Amino acid sequence of piguamerin, an antistasin‐type protease inhibitor from the blood sucking leech <i>Hirudo nipponia</i>

Kim, Dong Ryoung; Kang, Kae Won

doi:10.1046/j.1432-1327.1998.2540692.x

Cited by 30 publications

(23 citation statements)

References 4 publications

(5 reference statements)

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“…Some of the isolated putative serine protease inhibitors of this work display a clear similarity with other inhibitors of the antistasin family previously found in leeches, such as guamerin (44,45), piguamerin (46), bdellin A (47), and hirustasin (48) (see Table II). In Fig.…”

Section: Multidimensional Liquid Chromatography For the Separation Ansupporting

confidence: 66%

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

Yanes¹,

Villanueva

Querol

et al. 2005

Molecular & Cellular Proteomics

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The blood-feeding invertebrates are a rich biological source of drugs and lead compounds to treat cardiovascular diseases because they have evolved highly efficient mechanisms to feed on their hosts by blocking blood coagulation. In this work, we focused our attention on the leech Hirudo medicinalis. We performed, by "intensity fading" MALDI-TOF mass spectrometry, a comprehensive detection and functional analysis of pre-existent peptides and small proteins with the capability of binding to trypsin-like proteases related to blood coagulation. Combining "intensity fading MS" and off-line LC prefractionation allowed us to detect more than 75 molecules present in the leech extract that interact specifically with a trypsinlike protease over a sample profile of nearly 2,000 different peptides/proteins in the 2-20-kDa range. Moreover we resolved 232 individual components from the complex mixture, 13 of which have high sequence homology with previously described serine protease inhibitors. Our findings indicate that such extracts are much more complex than expected. Additionally, intensity fading MS, when complemented with LC separation strategies, seems to be a useful tool to investigate complex biological samples, establishing a new bridge between profiling, functional peptidomics, and subsequent drug discovery. Molecular & Cellular Proteomics 4:1602-1613, 2005.Although the task of identifying and characterizing genes in many sequenced genomes in the last years has been very intense, it is even more challenging when applied to the much higher complex field of the protein world (1). One difficult set of proteins for such analysis are those which we could term as "small" (i.e. below 15-20 kDa) (2) because of their variety and difficulty to be detected by several established proteomic approaches such as 2D 1 electrophoresis (3) or computational genomic scanning (4) among others. Therefore, the development of efficient and high throughput technologies for producing functionally related data of peptides and small proteins (2-20 kDa) is increasingly attracting attention. Some of these proteins have potential use for diagnostics or therapeutics (5-7). They include families of peptide hormones, neuropeptides, cytokines, growth factors, and enzyme inhibitors acting as biochemical messengers or modulators that organize the regulatory processes in all organisms (6). In fact, most of the important biopharmaceutical products approved for therapeutic applications are molecules within a molecular mass range of 1-40 kDa (8).The proteomic strategy focused on the analysis of peptides and small proteins from complex mixtures has been named "peptidomics" (9, 10). The primary proteomic tool, 2D gel electrophoresis with mass spectrometry, has been gradually enhanced, and sometimes replaced, by three main technologies: MALDI MS in combination with LC (off line) (9, 11, 16), ESI MS combined with nano-LC (on line) (12-16), and MALDI-TOF MS for in situ peptide profiling (17-21).One of the main goals of these technologies is to provide a compre...

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Section: Multidimensional Liquid Chromatography For the Separation Ansupporting

confidence: 66%

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

Yanes¹,

Villanueva

Querol

et al. 2005

Molecular & Cellular Proteomics

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“…Therin inhibits bovine trypsin with high affinity and specificity (Ki = 45 pM). This is much higher than other [82,83]), Hirudinaria nipponia (guamerins and piguamerin [84][85][86]) and the non-blood sucker leech Whitmania edentula (guamerin II, [87]) ( Table 3).…”

Section: Serine Protease Inhibitors In Leechesmentioning

confidence: 78%

A Review of the Most Important Classes of Serine Protease Inhibitors in Insects and Leeches

Clynen

Schoofs

Salzet

2005

MCRO

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The constant increase of life expectancy is associated with major aging of developed populations. This indicates that the new century will have one of most epidemic progressions of cardiovascular, cancer and inflammatory diseases. The high challenge for medical research is to compress such morbidity. Invertebrates have demonstrated to be truly useful models in drug discovery for such aging diseases. The last decade, drug discovery in leeches has opened the gate for new molecules to treat emphysema, coagulation, inflammation, dermatitis and cancer. Also other invertebrates, such as insects, harvest potential interesting molecules, such as serine protease inhibitors that can be exploited by the medical industry. In this review we discuss the most important classes of serine protease inhibitors in insects and leeches.

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“…Moreover, therostasin shows 31% sequence identity with the internal repeats 4 and 5 of Hydra antistasin, a Factor Xa inhibitor isolated from Hydra (18). In addition, therostasin shows 26-37% sequence identity with antistasintype proteins isolated from leeches including guamerin (19), hirustasin (20), and piguamerin (21). Sequence alignment of therostasin with these peptides revealed that identity is observed mainly in the C terminus of these inhibitors (Fig.…”

Section: Biochemical Characterization Of Therostasin-t Tessulatummentioning

confidence: 91%

Therostasin, a Novel Clotting Factor Xa Inhibitor from the Rhynchobdellid Leech, Theromyzon tessulatum

Chopin¹,

Salzet²,

Baert³

et al. 2000

Journal of Biological Chemistry

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Therostasin is a potent naturally occurring tightbinding inhibitor of mammalian Factor Xa (K i , 34 pM), isolated from the rhynchobdellid leech Theromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-␤-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.

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Amino acid sequence of piguamerin, an antistasin‐type protease inhibitor from the blood sucking leech Hirudo nipponia

Cited by 30 publications

References 4 publications

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

A Review of the Most Important Classes of Serine Protease Inhibitors in Insects and Leeches

Therostasin, a Novel Clotting Factor Xa Inhibitor from the Rhynchobdellid Leech, Theromyzon tessulatum

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