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. Inhibition of HSTβ by tryptogalinin.A two-phase exponential fit of the data for HSTβinhibition suggests a 2:1 binding ratio (A) for tryptogalinin and HSTβ (2 pM). Tryptogalinin tightly binds to HSTβ (B) – as the enzyme increases so does the IC50.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643938&req=5

pone-0062562-g003: A–B. Inhibition of HSTβ by tryptogalinin.A two-phase exponential fit of the data for HSTβinhibition suggests a 2:1 binding ratio (A) for tryptogalinin and HSTβ (2 pM). Tryptogalinin tightly binds to HSTβ (B) – as the enzyme increases so does the IC50.

Mentions: In Figure 3A we show that inhibition of HSTβ by tryptogalinin gave a two phase exponential fit indicating that tryptogalinin binds to HSTβ in 2:1 ratio (inhibitor:enzyme); this is further supported with the fact that we used 9.6 pM of tryptogalinin for a 50% inhibition of 5 pM of HSTβ (IC50). Figure 3B describes tryptogalinin as a tight binding inhibitor of HSTβ since the inhibition curves were different when we increased the amount of enzyme used in our assays. Finally, we show that there is a direct correlation between the amount of HSTβ used in the assay with the estimated IC50 (see Figure S1) – a typical characteristic of tight binding inhibitors [52]. The HSTβis a trypsin-like protease found in mast cells and a key player in inflammatory responses [58], therefore, our current study may prove useful for any future pharmaceutical studies. It is worth noting that the specificity of β-tryptase function and inhibition is due to its tetrameric structure [59]. To date, there are only two resolved crystal structures that are potent inhibitors of β-tryptase: TdPI and leech-derived tryptase inhibitor [60], [61].


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. Inhibition of HSTβ by tryptogalinin.A two-phase exponential fit of the data for HSTβinhibition suggests a 2:1 binding ratio (A) for tryptogalinin and HSTβ (2 pM). Tryptogalinin tightly binds to HSTβ (B) – as the enzyme increases so does the IC50.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062562-g003: A–B. Inhibition of HSTβ by tryptogalinin.A two-phase exponential fit of the data for HSTβinhibition suggests a 2:1 binding ratio (A) for tryptogalinin and HSTβ (2 pM). Tryptogalinin tightly binds to HSTβ (B) – as the enzyme increases so does the IC50.
Mentions: In Figure 3A we show that inhibition of HSTβ by tryptogalinin gave a two phase exponential fit indicating that tryptogalinin binds to HSTβ in 2:1 ratio (inhibitor:enzyme); this is further supported with the fact that we used 9.6 pM of tryptogalinin for a 50% inhibition of 5 pM of HSTβ (IC50). Figure 3B describes tryptogalinin as a tight binding inhibitor of HSTβ since the inhibition curves were different when we increased the amount of enzyme used in our assays. Finally, we show that there is a direct correlation between the amount of HSTβ used in the assay with the estimated IC50 (see Figure S1) – a typical characteristic of tight binding inhibitors [52]. The HSTβis a trypsin-like protease found in mast cells and a key player in inflammatory responses [58], therefore, our current study may prove useful for any future pharmaceutical studies. It is worth noting that the specificity of β-tryptase function and inhibition is due to its tetrameric structure [59]. To date, there are only two resolved crystal structures that are potent inhibitors of β-tryptase: TdPI and leech-derived tryptase inhibitor [60], [61].

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