Fluorogenic Peptide Substrates for Proteases in Blood Coagulation, Kallikrein-Kinin and Fibrinolysis Systems

Iwanaga, Sadaaki; Morita, Takashi; Kato, Harubumi; Harada, Tokuya; Adachi, Nami; Sugo, Teruko; Maruyama, Ikuro; Takada, Katsumi; Kimura, Takuo; Sakakibara, Shunsuke

doi:10.1007/978-1-4757-0926-1_15

Cited by 33 publications

(11 citation statements)

References 20 publications

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“…2D). These results are consistent with a previous report by Iwanaga et al, which found a preference for substrates with a P 2 Pro residue over those with a P 2 Ser, Thr, or Gly residue when studying a panel of 15 peptidyl coumarin substrates (21).…”

Section: Resultssupporting

confidence: 93%

“…substrates with a P 2 Phe residue to be more efficiently cleaved than those with P 2 Gly or Pro residues (21). Whereas it has been noted that extending substrate length beyond the P 2 site has little effect on turnover efficiency for some synthetic substrates (20), extension to at least the P 4 site is critical for the physiological functioning of plasma kallikrein (17).…”

Section: Resultsmentioning

confidence: 96%

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High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

153

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 96%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

153

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“…Plasminogen activator activities of the deacylated native and modified enzyme complexes were assayed with a synthetic fluorogenic substrate, Boc-Val-Leu-Lys-MCA, as follows [9]: enzyme complexes (0.5 pg) were preincubated with 50 pg human plasminogen in 1 ml of 20 mM L-lysine/lOO mM NaCI/SO mM Tris-HCl (pH 8.0) for 10 min at 37°C. Then, 1 ml of 0.1 mM Boc-Val-Leu-Lys-MCA was added and incubated for 20 min at 37°C.…”

Section: Plasminogen Activator Activitymentioning

confidence: 99%

Modification of acyl‐plasmin‐streptokinase complex with polyethylene glycol

et al. 1985

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Acyl‐plasmin‐streptokinase complex has advantages as a ‘site’ directed fibrinolytic agent with the active site protected from the plasma protease inhibitors. But, in clinical use, the fibrinolytic potential of this acylenzyme complex is modified or abolished by the presence of streptokinase antibodies in the patients. Therefore, better therapeutic agents are required. In this work, chemical modification of the acyl‐plasmin‐streptokinase complex with polyethylene glycol was found to result in marked resistance to neutralization with streptokinase antibodies.

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“…Substrate specificity of F.XIa was examined using various synthetic peptidyl fluorogenic substrates and was compared with that of bovine (Iwanaga et al, 1979) or human (Kawabata et al, 1988) F.XIa. The coagulation assay with human deficiency plasma clearly indicates that porcine F.XIa can activate human F.XI.…”

mentioning

confidence: 99%

“…ER: Essentially resistant; ND: Not detectable; NE: Not examined. a Data were taken from Iwanaga et al (1979). b Data were taken from Kawabata et al (1988), and relative activity was recalculated (Boc-Phe-Ser-Arg-NH-Mec = 100%).…”

mentioning

confidence: 99%

Short Communication

1994

Biological Chemistry Hoppe-Seyler

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Porcine factor XI and activated factor XI were purified by the introduction of affinity Chromatography on high molecular mass kininogen. On the affinity Chromatography, it was observed that high affinity exsists between porcine factor XI and high molecular mass kininogen. In the preparation, however, factor XII, plasma prekallikrein and high molecular mass kininogen were not detected. The factor XI forms a dimer, and is a heterogeneous molecule, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity of activated factor XI and inhibition profile of activated factor XI against proteinase inhibitors were investigated by comparison with those of bovine and human activated factor XI. From these results, the properties of porcine activated factor XI show great similarities with those of bovine and human activated factor XI.

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Fluorogenic Peptide Substrates for Proteases in Blood Coagulation, Kallikrein-Kinin and Fibrinolysis Systems

Cited by 33 publications

References 20 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Modification of acyl‐plasmin‐streptokinase complex with polyethylene glycol

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