The Extended Cleavage Specificity of Human Thrombin

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

Sculpted through Time : Evolution and Function of Serine Proteases from the Mast Cell Chymase Locus

Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of granule serine proteases, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display

Amino acid summary of thrombin cleavage sequences.

<p>Amino acids shown in bold and larger font are deviations from the preferred thrombin consensus sequence. The cleavage efficiency compared to the consensus is shown as a percentage.</p

Amino acid frequency in positions P4 to P4′ of thrombin-susceptible phage sequences.

<p>This analysis is based on the alignments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031756#pone-0031756-g001" target="_blank">Figure 1A and 1B</a>. For clarity, amino acids are displayed in functional groups, starting to the left with aromatic residues, and ending with acidic residues to the right.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Comparison of selected studies since 1981 establishing the substrate recognition sequence of thrombin.

<p>Letters in bold indicates investigated positions, residues that were held constant are in parentheses. The preferred amino acids are denoted in the order of preference. Equally favorable residues are indicated by the absence of a slash (/). n.d., not determined; -, not applicable; pNA, para-nitroanilide.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Analysis of the cleavage specificity by the use of new types of recombinant protein substrate.

<p>Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by thrombin. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His₆-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one <i>Bam HI</i> and one <i>SalI</i> site, which are indicated in the bottom of panel A. Panels B to E shows the cleavage of a number of substrates by thrombin, where individual amino acids has been changed from the thrombin consensus sequence. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage (in minutes) is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Alignment of sequences obtained after five selection rounds with 1 U of thrombin or 0.2 U of thrombin, compared to natural substrates.

<p>Panel A shows the result with 1 U of thrombin, panel B the result with 0.2 U of thrombin and panel C a panel of natural substrates. The P1 residue in natural substrates (after which cleavage occurs) is denoted in parentheses. Substrate sequences refer to <i>Homo sapiens</i> where not indicated otherwise.! marks phage sequences that have LTP<u>R</u>G instead of LTP<u>G</u>G in the N-terminal flank. Residues from the non-randomized phage region are in italics. *, from <i>Rattus norvegicus</i>; IGFBP, insulin-like growth factor-binding protein; PAR, protease-activated receptor. The cleavage site of thrombin in the natural substrates listed in panel C is numbered from the N terminal of the pre-pro protein, from the first methionine. This list of natural substrates is a selection of a few of the most well known substrates of this enzyme. However, the list of potential in vivo substrates is much longer and includes many other proteins such as protein S, TAFI, antithrombin, heparin cofactor II and nexin I.</p