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What is the function of human Trypsin Inhibitor if trypsin is secreted in the inactive form of Trypsinogen?

What is the function of human Trypsin Inhibitor if trypsin is secreted in the inactive form of Trypsinogen?


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I was reading about pancreatic digestive enzymes in a Textbook of Medical Physiology and I came across Trypsin Inhibitor. The text stated that:

It is important that the proteolytic enzymes of the pancreatic juice not become activated until after they have been secreted into the intestine because the trypsin and the other enzymes would digest the pancreas itself… substance called trypsin inhibitor. This substance is formed in the cytoplasm of the glandular cells, and it prevents activation of trypsin both inside the secretory cells and in the acini and ducts of the pancreas.

It also states that:

When first synthesized in the pancreatic cells, the proteolytic digestive enzymes are in the inactive forms trypsinogen, chymotrypsinogen, and procarboxypolypeptidase, which are all inactive enzymatically. They become activated only after they are secreted into the intestinal tract.

My question is, why is Trypsin Inhibitor secreted if trypsin is already secreted as trypsinogen which can only be activated in the intestine?


Trypsin

Trypsin (EC 3.4.21.4) is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. [2] [3] Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinization, and proteins that have been digested/treated with trypsin are said to have been trypsinized. [4] Trypsin was discovered in 1876 by Wilhelm Kühne and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin. [5]


Pancreas

Trypsinogen Activation Peptide

When trypsinogen is activated to trypsin, a small peptide, TAP, is split from the trypsinogen molecule. Under normal conditions, activation of trypsinogen takes place only in the small intestine and TAP is undetectable in the blood. During pancreatitis, trypsinogen is activated prematurely in pancreatic acinar cells and TAP is released into the vascular space. 71 Urine TAP assays have shown some promise in experimental models of feline pancreatitis, 74 but serum and urine TAP assays are less promising in clinical studies. 75 Evidence-based data is needed to determine the true specificity and sensitivity of this assay.


Results and discussion

Molecular docking identifies candidate molecules with prospective mesotrypsin inhibitory activity

An ensemble docking-based virtual screening [27] of compounds from the FDA and NPD databases was conducted using three crystal structures of mesotrypsin (PDB IDs: 3P92 [22], 3P95 [22], and 1H4W [2]). Positive hits were chosen based on low docking scores to mesotrypsin and crucial hydrogen bond interactions observed with mesotrypsin binding pocket residues such as Asp-189, Ser-190, Gln-192, Arg-193, and Gly-216 [22]. The docking scores were formulated on the “extra-precision” XP Glide software algorithm which considers factors such as: lipophilicity, displacement of water, hydrogen bonding and electrostatic interactions, and metal ion/ligand interactions as favorable interactions, while the desolvation of polar or charged groups, restriction of motion, and the entropic cost of binding adversely affect docking score [28, 29]. Twenty-eight top compounds were computationally predicted to exhibit activity towards mesotrypsin based on the virtual screening details of selection criteria are described in Materials and Methods.

Virtual screening and experimental validation identify bis-benzamidine compounds with mesotrypsin inhibitory activity

From the twenty-eight top compounds identified in the virtual screen, twelve compounds were found to be readily commercially available and were therefore procured and evaluated for inhibitory activity towards mesotrypsin (Fig 1, S1 Table). Compounds were tested by an in vitro activity assay utilizing the colorimetric peptide reporter substrate N-Cbz-Gly-Pro-Arg-pNA. The assay monitors the rate of para-nitroaniline production after substrate cleavage by mesotrypsin. Of the twelve compounds tested, ten displayed no inhibitory activity towards mesotrypsin into the low millimolar range. Conversely, two compounds, diminazene (CID 22956468) and hydroxystilbamidine (CID 16212515) exhibited inhibitory activity towards mesotrypsin in the low micromolar range (Fig 2A and 2B).


Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function.

Keywords: chronic pancreatitis chymotrypsin C mesotrypsin pancreas protease inhibitor serine protease serine protease inhibitor trypsin.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.


Discussion

In the present study we used phage display technology to investigate the significance of tyrosine sulfation in the substrate binding specificity of human cationic and anionic trypsins. Our findings demonstrate that the negatively charged sulfate group on Tyr154 modifies the P2′ selectivity of trypsins it slightly inhibits binding of hydrophobic side chains (Ala, Ile, Leu), whereas it maintains an essentially unaltered affinity for positively charged Arg and Lys residues. The overall effect of sulfation is a 6�-fold increase in selectivity towards basic P2′ residues. This conclusion is consistent with structural modeling showing steric proximity between Tyr154 and the P2′ side chain of bound inhibitor (see Figure 1 ).

We also found that human cationic trypsin favored residues with short side chains (Ala, Gly, or Ser) at P1′ and acidic residues (Asp or Glu) at P3′ these selection patterns were independent of sulfation. These observations differ from previous studies mapping the S1′ subsite of bovine and rat trypsins using acyl-transfer experiments, which found that the S1′ site exhibited broad specificity with an apparent preference toward hydrophobic side chains rather than Ala/Gly/Ser as observed here [29]–[33]. Similarly broad specificity without selectivity for Asp/Glu was observed for the S3′ site in rat trypsin [30]. The different selection pattern in our experiments may be related to several differences between the acyl-transfer experiments and the phage display approach taken here. An important difference is that preferences revealed in acyl-transfer experiments encompass both binding affinity and catalytic competence for ligation a successful nucleophile must not only bind to the prime side subsites of the enzyme, but also carry out productive nucleophilic attack on a second substrate occupying the nonprime side subsites of the enzyme. By contrast, phage display selection unmasks the binding preferences of the enzyme uncoupled from catalytic rates. Another contributing factor may be the conformational constraints of the relatively rigid BPTI scaffold. Indeed, mutational analysis of the P1′ position of BPTI found Ala, Gly and Ser as the preferred residues for tight binding to trypsin, chymotrypsin and plasmin [34]. Of interest, another study mapping binding preferences of bovine trypsin using fluorescence-quenched substrates found a pattern of specificity more closely approximating our results, in which Ser and Ala (in addition to Arg) were preferred residues at P1′, and Asp was among the more favored residues at P3′ [35].

The P1′ and P3′ specificities observed may be interrelated, as these subsites often have a cooperative relationship. Owing to the extended (canonical) backbone conformation of bound inhibitors and substrates, side chains at the P1′ and P3′ positions point in the same direction and their interactions with the S1′ and S3′ subsites are contiguous. Crystal structures indicate that Lys66 (corresponding to Lys60 in conventional chymotrypsin numbering) may be a determinant of S1′ and S3′ specificity [36], and the Asp/Glu selection at P3′ may be explained by a favorable electrostatic interaction. This interaction, in turn, may partially obstruct the S1′ subsite, resulting in the selection of small volume side chains at P1′ instead of larger hydrophobic residues. A similarly restricted P1′ preference is also characteristic of thrombin, where Lys60f (chymotrypsin numbering) occludes the S1′ subsite and limits its specificity to amino-acids with small side-chains [37], [38].

The biological significance of the observed P2′ selectivity for basic amino acids in sulfated human trypsins remains unclear. As detailed in the introduction, previous studies were unable to identify a convincing role for tyrosine sulfation in trypsin function. The large majority of vertebrate trypsins do not appear to be sulfated, as judged by the absence of Tyr154 or the required sulfation motif (see Table 1 in reference [6]). This suggests that trypsin sulfation in humans may have evolved to facilitate the digestion of specialized substrates present in the primate diet only. Alternatively, the true evolutionary driving force of trypsin sulfation may have been unrelated to catalytic activity and the relatively small changes in substrate specificity may be inconsequential in the digestive function of trypsins. Yet a third possibility is that sulfation may enhance the catalytic capability of trypsins toward protein substrates by weakening prime side interactions. For protein substrates, strong affinity between the prime side residues and corresponding protease subsites will have the effect of retarding the deacylation step of the reaction. For good substrates of trypsin, deacylation can be the rate determining step in the overall reaction [39], [40]. As a consequence, by diminishing prime side affinity for the majority of protein substrates the effect of Tyr154 sulfation may be to accelerate enzyme turnover. Interestingly, rat anionic trypsin-2 contains a Glu residue in place of Tyr154 and this negatively charged side chain may mimic the function of a sulfated Tyr154. Indeed, a salt bridge formed between Glu154 and the P2′ Arg of the bound inhibitor is evident in the crystal structure of rat anionic trypsin-2 with BPTI [36]. Furthermore, prime side mapping of rat trypsin using acyl-transfer experiments demonstrated an S2′ preference for positively charged residues [30].

In summary, we demonstrated that sulfation of human anionic and cationic trypsins on Tyr154 increases selectivity towards basic versus apolar residues at the P2′ position of inhibitors that bind in a substrate-like fashion. Although the increase in selectivity is relatively small, we speculate that this post-translational change in substrate specificity may facilitate digestion of a broader range of dietary proteins.


Trypsin inhibitor

In order to prevent the action of active trypsin in the pancreas which can be highly damaging, inhibitors such as BPTI and SPINK1 in the pancreas and α1-antitrypsin in the serum are present as part of the defense against its inappropriate activation. Any trypsin prematurely formed from the inactive trypsinogen would be bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest found, and trypsin is bound by some of its pancreatic inhibitors essentially irreversibly. [13] In contrast with nearly all known protein assemblies, some complexes of trypsin bound by its inhibitors do not readily dissociate after treatment with 8M urea. [14]


Paneth cell trypsin is the processing enzyme for human defensin-5

The antimicrobial peptide human α-defensin 5 (HD5) is expressed in Paneth cells, secretory epithelial cells in the small intestine. Unlike other characterized defensins, HD5 is stored in secretory vesicles as a propeptide. The storage quantities of HD5 are ∼ 90–450 μg per cm 2 of mucosal surface area, which is sufficient to generate microbicidal concentrations in the intestinal lumen. HD5 peptides isolated from the intestinal lumen are proteolytically processed forms—HD5(56–94) and HD5(63–94)—that are cleaved at the Arg 55 -Ala 56 and Arg 62 -Thr 63 sites, respectively. We show here that a specific pattern of trypsin isozymes is expressed in Paneth cells, that trypsin colocalizes with HD5 and that this protease can efficiently cleave HD5 propeptide to forms identical to those isolated in vivo. By acting as a prodefensin convertase in human Paneth cells, trypsin is involved in the regulation of innate immunity in the small intestine.


What is the function of human Trypsin Inhibitor if trypsin is secreted in the inactive form of Trypsinogen? - Biology

To prevent the action of active trypsin in the pancreas, which can be highly damaging, inhibitors such as BPTI and SPINK1 in the pancreas and α1-antitrypsin in the serum are present as part of the defense against its inappropriate activation. Any trypsin prematurely formed from the inactive trypsinogen is then bound by the inhibitor. The protein-protein interaction between trypsin and its inhibitors is one of the tightest bound, and trypsin is bound by some of its pancreatic inhibitors nearly irreversibly. In contrast with nearly all known protein assemblies, some complexes of trypsin bound by its inhibitors do not readily dissociate after treatment with 8M urea.

Trypsin can also be used to dissociate dissected cells (for example, prior to cell fixing and sorting).

In the duodenum, trypsin catalyzes the hydrolysis of peptide bonds, breaking down proteins into smaller peptides. The peptide products are then further hydrolyzed into amino acids via other proteases, rendering them available for absorption into the blood stream. Tryptic digestion is a necessary step in protein absorption, as proteins are generally too large to be absorbed through the lining of the small intestine.

Trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form.

Trypsin can be used to break down casein in breast milk. If trypsin is added to a solution of milk powder, the breakdown of casein causes the milk to become translucent. The rate of reaction can be measured by using the amount of time needed for the milk to turn translucent.

Trypsin is produced as the inactive zymogen trypsinogen in the pancreas. When the pancreas is stimulated by cholecystokinin, it is then secreted into the first part of the small intestine (the duodenum) via the pancreatic duct. Once in the small intestine, the enzyme enteropeptidase activates trypsinogen into trypsin by proteolytic cleavage.

The enzymatic mechanism is similar to that of other serine proteases. These enzymes contain a catalytic triad consisting of histidine-57, aspartate-102, and serine-195. This catalytic triad was formerly called a charge relay system, implying the abstraction of protons from serine to histidine and from histidine to aspartate, but owing to evidence provided by NMR that the resultant alkoxide form of serine would have a much stronger pull on the proton than does the imidazole ring of histidine, current thinking holds instead that serine and histidine each have effectively equal share of the proton, forming short low-barrier hydrogen bonds therewith. By these means, the nucleophilicity of the active site serine is increased, facilitating its attack on the amide carbon during proteolysis. The enzymatic reaction that trypsin catalyzes is thermodynamically favorable, but requires significant activation energy (it is "kinetically unfavorable"). In addition, trypsin contains an "oxyanion hole" formed by the backbone amide hydrogen atoms of Gly-193 and Ser-195, which through hydrogen bonding stabilize the negative charge which accumulates on the amide oxygen after nucleophilic attack on the planar amide carbon by the serine oxygen causes that carbon to assume a tetrahedral geometry. Such stabilisation of this tetrahedral intermediate helps to reduce the energy barrier of its formation and is concomitant with a lowering of the free energy of the transition state. Preferential binding of the transition state is a key feature of enzyme chemistry.

Trypsin is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins. Trypsin is formed in the small intestine when its proenzyme form, the trypsinogen produced by the pancreas, is activated. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. It is used for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinisation, and proteins that have been digested/treated with trypsin are said to have been trypsinized. Trypsin was discovered in 1876 by Wilhelm Kühne and was named from the Ancient Greek word for rubbing since it was first isolated by rubbing the pancreas with glycerin.

Also one of the first exoenzymes to be discovered, trypsin was named in 1876, forty years after pepsin. This enzyme is responsible for the breakdown of large globular proteins and its activity is specific to cleaving the C-terminal sides of arginine and lysine amino acid residues. It is the derivative of trypsinogen, an inactive precursor that is produced in the pancreas. When secreted into the small intestine, it mixes with enterokinase to form active trypsin. Due to its role in the small intestine, trypsin works at an optimal pH of 8.0.

Trypsin is available in high quantity in pancreases, and can be purified rather easily. Hence, it has been used widely in various biotechnological processes.

As a protein, trypsin has various molecular weights depending on the source. For example, a molecular weight of 23.3 kDa is reported for trypsin from bovine and porcine sources.

These human genes encode proteins with trypsin enzymatic activity: Other isoforms of trypsin may also be found in other organisms.

Trypsin is commonly used in biological research during proteomics experiments to digest proteins into peptides for mass spectrometry analysis, e.g. in-gel digestion. Trypsin is particularly suited for this, since it has a very well defined specificity, as it hydrolyzes only the peptide bonds in which the carbonyl group is contributed either by an arginine or lysine residue.

Trypsin is a serine protease that cleaves protein substrates after lysine or arginine residues using a catalytic triad to perform covalent catalysis, and an oxyanion hole to stabilise charge-buildup on the transition states.

Trypsin should be stored at very cold temperatures (between &minus20 and &minus80 °C) to prevent autolysis, which may also be impeded by storage of trypsin at pH 3 or by using trypsin modified by reductive methylation. When the pH is adjusted back to pH 8, activity returns.

Human trypsin has an optimal operating temperature of about 37 °C. In contrast, the Atlantic cod has several types of trypsins for the poikilotherm fish to survive at different body temperatures. Cod trypsins include trypsin I with an activity range of 4 to 65 °C (40 to 150 °F) and maximal activity at 55 °C (130 °F), as well as trypsin Y with a range of 2 to 30 °C (36 to 86 °F) and a maximal activity at 21 °C (70 °F).

The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is, thus, responsible for the specificity of the enzyme. This means that trypsin predominantly cleaves proteins at the carboxyl side (or "C-terminal side") of the amino acids lysine and arginine except when either is bound to a C-terminal proline, although large-scale mass spectrometry data suggest cleavage occurs even with proline. Trypsin is considered an endopeptidase, i.e., the cleavage occurs within the polypeptide chain rather than at the terminal amino acids located at the ends of polypeptides.

Activation of trypsin from proteolytic cleavage of trypsinogen in the pancreas can lead to a series of events that cause pancreatic self-digestion, resulting in pancreatitis. One consequence of the autosomal recessive disease cystic fibrosis is a deficiency in transport of trypsin and other digestive enzymes from the pancreas. This leads to the disorder termed meconium ileus, which involves intestinal obstruction (ileus) due to overly thick meconium, which is normally broken down by trypsin and other proteases, then passed in feces.

In a tissue culture lab, trypsin is used to resuspend cells adherent to the cell culture dish wall during the process of harvesting cells. Some cell types adhere to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to cleave proteins holding the cultured cells to the dish, so that the cells can be removed from the plates.

The activity of trypsin is not affected by the enzyme inhibitor tosyl phenylalanyl chloromethyl ketone, TPCK, which deactivates chymotrypsin. This is important because, in some applications, like mass spectrometry, the specificity of cleavage is important.


Identification of novel peptide inhibitors for human trypsins

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.

Journal

Biological Chemistry &ndash de Gruyter

Published: Feb 1, 2010

Keywords: computer modeling inhibitors peptidases phage display serine proteases


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