate of trypsin synthesis. Besides, researchers found that juvenile hormone regulates early trypsin synthesis by controlling transcription in the same species (Noriega and Wells 1999). Early trypsin mRNA is absent in larvae, pupae, and newly emerged females. The advent of the juvenile hormone is the signal for the transcription of the early trypsin gene when the organism reaches maturation. Thus, protein in the blood meal is responsible for translation (Noriega et al. 1996).

In mosquitoes, flies, fleas, and biting midges, the trypsin-modulating oostatic factor, TMOF, a decapeptide that functions as a physiological signal that terminates trypsin biosynthesis, was added to feed the citrus weevil D. abbreviatus. This stopped trypsin activity in the midgut gland, as well as significantly decreasing larval weight, growth, and survival. This suggests that trypsin synthesis decreases during translation, probably under the influence of TMOF-like hormones (Yan et al. 1999; Borovsky 2003).

In decapod crustaceans, a neuropeptide, the crustacean hyperglycemic hormone (CHH), is synthesized by neurosecretory somata, grouped together in the X-organ. CHH is a pleiotropic hormone that plays a central role in regulation of glycemia (Ollivaux and Soyes 2000). Sedlemeier (1988) showed that CHH exhibits a secretagogue effect on digestive enzymes, particularly controlling amylase release by the midgut gland of the crayfish Orconectes limosus. To date, a possible regulatory effect of CHH over trypsin synthesis remains unknown.

In penaeids, neuroendocrine regulation of trypsin activity was suggested by Gibson and Barker (1979) and Sedlmeier (1988). Release of pancreatic enzymes requires participation of gastrointestinal hormones, such as cholecystokinin (CCK), gastrin, secretin, and bombesin. In mammals, regulation by hormones plays a role in appetite satiation, pain perception, and neuronal transmission (Gibbs et al. 1979). In insects, some substances of the CCK-gastrin family have been identified, but efforts to purify gastrointestinal hormones from crustaceans have failed and knowledge of these substances is still limited. Since Larson and Vigna (1983) proposed that CCK-gastrin evolved in invertebrates as a neural peptide and was subsequently used by vertebrates as a regulatory peptide in the nervous and gastrointestinal endocrine systems, we might expect these gastrointestinal materials in crustaceans would be widespread. These authors found that the crab Cancer magister had structurally similar peptides to the bioactive C-terminal amino acid sequence common to gastrin and CCK. Also, strong recognition for CCK-like peptides was detected when using antibodies generated against mammalian CCK8 in rock crabs Cancer borealis (Christie et al. 1995). Sulfakinins were recently discovered as multi-functional peptides with homologies to G-CCK peptides, which have, so far, been found only in insects and crustaceans; they inhibit food intake and stimulate release of enzymes, such as α-amylase, among others (Nichols et al. 1988; Torfs et al. 2002; Meyering-Vos and Müller 2007). Johnsen et al. (2000) purified three novel members of the sulfakinin family from an extract of the central nervous system of the giant tiger prawn P. monodon. Immuno-cytochemical studies with sulfakinin antisera showed a sparse neuronal distribution pattern, similar to that of insects. This suggests a role in neurotransmission or neuromodulation, although a direct role in digestive system physiology remains to be clarified, as has been shown by in vitro assays of the sulfakinins in orthopteran and blattodean insects (Johnsen et al. 2000; Meyering-Vos and Müller 2007). In the American crayfish O. limosus, Resch-Sedlmeier and Sedlmeier (1999) detected proteinase and amylase activity in the midgut gland after incubation with CCK-8, gastrin, secretin, and bombesin. These results may support the assumption that Crustacea may possess endogenous factors similar to vertebrate hormones, at least, that the pertinent receptors have the ability to recognize some hormones.

Inhibition

One of the most important mechanisms regulating proteolysis of autologous proteins is the synthesis of endogenous, cognate inhibitors that maintain inactivity in spontaneously activated enzymes. Inhibitors play a major role in regulating enzyme activity in plants, animals, and microorganisms. Avoiding unwanted autohydrolysis is so essential to the organism that considerable energy must be invested in producing proteinase inhibitors. Some data reveal the importance of inhibitors in a broad range of processes. Inhibitors hamper proteinase activity by forming inhibitor–enzyme complexes. The resulting inhibitor–proteinase complex is inactive because the catalytic site of the enzyme is blocked. According to Krowarsch et al. (2003), three mechanisms evolved for inhibitors to block access of the substrate to the enzyme. (1) Canonical inhibitors bind to the enzyme through an exposed, convex-binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of the ideal substrate. (2) Non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. (3) Serpins, similar to canonical inhibitors, interact with their target proteinases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

Given the importance of proteinase inhibitors in modulating biological processes, some invertebrate trypsin inhibitors are intended for practical medical applications. Examples are the recombinant leech-derived tryptase inhibitor-2PL (LDTI-2PL) or the tick Boophilus microplus trypsin inhibitors (BmTIs) that are intended to study the effects of serine proteinase inhibitors on the inflammatory process induced by administration of carrageen into the rat pleural cavity and on release of kinins in pleural exudates (Malavazi-Piza et al. 2004). Proteinase inhibitors are extensively used in food technology. An example is surimi production, where hampering the muscle proteinases affects gel formation. (Comprehensive reviews of proteinase inhibitors in food technology can be found in García-Carreño et al. (2000) and García-Carreño and Hernández-Cortés (2000)). Because some invertebrate trypsin inhibitors are short polypeptides, ∼35 amino acid residues, they are studied to modify proteinases of practical interest, either depressing or enhancing function, for noxious or useful proteinases (Boigegrain et al. 1994). Some insect species frequently feed on plants, synthesizing and storing proteinase inhibitors to disrupt digestive enzymes of pests and predators; thus proteinase inhibitors play a significant role in the regulation of proteolysis, whether the target enzymes are of exogenous or endogenous origin; hence, some researchers suggested that insect digestive systems adapt to the presence of inhibitors (Cherqui et al. 2001; De Leo et al. 2001; Diaz-Mendoza et al. 2005).

Inhibitors of proteinases are exceptional molecules because, in spite of being proteins, they are negligibly hydrolyzed by the specific cognate enzyme. In many arthropod species, serine proteinase inhibitors have been identified (Cherqui et al. 2001). The serine proteinase inhibitors produced by plants and specific for invertebrate trypsins include inhibitors belonging to the Kunitz family as soybean trypsin inhibitor (SBTI) and soybean Kunitz trypsin inhibitor (SKTI) and the Kazal family in Schistocerca gregaria, A. leptodactylus, A. fluviatilis, Pascifastacus leniusculus, and Helicoverpa zea (Volpicella et al. 2003; Fodor et al. 2005) in arthropods. Such inhibitors are highly specific, possessing, at their surface, one or more peptide bonds made with the α-carboxylic side of Arg known as reactive sites. The reactive site specifically interacts with the active site of the cognate enzyme. Both natural and synthetic inhibitors inhibit trypsin. The extent of the inhibition (K i = molar) for trypsin goes from mM (millimolar) to fM (femtomolar), indicating that quite different affinities between them are possible (Zollner 1993).

As proteases, inhibitors for digestive enzymes seem to have arisen early in evolution in invertebrates. The structure of inhibitors has been described and categorized. Inhibitors belong to ten families comprised of molecules with homologies, both in sequence, but more importantly, in the number and array of disulfide bridges that yield a particular topology. Each family has a number and position of conserved disulfide bridges. Several inhibitors for trypsin have been described in a sea anemone Anemonia sulcata (Tschesche and Kolkenbrock 1984). For a comprehensive review of inhibitors, see Laskowski and Kato (1980).

The presence of proteinaceous inhibitors for digestive proteinases gives some clues about mechanisms of regulation of activity of digestive enzymes. Some inhibitors have been described in mammals and are thought to control the enzyme activity in spontaneous activation of the matching proteinase before secretion to avoid the unwanted hydrolysis of proteinaceous intracellular components. In crustaceans, an inhibitor for trypsin isolated from the midgut gland of the whiteleg shrimp P. vannamei was described (García-Carreño et al. 1998). Inhibitors for trypsin have been found in four species of this penaeid genus (de Albuquerque et al. 2002). These inhibitors constrain, to different degrees, trypsins in the same species and, at some lesser rate, trypsins in related species. In penaeid species, the presence of inhibitors synthesized in the same organ as trypsin recall the mechanism of regulation of digestive enzymes in mammals.

Trypsin inhibitors in penaeids reduced the activity of paralogous and orthologous trypsins. In this review, we used the nomenclature of Southan (2000), where paralogues are homologue enzymes or inhibitors co-synthesized in the same organisms and orthologues are homologue enzymes or inhibitors found within other taxa. Inhibitors in four penaeid species are low molecular weight proteins and inhibition of autologous and orthologous trypsins are proportional to the concentration of the inhibitor (de Albuquerque et al. 2002). Although the four closely related shrimp species have similar total protein and total proteinase activity, the composition and molecular masses of the enzymes were distinct. The differences were more conspicuous when comparing protein composition in the midgut gland.

Trypsins as team players

When studying digestive physiology, new questions arise. Why does an organism need several different proteinases? Why is one class or specificity not enough? Why do animals evolve with several classes and specificities? Of course, the simple answer is: to increase its biological fitness. The formal cause is that organisms having multiple classes of proteinases, such as trypsin and chymotrypsin, can enhance hydrolysis of food protein, exposing a variety of essential amino acids to the action of carboxypeptidases, which will eventually be released for absorption. Thus, the mere fact of having a wider spectrum of proteinases increases the efficient use of food protein.

The advantage of having isotrypsins was already discussed. But, a deeper analysis of the presence of several classes of proteinases is required. Teschke and Saborowski (2005) found that in two caridean shrimp, contrary to expectations, trypsin activity represented a small portion of proteinases activity, where maximum activity was reached at acid pH, far from the pH for serine proteinases. Moreover, a small percentage of the individuals had high trypsin activity in the midgut gland and a large percentage had low trypsin activity. In individuals with low trypsin activity, a cysteine proteinase was the substitute contributor to total proteinase activity. Researchers concluded that eucarids may have different traits for using food protein. For example, the California spiny lobster Panulirus interruptus contained isotrypsins and isochymotrypins as major proteinases, but another band of activity when analyzing activity by gel electrophoresis at acid pH (Celis-Guerrero et al. 2004). At that time, this seemed unusual, but was later supported by evidence obtained from the European lobster Homarus gammarus, where most of the proteinolytic activity was an enzyme working at acid pH that was completely inhibited by pepstatin A (Navarrete del Toro et al. 2006).

Astacin family members contain proteases that are widely distributed in nature, in bacteria, crustaceans, nematodes, cnidaries, mollusks, and several vertebrates). The family received its name after purification, biochemical characterization, and sequencing of a protease from the noble crayfish Astacus astacus L. (EC 3.4.24.21) (Titani et al. 1987). This enzyme is synthesized in the crayfish midgut gland (Vogt et al. 1989). It is stored extracellularly as an active proteinase in the stomach-like cardia, and is thought to be a digestive enzyme in a variety of crustaceans. Because of the fact that astacins are present in a tissue-specific manner in mature animals and are temporally and spatially present in developing systems, it has being proposed that its role is in mature and developing animals. Exploration of the structure, function, and regulation of these enzymes is at an early stage. A similar situation is found with other enzymes, such as cathepsins, which are distributed in several phyla. However, to date there is no evidences of a close link between trypsin functions and astacin or cathepsin functions, and if such a connection exists, it remains to be understood.

An interesting example of a coordinated network between proteinases occurs in the flatworm Schistosoma mansoni. Delcroix et al. (2006) used class-specific inhibitors and RNA interference techniques and determined “…that initial degradation of host blood proteins is ordered, occasionally redundant, and substrate specific…” in a proteinase network in which major proteinases function. They suggest that the protease network is conserved throughout invertebrate evolution.

Digestive proteinases do not work alone, with some exceptions, such as porcine pepsin B. Proteinases participate in food protein digestion in a coordinated manner, at least for endopeptidases, followed by exopeptidases. Other types or coordination among proteinases remain to be fully understood.

Trypsins from extremophiles

Temperature is one of the most important environmental factors sustaining life on Earth. Low temperatures do not favor chemical reaction rates catalyzed by enzymes (D’Amico et al. 2002). Organisms living under cold conditions require a variety of fundamental adaptations, including expression of enzymatic activities at appropriate levels, which must work under increased viscosity of the medium induced by low temperatures to allow the organism metabolic fluxes more or less comparable to those exhibited by closely related mesophilic species (Zecchinnon et al. 2001). Several taxa are devoid of temperature regulation, living close to 0 °C, referred as psychrophiles, such as bacteria, yeast algae, plants, insects, marine and terrestrial invertebrates, and fish (Feller and Gerday 2003). In terms of species diversity, microorganisms are the most abundant psychrophiles.

Even though the largest proportion of biomass on Earth is generated at cold temperatures, to our knowledge, there are only some reports related to invertebrate psychrophilic enzymes, for example: lysozyme from the tobacco hornworm moth M. sexta (Sotelo-Mundo et al. 2007), DNAse II from the Iceland scallop Chlamys islamica (Øverbø and Myrnes 2006), citrate synthase from the isopods Idotea baltica and Idotea emarginata (Salomon and Buchholz 2000), alkaline phosphatase from the Arctic shrimp Pandalus borealis (de Backer et al. 2002), and glutathion S-transferase from the Icelandic scallop Chlamys islandica (Myrnes and Nielsen 2007).

With the interest for novel industrial applications in recent years, attention has focused on cold-adapted trypsins. However, the molecular basis of enzyme adaptation to low temperatures is still poorly understood, probably because of the limited number of available amino acid sequences and structural data. Leiros et al. (2000) reported an alignment of 27 vertebrate trypsin amino acid sequences and suggested that the increased substrate affinity of the psychrophilic trypsins compared to mesophilic forms is probably achieved by the optimization of electrostatic complementarity between the binding site and substrate. The generally reduced electrostatic surface potential of all cold trypsins, especially in the C-terminal domain, is also suggested as a factor that increases the substrate affinity by a more efficient guidance and orientation of the substrate prior to binding (Smalas et al. 1994).

Additionally, brachyurins (serine proteases isolated from several decapod crustaceans) show chymotrypsin-like, trypsin-like, and elastase-like activities towards synthetic substrates (Kristjáansdóttir and Gudmundsdóttir 2000) plus all the characteristics of cold-adapted enzymes (Kristjáansdóttir 1999). These enzymes are members of Clan SA of the S1 family of serine endopeptidases and include the euphaulysin from krill (Turkiewicz et al. 1991). Two distinguishing features of euphaulysins are their ability to cleave collagen and its sensitivity to autolysis (Gudmundsdóttir 2002). Considering, the recent finding of this enzyme and its commercial interest, it would not be surprising if new reports on the isolation and characterization of trypsins from invertebrates inhabiting cold environments are published in the near future.

After the discovery of a new family of crabs and dense aggregations of bresilioid shrimp at hydrothermal vents in the eastern Pacific Ocean (Williams 1980; Williams and Chace 1982) the number of publications dealing with taxonomy, ecological physiology, and distribution of invertebrates at these sites in the deep sea has increased markedly (Martin and Haney 2005). It is now known that the invertebrate fauna, distinguished by its extreme temperatures, low oxygen, and toxic hydrogen sulfide and heavy metals, is extraordinarily complex. However, to our knowledge only a few studies have been devoted to understanding the biochemical characteristics of enzymes from invertebrates living in such remote and extreme biotopes. References to enzymes of organisms in extreme environments have largely focused on hyperthermophilic microorganisms. As in the case of cold-adapted enzymes, in the near future, we would expect publications related to the isolation, purification, and biochemical characterization of enzymes from invertebrates adapted to severe water environments.

Discussion

According to Neurath (2001), “Trypsin has fascinated biochemists, enzymologists, and biologists because it was one of first proteases to be identified and it plays an important digestive and regulatory role in a variety of physiological processes”. Trypsin possesses two main functions, hydrolysis of food protein and activation of zymogens of trypsin and other digestive proteases. Understanding trypsin functions allowed us to learn about active site mechanisms, domain organization, evolution, phylogeny, and irreversible signal transduction, as well as zymogen activation. Learning about trypsins is possible with the development of new techniques. The earliest approach was biochemical characterization. Mammal trypsin was the first protease crystallized for understanding molecular characterization. Also, characterization of biochemical specificities was done at that moment by synthesizing specific substrates and inhibitors. With the advent of electrophoresis, researchers discovered that trypsins were synthesized as isoforms, which lead to a paradigm shift that several genes were involved in isotrypsins synthesis. Also, evolution and phylogeny of trypsin could be assessed by a combination of electrophoresis techniques and specific antibodies that elicited reactions to one enzyme and compared cross-reactions with others. Of course, several questions have arisen about the function of isoenzymes. During the era of gene cloning, another paradigm shift occurred in our understanding the genetics of trypsin when the number of genes expanded beyond the ones inferred from isoenzyme studies. Development of molecular biology techniques also opened the possibility of understanding the multi-gene families of proteases. Techniques, such as screening genomic libraries, cloning genes, and analyzing complete genomes, helped make important contributions in finding new functions for proteases genes (Heger and Ponting 2008).

Since the dawn of genomics, the picture of molecular genetics of trypsin continued to change as researched realized that the number of trypsin genes was even greater and that some transcribe in the digestive system cells while others transcribe in other tissues. This increased the opportunity of finding and studying specific domains in genes and their deduced proteins, and establishing integrative studies, such as that developed by Shah et al. (2008) for structural-functional analyses of three dimensional structures. Microarray gene expression profiles, protein–protein interactions, protein and genes databases, and bioinformatic tools allowed the introduction of functional residue clustering (FRC) as a new tool for transferring, in vivo, substrate specificity in a quantitative manner that is highly sensitive and specific. This novel method assigns putative inhibitor relationships for proteases, providing evidence that explains evolution of new functions for serine-protease homologues in immune response by using high-throughput data. Trypsin studies moved from crude extracts to pure proteins, to characterization of molecules and isoforms, to characterization of genes. In doing this, the vision changed and knowledge increased, each time supported by more analytical and explicative methods.

Proteinases evolved to fulfill advanced physiological functions; we now know that trypsins are involved in a plethora of activities in invertebrates, such as activation of prophenoloxidase synthesized in hemocytes and as a defense response to invading microorganisms in the so-called innate immunity of invertebrates that uses melanin to encapsulate invaders.

Obviously, the key role that trypsin plays in living organisms is not restricted to the more studied species; in fact, there are incalculable insect and crustacean species that show special proteins. For example, insects are agents of agricultural or medical problems and targets of several studies, including gene regulation. For crustaceans, many species can synthesize highly efficient proteinases adapted to extreme conditions. Still, several problems in studying proteinases in invertebrates must be addressed. It is difficult to obtain information about these enzymes because of the small size of invertebrates, the molting process, and the lack of species-specific substrates suitable for some evaluations of enzyme activity. A limitation in genetic studies is the absence of adequate in vivo expression systems to evaluate crustacean proteinases. The importance of several insect species in commercial production, plague control, and human diseases and crustaceans in ecology and as food for human consumption has increased interest in the science community and government organizations to find a deeper comprehension of factors involved in digestion.

In summary, digestive trypsins are members of a network of proteinases that act in a coordinated manner. They serve to activate zymogens, start digestion with endopeptidases and exopeptidases; some work where others are inhibited by specific inhibitors in food and some specifically hydrolyze some protein substrates. More complex networks and coordination types are expected to be found in the near future. Perhaps, Hans Neurath (1994) was right when he announced that the study of proteinases was in a second golden era.