Residue determinants and sequence analysis of cold-adapted trypsins

The digestive enzyme trypsin is among the most extensively studied proteins, and its structure has been reported from a large number of organisms. This article focuses on the trypsins from vertebrates adapted to life at low temperatures. Cold-adapted organisms seem to have compensated for the reduced reaction rates at low temperatures by evolving more active and less temperature-stable enzymes. We have analyzed 27 trypsin sequences from a variety of organisms to find unique attributes for the cold-adapted trypsins, comparing trypsins from salmon, Antarctic fish, cod, and pufferfish to other vertebrate trypsins. Both the "cold" and the "warm" active trypsins have about 50 amino acids that are unique and conserved within each class. The main unique features of the cold-adapted trypsins attributable to low-temperature adaptation seem to be (1) reduced hydrophobicity and packing density of the core, mainly because of a lower (Ile + Leu)/(Ile + Leu + Val) ratio, (2) reduced stability of the C-terminal, (3) lack of one warm trypsin conserved proline residue and one proline tyrosine stacking, (4) difference in charge and flexibility of loops extending the binding pocket, and (5) different conformation of the "autolysis" loop that is likely to be involved in substrate binding. Received: January 14, 1999 / Accepted: March 31, 1999     

Atlantic Cod Trypsins: From Basic Research to Practical Applications

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.     

Isolation and Primary Structure of Trypsin from the King Crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the crab Paralithodes camtschaticuswas isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. A cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the shrimp Penaeus vannamei appeared to be closest in primary structure to that of the king crab (65% identity).     

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I — Kinetic parameters,autolysis and thermal stability

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k_cat/K_m) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 °C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.     

Stabilization of trypsin by modification with comb-shaped copolymers of poly(ethylene glycol) derivative and maleic anhydride

Summary Trypsin from bovine pancreas was coupled with copolymers of poly(ethylene glycol) derivative and maleic anhydride with the molecular weights of 13 kDa and 100 kDa (activated PM₁₃ and PM₁₀₀). The modified trypsins were more stable towards autolysis and heat- or urea-treatment than nonmodified trypsin. Stabilization of trypsin caused by the chemical modification with activated PMs is discussed in relation to the protein conformation.     

Isolation and primary structure of trypsin from the red king crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the Paralithodes camtschaticus crab was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. The cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the Penaeus vannamei shrimp appeared to be the most close in its primary structure to that of the king crab (70% identity).     

Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency

It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.     

Optimal foraging versus shared doom effects: interactive influence of mussel size and epibiosis on predator preference

In the western Baltic Sea, the highly competitive blue mussel Mytilus edulis tends to monopolize shallow water hard substrata. In many habitats, mussel dominance is mainly controlled by the generalist predator Carcinus maenas. These predator-prey interactions seem to be affected by mussel size (relative to crab size) and mussel epibionts.There is a clear relationship between prey size and predator size as suggested by the optimal foraging theory: Each crab size class preferentially preys on a certain mussel size class. Preferred prey size increases with crab size.Epibionts on Mytilus, however, influence this simple pattern of feeding preferences by crabs. When offered similarly sized mussels, crabs prefer Balanus-fouled mussels over clean mussels. There is, however, a hierarchy of factors: the influence of attractive epibiotic barnacles is weaker than the factor ‘mussel size’. Testing small mussels against large mussels, presence or absence of epibiotic barnacles does not significantly alter preferences caused by mussel size. Balanus enhanced crab predation on mussels in two ways: Additional food gain and, probably more important, improvement in handling of the prey. The latter effect is illustrated by the fact that artificial barnacle mimics increased crab predation on mussels to the same extent as do live barnacles.We conclude that crab predation preferences follows the optimal foraging model when prey belong to different size classes, whereas within size classes crab preferences is controlled by epibionts.     

Isolation and characteristics of trypsin inhibitor from the hepatopancreas of a squid (Todarodes pacificus)

Trypsin inhibitor was purified from the hepatopancreas of squid (Todarodes pacificus). The final inhibitor preparation was nearly homogeneous by SDS-PAGE with an estimated molecular weight of approximately 6300. The squid trypsin inhibitor was acid- and heat-stable, and active against trypsins from the pyloric ceca of starfish (Asterias amurensis) and saury (Cololabis saira) and porcine pancreatic trypsin. Amino acid composition of the squid trypsin inhibitor was compared with other invertebrate trypsin inhibitors. The squid trypsin inhibitor inhibited the autolysis of walleye pollock (Theragra chalcogramma) myofibrillar proteins.     

The midgut trypsins of shrimp (Penaeus monodon). High efficiency toward native protein substrates including collagens

Two shrimp trypsins have been purified from the midguts of Penaeid shrimps by various chromatographies and HPLC. The molecular masses of them are 27 and 29 kDa, respectively. They show the typical specificity of trypsin for substrates and inhibitors, and their N-terminal amino-acid sequences are homologous to those of other trypsins. The shrimp enzymes are very acidic (pI less than or equal to 2.4), and show distinctively low Km for the synthetic amide substrates. They also hydrolyse various native proteins more efficiently than bovine trypsin in vitro. However, the anionic shrimp trypsins do not have special preference for basic protein substrates over the acidic one. Collagenolytic activity of the midgut extract was mainly due to serine proteases. The collagenolytic activity of the purified shrimp trypsin was confirmed by assays with either soluble or insoluble native type I collagens. In comparison with the other trypsins from the Crustacean decapods, the shrimp enzymes have four pairs of disulfide bonds, intermediary between the crayfish trypsin (three pairs) and the crab trypsin (five pairs), and are immunochemically different from them.     

Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site

One of ostrich (Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen). Ostrich trypsinogen undergoes bovine enterokinase activation and autoactivation much faster than human trypsinogen 1 and exhibits on a synthetic substrate a somewhat higher enzymatic activity than the latter one. The most interesting property of ostrich trypsin is its relatively fast autolysis that can be explained via a mechanism different from the common mechanism for rat and human 1 trypsins. The latter proteases have a site, Arg117-Val118, where the autolysis starts and then goes on in a zipper-like fashion. This is absent from ostrich trypsin. Instead it has a couple of cleavage sites within regions 67-98, including two unusual ones, Arg76-Glu77 and Arg83-Ser84. These appear to be hydrolysed fast in a non-consecutive manner. Such an autolysis mechanism could not be inhibited by a single-site mutation which in humans is proposed to lead to pancreatitis.     

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

d-glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 d-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T_50% was increased by 7 °C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25 °C (without stabilizing Ca²⁺ ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (k_cat/K_m) than unmodified trypsin against the substrate N-α-benzoyl-l-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.     

Purification and Characterization of Cold Adapted Trypsins from Antarctic krill (Euphausia superba)

Three trypsins (TRY-ES) were purified from Antarctic krill (Euphausia superba) by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography, with relative molecular mass of 28.7, 28.8 and 29.2 kDa respectively. The TRY-ES was inhibited by specific trypsin inhibitors (benzamidine, STI, CHOM and TLCK), with optimum temperature at 40 (Trypsin I), 45 (Trypsin II) and 40 °C (Trypsin III) repetitively. The TRY-ES was stabled between 5 and 40 °C, which was consistent with the red shift in fluorescence intensity peak at 40 °C (Trypsin I) and 45 °C (Trypsin II and Trypsin III) and blue shift at 40 °C (Trypsin II and Trypsin III). The K _cat/K _m values of the TRY-ES was 14.28, 9.46 and 5.93 mM^?1s^?1 respectively, 1.1–10.2 folds higher than trypsins from other crustacean and mammal, which was supported by the differences in thermodynamics parameters, the free energy, enthalpy, and entropy of benzamidine and the TRY-ES system.     

Cold Adaptation,Ca2+ Dependency and Autolytic Stability Are Related Features in a Highly Active Cold-Adapted Trypsin Resistant to Autoproteolysis Engineered for Biotechnological Applications

Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca²⁺ dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10^° C and at least 50% more active than pig trypsin up to 50^° C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca²⁺ concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca²⁺ dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.     

New Host Range for Hematodinium in Southern Australia and Novel Tools for Sensitive Detection of Parasitic Dinoflagellates

Hematodinium is a parasitic dinoflagellate and emerging pathogen of crustaceans. It preferably manifests in haemolymph of marine decapod crustaceans, killing a large variety of genera with significant impacts on fisheries worldwide. There is, however, evidence that some crustacean stocks harbor high prevalence, low intensity infections that may not result in widespread host mortality and are therefore hard to detect. The most widely used methods for detection of Hematodinium are conventional blood smears and polymerase chain reaction (PCR) against ribosomal RNAs. Blood smears demand a trained investigator, are labor intensive and not readily scalable for high-throughput sampling. PCRs only detect parasite DNA and can also suffer from false negatives and positives. In order to develop alternative detection tools for Hematodinium cells in decapod crustaceans we employed an immunological approach against a newly identified, abundant dinoflagellate-specific nuclear protein—Dinoflagellate/Viral NucleoProtein (DVNP). Both immunofluorescence assay (IFA) and Western blot methods against DVNP showed high sensitivity of detection. The Western blot detects Hematodinium parasites to levels of 25 parasites per milliliter of crustacean haemolymph, with the potential for sample pooling and screening of large samples. Using both PCR and these new tools, we have identified Hematodinium cells present in three new host crab taxa, at high prevalence but with no sign of pathogenesis. This extends the known range of Hematodinium to southern Australia.     

Distribution and ecological relations among the alien crab, <Emphasis Type="Italic">Percnon gibbesi</Emphasis> (H. Milne-Edwards 1853) and autochthonous species,in and out of an SW Mediterranean MPA

The distribution of the allochthonous crab Percnon gibbesi and its relationships with other benthic invertebrate species was assessed inside the marine reserve of Cabo de Palos—Islas Hormigas (Mediterranean sea, Spain) and neighbouring non-protected sites. Although a significant spatial variability was detected at finer spatial scale, there was no influence of protection measures or insularity on the abundance of P. gibbesi. The presence of small holes, encrusting algae and low slope favour the colonization success of this crustacean, and the spatial distribution of these habitat features could explain the observed pattern. The abundance of P. gibbesi was similar to that of native crab species; however, a non-significant negative relationship between the abundance of P. gibbesi and native crabs (Pachygrapsus marmoratus and Eriphia verrucosa), urchins (Arbacia lixula and Paracentrotus lividus) and a snail (Phorcus turbinatus) was observed. This work highlights the importance of monitoring alien crab population densities taking structural habitat and other potentially influential factors into account and the likely effect of this alien species on the native ones.     

Human cationic trypsinogen. Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis

Mutation Asn-21 --> Ile in human cationic trypsinogen (Tg-1) has been associated with hereditary pancreatitis. Recent studies with rat anionic Tg (Tg-2) indicated that the analogous Thr-21 --> Ile mutation stabilizes the zymogen against autoactivation, whereas it has no effect on catalytic properties or autolytic stability of trypsin (Sahin-Tóth, M. (1999) J. Biol. Chem. 274, 29699-29704). In the present paper, human cationic Tg (Asn-21-Tg) and mutants Asn-21 --> Ile (Ile-21-Tg) and Asn-21 --> Thr (Thr-21-Tg) were expressed in Escherichia coli, and zymogen activation, zymogen degradation, and trypsin autolysis were studied. Enterokinase activated Asn-21-Tg approximately 2-fold better than Ile-21-Tg or Thr-21-Tg, and catalytic parameters of trypsins were comparable. At 37 degrees C, in 5 mm Ca(2+), all three trypsins were highly stable. In the absence of Ca(2+), Asn-21- and Ile-21-trypsins suffered autolysis in an indistinguishable manner, whereas Thr-21-trypsin exhibited significantly increased stability. In sharp contrast to observations with the rat proenzyme, at pH 8.0, 37 degrees C, autoactivation kinetics of Asn-21-Tg and Ile-21-Tg were identical; however, at pH 5. 0, Ile-21-Tg autoactivated at an enhanced rate relative to Asn-21-Tg. Remarkably, at both pH values, Thr-21-Tg showed markedly higher autoactivation rates than the two other zymogens. Finally, autocatalytic proteolysis of human zymogens was limited to cleavage at Arg-117, and no digestion at Lys-188 was detected. The observations indicate that zymogen stabilization by Ile-21 as observed in rat Tg-2 is not characteristic of human Tg-1. Instead, an increased propensity to autoactivation under acidic conditions might be relevant to the pathomechanism of the Asn-21 --> Ile mutation in hereditary pancreatitis. In the same context, faster autoactivation and increased trypsin stability caused by the Asn-21 --> Thr mutation in human Tg-1 might provide a rationale for the evolutionary divergence from Thr-21 found in other mammalian trypsinogens.     

Relating Trypsin Enzymatic Properties with Amino Acid Composition

In this paper, the DNA fragment of trypsin genes from eight crustaceans were cloned and sequenced. The amino acid composition of the 24 deduced and 42 selected trypsin sequences were compared. Low arginine, methionine, and proline content and high aspartic acid, glutamic acid, and isoleucine content attributed to the distinct catalytic efficiency of crustacean trypsins.     

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI‐induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI‐induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.