Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

10.1038/72642 ◽  
2000 ◽  
Vol 18 (2) ◽  
pp. 187-193 ◽  
Author(s):  
Bradley J. Backes ◽  
Jennifer L. Harris ◽  
Francesco Leonetti ◽  
Charles S. Craik ◽  
Jonathan A. Ellman
Keyword(s):  
Substrate Specificity ◽  
Peptide Substrates ◽  
Positional Scanning ◽  
Fluorogenic Peptide ◽  
Positional Scanning Libraries

scholarly journals Human and mouse granzyme M display divergent and species-specific substrate specificities

2011 ◽  
Vol 437 (3) ◽  
pp. 431-442 ◽  
Author(s):  
Stefanie A.H. de Poot ◽  
Marijn Westgeest ◽  
Daniel R. Hostetter ◽  
Petra van Damme ◽  
Kim Plasman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Specific Substrate ◽  
Structural Determinants ◽  
Cytotoxic Lymphocyte ◽  
Potent Inducer ◽  
Methionine Residue ◽  
Positional Scanning ◽  
Methionine Residues ◽  
Species Specific ◽  
Positional Scanning Libraries

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

scholarly journals Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates

2017 ◽  
Vol 117 (09) ◽  
pp. 1750-1760 ◽  
Author(s):  
Emrah Kara ◽  
Dipankar Manna ◽  
Åge Løset ◽  
Eric L. Schneider ◽  
Charles S. Craik ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Phage Display ◽  
Factor Vii ◽  
Small Peptides ◽  
Inhibitory Antibody ◽  
Fluorescent Substrate ◽  
Complex Biological System ◽  
Peptide Substrates ◽  
Positional Scanning

SummaryFactor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1’). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histonetreated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization

2003 ◽  
Vol 370 (1) ◽  
pp. 57-67 ◽  
Author(s):  
John M.U. HAMILTON ◽  
David J. SIMPSON ◽  
Stefan C. HYMAN ◽  
Bongani K. NDIMBA ◽  
Antoni R. SLABAS
Keyword(s):  
Arabidopsis Thaliana ◽  
Substrate Specificity ◽  
Proteolytic Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Cell Suspension Cultures ◽  
Immunogold Labelling ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
Acidic Conditions ◽  
Fluorogenic Peptide

A C-terminal portion of Ara12 subtilisin-like protease (residues 542—757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1′ position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

PROTEOMICS ◽  
2005 ◽  
Vol 5 (5) ◽  
pp. 1292-1298 ◽  
Author(s):  
Dhaval N. Gosalia ◽  
Cleo M. Salisbury ◽  
Dustin J. Maly ◽  
Jonathan A. Ellman ◽  
Scott L. Diamond
Keyword(s):  
Substrate Specificity ◽  
Serine Protease ◽  
Solution Phase ◽  
Peptide Microarrays ◽  
Protease Substrate ◽  
Fluorogenic Peptide
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