Thrombin specificity

Chang, Jui‐Yoa; Alkan, Şefik Ş.; Hilschmann, Norbert; Braun, Dietmar

doi:10.1111/j.1432-1033.1985.tb09092.x

Cited by 29 publications

(15 citation statements)

References 35 publications

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“…2) and, as stated above, thrombin's long-known requirement for arginine in position P1 was reproduced [8], [9], [12], [35]. In position P2, the well-established proline [7], [8], [11], [12], [36], [37] dominated (71%), but also aliphatic aa were tolerated. P2 glycine, valine or isoleucine were together present in 23% of the sequences.…”

Section: Resultsmentioning

confidence: 71%

The Extended Cleavage Specificity of Human Thrombin

Gallwitz¹,

Enoksson²,

Thorpe³

et al. 2012

PLoS ONE

114

126

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Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.

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Section: Resultsmentioning

confidence: 71%

The Extended Cleavage Specificity of Human Thrombin

Gallwitz¹,

Enoksson²,

Thorpe³

et al. 2012

PLoS ONE

114

126

View full text Add to dashboard Cite

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“…Thrombin is a protease that recognizes LVPR^GS and cleaves between arginine and glycine [190][191][192]. Although thrombin cleavage at the designated sequence is relatively specific, there have been reports of thrombin cleavage at alternative sites [193].…”

Section: Thrombinmentioning

confidence: 99%

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Young

Britton

Robinson

2012

Biotechnology Journal

401

267

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Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

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“…Eel calcitoninb PTH fragment Antibody K-chain [59] Antibody i-chain [59] Antibody i-chain [59] Antibody A-chain [59] Antibody A-chain [59] P [54] Human factor XI11 [57] Human factor XI11 [57] Gastrin-releasing peptide Bovine &-thrombin [33] Antithrombin TIT [52] Rabbit myelin basic protein [55] Rabbit myelin basic protein [55] Cholecystokinin [26] Antibody i-chain [59] Antibody L-chain [59] Secretin [25] Myoglobin [48] Chymotrypsinogen A [48] Trysinogen [48] Human m-thrombin [34] Troponin [51] Rabbit myelin basic protein [55] Cholecystokinin their optimal P2-P4 structures. This effect is best demonstrated by the significant difference of thrombin susceptibility between salmon and eel calcitonins.…”

Section: Salmon Calcitoninbmentioning

confidence: 99%

Thrombin specificity

Chang

1985

European Journal of Biochemistry

Self Cite

202

108

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a-Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative aminoterminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynorphin A, somatostatins, gastrin-releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of a-thrombin. All selected polypeptides have one single major cleavage site and both Arg-Xaa and Lys-Xaa bonds were found to be selectively cleaved by a-thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit a-thrombin in 20 pl of 50 mM ammonium bicarbonate at 25 "C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for a-thrombin has the structures of (a) P4-P3-Pro-Arg-P 1'-P2', where P3 and P4 are hydrophobic amino acid and PI', P2' are nonacidic amino acids and (b) P2-Arg-P l', where P 2 or P 1' are Gly. The requirement for hydrophobic P 3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin-releasing peptide and calcitonins after chemical removal of hydrophobic P 3 and P4 residues. The requirement for nonacidic P1' and P2' residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P 1' and P2' residues. These findings confirm the importance of hydrophobic P2-P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1' and P2' residues are also crucial for thrombin specificity. It is concluded that specific cleavage of polypeptides by a-thrombin can be SedSOnably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage. a-Thrombin is a trypsin-like serine proteinase which preferentially cleaves polypeptide substrates at Arg/Lys-Xaa bonds [I -31. Unlike trypsin, a-thrombin displays highly restricted specificity toward protein substrates. This specificity is best known by its selective cleavage of two Arg-Gly bonds from amongst 181 Arg/Lys-Xaa bonds in fibrinogen [4]. Attempt to delineate the structural element in fibrinogen which accounts for restricted thrombin cleavage has mostly focussed on the fibrinogen Aa chain [5 -81 and has led to the findings that (a) the Aa (1-23) fragment contains a fundamental structure element for thrombin binding Table 1 for numbering of residues.) The recognization of the importance of hydrophobic P2 and P9 residues has further led to the development of a series of useful tripeptide substrates [9 -131 and inhibitors [I 4, 151. Another source of understanding of thrombin specificity comes from thrombin cleavage at non-fibrinogen polypeptides. A number of peptides and proteins have been Abbreviations. DABS-GI, dimethylaminoazobenzene sulfonyl chloride;...

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Thrombin specificity

Cited by 29 publications

References 35 publications

The Extended Cleavage Specificity of Human Thrombin

The Extended Cleavage Specificity of Human Thrombin

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

Thrombin specificity

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