-
Journal of Comparative Physiology. B,... Jan 2015Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and... (Review)
Review
Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Evolution, Molecular; Feeding Behavior; Gene Expression Regulation, Enzymologic; Isoenzymes; Models, Molecular; Open Reading Frames; Palinuridae; Phylogeny; Trypsin
PubMed: 25192870
DOI: 10.1007/s00360-014-0851-y -
Vie Medicale (Paris, France : 1920) Jun 1955
Topics: Trypsin
PubMed: 14397247
DOI: No ID Found -
Journal de Medecine de Bordeaux Et Du... Dec 1954
Topics: Humans; Trypsin
PubMed: 14354380
DOI: No ID Found -
Biological Chemistry Sep 2018In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically...
In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1' position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1' and P2' preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2' is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.
Topics: Amino Acid Sequence; Fluorescent Dyes; Humans; Isoenzymes; Models, Molecular; Sequence Alignment; Substrate Specificity; Trypsin
PubMed: 29883318
DOI: 10.1515/hsz-2018-0107 -
La Riforma Medica Nov 1959
Topics: Anti-Inflammatory Agents; Peripheral Vascular Diseases; Thrombophlebitis; Trypsin; Vascular Diseases
PubMed: 13792894
DOI: No ID Found -
Annales Pharmaceutiques Francaises Apr 1961
Topics: Trypsin
PubMed: 14448186
DOI: No ID Found -
Biochimica Et Biophysica Acta. Proteins... Jan 2017Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical...
Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (k/K) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.
Topics: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cloning, Molecular; Gadus morhua; Isoenzymes; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin
PubMed: 27742554
DOI: 10.1016/j.bbapap.2016.10.005 -
Biochemistry Feb 2004Alpha-synuclein modulates dopamine homeostasis in dopamine-producing neurons of substantia nigra, partly through regulation of human dopamine transporter (hDAT)...
Alpha-synuclein modulates dopamine homeostasis in dopamine-producing neurons of substantia nigra, partly through regulation of human dopamine transporter (hDAT) activity. To identify the underlying mechanisms, we disrupted the modulation of hDAT activity by wild-type (wt) alpha-synuclein, and its familial Parkinson's disease linked mutants A30P and A53T, by mild trypsinization (0.1%, 30 s) of Ltk(-) cotransfected cells. Trypsin completely reversed the attenuation of hDAT function mediated by wt and the A30P mutant. In A53T coexpressing cells, where DAT activity is not downregulated, trypsinization did not induce any changes. These effects of trypsin were mimicked by collagenase I and Dispase (0.1%, 1 min each) but not by chymotrypsin, Pronase, or papain (0.1%, up to 2 min each). Trypsin increased dopamine uptake in rat primary mesencephalic neurons, suggesting that DAT activity is also subjected to modulation by alpha-synuclein in these neurons that endogenously coexpress both proteins. In trypsinized cells, dopamine accelerated both production of reactive oxygen species and cell death in hDAT and wt or A30P, but not A53T, coexpressing cells, compared to nontrypsinized cells. Paradoxically, trypsin increased the protein-protein interactions between the synuclein variants and hDAT, without any noticeable proteolysis of these proteins. hDAT-alpha-synuclein protein-protein interactions occurred through residues 58-107 (NAC domain) of the alpha-synuclein variants and residues 598-620 of the carboxy-terminal tail of hDAT, in both trypsinized and nontrypsinized cells. Confocal microscopy and biotinylation studies show that, in cells expressing the wt or A30P variants, but not the A53T mutant, hDAT is sequestered away from the plasma membrane into the cytoplasm, an effect that is reversed by trypsin. These results show that alpha-synuclein modulates hDAT function through trafficking of the transporter in a process that can be disrupted by trypsin.
Topics: Alanine; Amino Acid Substitution; Animals; Biotinylation; Cell Death; Cell Line; Cell Line, Tumor; Cells, Cultured; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Membrane Glycoproteins; Membrane Transport Modulators; Membrane Transport Proteins; Mice; Nerve Tissue Proteins; Proline; Protein Transport; Rats; Synucleins; Transfection; Trypsin; alpha-Synuclein
PubMed: 14756560
DOI: 10.1021/bi035308s -
International Journal of Biological... Dec 2020Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are...
Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.
Topics: Animals; Cold Temperature; Enzyme Activation; Enzyme Stability; Fish Proteins; Fishes; Hydrogen Bonding; Models, Molecular; Molecular Docking Simulation; Mutation; Protein Structure, Secondary; Trypsin
PubMed: 32827617
DOI: 10.1016/j.ijbiomac.2020.08.136 -
Rapid Communications in Mass... Apr 2016Trypsin is an important digestive enzyme in peptide sample preparation for proteomics. It digests proteins at the C-terminal of Arg or Lys residues. The majority of...
RATIONALE
Trypsin is an important digestive enzyme in peptide sample preparation for proteomics. It digests proteins at the C-terminal of Arg or Lys residues. The majority of commercial products are obtained from animal sources. In a previous study, we reported the production process for recombinant trypsin (r-trypsin) and acetylated trypsin (r-Ac-trypsin). In this paper, we want to evaluate whether the r-trypsin and r-Ac-trypsin are suitable for proteomics research.
METHODS
The trypsins used in this research were first normalized to the same concentration and used for further evaluation. The stability and buffer compatibility (2M urea, 0.1% SDS and 10% acetonitrile) were compared and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The digestion efficiency and specificity were compared based on a simple protein substrate, human serum albumin (HSA) and a complex proteomic sample, yeast lysate. The acquisition of proteomics data was achieved by ultra-high performance liquid chromatography (UPLC) connected to an LTQ Orbitrap Velos mass spectrometer.
RESULTS
r-Ac-trypsin demonstrated similar tolerance to 2 M urea and 10% acetonitrile but weaker 0.1% SDS tolerance than commercial trypsins. Based on simple protein sample HSA, the activity and specificity of r-Ac-trypsin were similar to that of commercial trypsins. However, it demonstrated superior activity and specificity on complicated samples like yeast lysate. More interestingly, the newly developed r-Ac-trypsin was more resistant to autolysis, which enabled more complete digestion of proteomic samples.
CONCLUSIONS
The r-Ac-trypsin described here is a recombinant product. In addition it showed similar or superior properties such as stability activity and specificity to commercial products. It can be used in peptide sample preparation in proteomics studies.
Topics: Enzyme Stability; Mass Spectrometry; Proteomics; Recombinant Proteins; Trypsin
PubMed: 27003043
DOI: 10.1002/rcm.7535