Limits...
Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

Valdés JJ, Schwarz A, Cabeza de Vaca I, Calvo E, Pedra JH, Guallar V, Kotsyfakis M - PLoS ONE (2013)

Bottom Line: Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin.The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic. valdjj@gmail.com

ABSTRACT

Background: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions.

Results: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).

Conclusions: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

Show MeSH
A–B. Titration of the targeted serine proteases with the recombinant protein.An inhibition screening (A) using 14 vertebrate serine proteases – the asterisk denotes with p-values ≤ 0.05 and ≤50% inhibition. Inhibition curves (B) were produced for the targeted enzymes by plotting the concentration of the inhibitor and the estimated percentage of the enzymatic activity. Since the amount of enzyme used in the assay varies for each protease, we represent the molar excess of the I. scapularis Kunitz peptide to achieve 50% inhibition of the respective enzyme, when compared to the amount of enzyme used in each assay (also see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643938&req=5

pone-0062562-g002: A–B. Titration of the targeted serine proteases with the recombinant protein.An inhibition screening (A) using 14 vertebrate serine proteases – the asterisk denotes with p-values ≤ 0.05 and ≤50% inhibition. Inhibition curves (B) were produced for the targeted enzymes by plotting the concentration of the inhibitor and the estimated percentage of the enzymatic activity. Since the amount of enzyme used in the assay varies for each protease, we represent the molar excess of the I. scapularis Kunitz peptide to achieve 50% inhibition of the respective enzyme, when compared to the amount of enzyme used in each assay (also see Materials and Methods).

Mentions: Compared to vertebrate Kunitz inhibitors that play a role in inflammatory responses, invertebrate Kunitz peptides possess a diverse inhibitory effect on protease activity [46]. To date, hundreds of tick Kunitz peptides have been discovered from sequencing projects [13], [47], [48], [49], [50]; however, as summarized below, roughly a dozen of tick Kunitz peptides are functionally described. Therefore, we overexpressed the divergent salivary Kunitz peptide in order to characterize its potential inhibitory activity against several vertebrate serine proteases. After HPLC purification, the purity of the recombinant peptide exceeded 95% (coomassie stained gel – data not shown). We then tested the recombinant peptide against 14 vertebrate serine proteases. Figure 2A depicts the results showing a statistically significant inhibition for trypsin, α-chymotrypsin, HSTβ, plasmin, matriptase and elastase. Figure 2B shows standard inhibition curves for all the targeted enzymes to further verify that the recombinant protein inhibits the above-mentioned enzymes [51]. Based on the data from these inhibition curves and calculating the Km of the substrates under the assay conditions (data not shown), we were able to calculate the apparent Ki (Ki (app)) for the targeted enzymes (see Materials and Methods) revealing a low pM Ki(app) for HSTβ (Table 1).


Tryptogalinin is a tick Kunitz serine protease inhibitor with a unique intrinsic disorder.

Valdés JJ, Schwarz A, Cabeza de Vaca I, Calvo E, Pedra JH, Guallar V, Kotsyfakis M - PLoS ONE (2013)

A–B. Titration of the targeted serine proteases with the recombinant protein.An inhibition screening (A) using 14 vertebrate serine proteases – the asterisk denotes with p-values ≤ 0.05 and ≤50% inhibition. Inhibition curves (B) were produced for the targeted enzymes by plotting the concentration of the inhibitor and the estimated percentage of the enzymatic activity. Since the amount of enzyme used in the assay varies for each protease, we represent the molar excess of the I. scapularis Kunitz peptide to achieve 50% inhibition of the respective enzyme, when compared to the amount of enzyme used in each assay (also see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643938&req=5

pone-0062562-g002: A–B. Titration of the targeted serine proteases with the recombinant protein.An inhibition screening (A) using 14 vertebrate serine proteases – the asterisk denotes with p-values ≤ 0.05 and ≤50% inhibition. Inhibition curves (B) were produced for the targeted enzymes by plotting the concentration of the inhibitor and the estimated percentage of the enzymatic activity. Since the amount of enzyme used in the assay varies for each protease, we represent the molar excess of the I. scapularis Kunitz peptide to achieve 50% inhibition of the respective enzyme, when compared to the amount of enzyme used in each assay (also see Materials and Methods).
Mentions: Compared to vertebrate Kunitz inhibitors that play a role in inflammatory responses, invertebrate Kunitz peptides possess a diverse inhibitory effect on protease activity [46]. To date, hundreds of tick Kunitz peptides have been discovered from sequencing projects [13], [47], [48], [49], [50]; however, as summarized below, roughly a dozen of tick Kunitz peptides are functionally described. Therefore, we overexpressed the divergent salivary Kunitz peptide in order to characterize its potential inhibitory activity against several vertebrate serine proteases. After HPLC purification, the purity of the recombinant peptide exceeded 95% (coomassie stained gel – data not shown). We then tested the recombinant peptide against 14 vertebrate serine proteases. Figure 2A depicts the results showing a statistically significant inhibition for trypsin, α-chymotrypsin, HSTβ, plasmin, matriptase and elastase. Figure 2B shows standard inhibition curves for all the targeted enzymes to further verify that the recombinant protein inhibits the above-mentioned enzymes [51]. Based on the data from these inhibition curves and calculating the Km of the substrates under the assay conditions (data not shown), we were able to calculate the apparent Ki (Ki (app)) for the targeted enzymes (see Materials and Methods) revealing a low pM Ki(app) for HSTβ (Table 1).

Bottom Line: Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin.The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

View Article: PubMed Central - PubMed

Affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic. valdjj@gmail.com

ABSTRACT

Background: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions.

Results: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases).

Conclusions: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

Show MeSH