The importance of exosite interactions for the cleavage efficiency of cleavage sites in fibrinogen α chain (panel A), β chain (panel C) and protein C (panel E).

<p>The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels B, D and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.</p

Analyses of the importance of Phe8, Gly12 and three negatively charged residues in the N-terminal region of fibrinogen α chain for the cleavage by thrombin.

<p>The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. The mutations are marked in green in the sequences above each gel. Panels B and D shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.</p

The FcαR locus and neighboring genes.

<p>Panel A shows a schematic drawing of chromosome 19, panel B a schematic drawing of the LRC locus, panel C the domain structure of the LILRs of this locus (an ‘R’ represents arginine in the transmembrane region) and panel D the domain structure of the KIRs. In the detailed map of the region encoding the IgA receptor each horizontal line corresponds to a chromosome on which different Fc receptor genes are located. Genes are color coded. The IgA receptor in red, the KIRs and LILRs in yellow, the neighboring genes that are used as markers for the chromosomal region, NCR1 in dark green, the NACHT in orange, the NLRP7 in light blue, the PGRL1 in light green and the FQD1-FB1 in dark blue. The overall structure of the LRC locus and the protein domain structures have been adopted from Espeli et al 2010 and Brown et al 2004 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096903#pone.0096903-Espeli1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096903#pone.0096903-Brown1" target="_blank">[68]</a>.</p

Analysis of the cleavage by active rMCP-2 of a number of recombinant cell adhesion and cell junctional proteins.

<p>Panel A shows schematic drawings of the basic structure of occludin and claudin. The sequence of the surface loops of occludin and claudin 7 are shown in panel B. The extracellular region is shown in black where the potential rMCP-2 cleavage sites (Tyr and Phe) have been marked in red. The yellow region shows the short region of the membrane spanning region remaining in these clones and the purple region is the kinker including a Cys that has been inserted to form a loop structure of the extracellular loops in the 2xTrx construct. In panel C the cleavage pattern of the rMCP-2 consensus 2xTrx substrate (VVLFSAVL), as positive control, occluding loop 1, occluding loop 2 and claudin 7 loop 1 is depicted. In panel D the cleavage of rat cadherin 17, rat E-cadherin and human protocadherin alpha 4 is presented where the protein is marked above each cleavage reaction and the time of cleavage in minutes is depicted above each lane. These three latter recombinant proteins were produced in mammalian cells. As can be seen from the figure cadherin 17 and protocadherin alpha 4 are very sensitive to cleavage by rMCP-2 whereas almost no cleavage is seen for E-cadherin.</p

Analyses of the minimal sites for three thrombin cleavage sites in FVIII by the use of recombinant protein substrates.

<p>Panel A shows the overall structure of the recombinant protein substrates used for analysis. In these substrates, two thioredoxin (trx) 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 trx molecules with the use of two unique restriction sites, one <i>BamHI</i> and one <i>SalI</i> site, which are indicated in the bottom of panel A. Panels B shows a schematic representation of a cleavage reaction. 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. Panel C shows a comparative analysis of the cleavage efficiency of the thrombin consensus sequence with the P4-P4’ sequences from three cleavage sites in FVIII. 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 their corresponding lanes. In panel D we show an SDS-PAGE density summary of cleaved substrates. All protein gels were analyzed using Image Quant TL 1D gel density software (v8.1) from GE Life Science (Piscataway, NJ USA) or the UN-SCAN-IT Gel Analysis Software from Silk Scientific Inc. (Orem, Utah USA). Individual bands from the full-length constructs (top bands) were analyzed with manual lane editing and minimum profile. Bands were detected automatically and gating adjusted to compare cleavage over the time course. Figures show percentage cleavage of the original construct (time point 0 minutes). Standard deviation of the time points are shown (mean +- standard deviation). Statistical analyses were performed using the Mann-Whitney test with two-tailed P value.</p

Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.

<p>Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.</p

Analysis of the cleavage specificity by the use of recombinant protein substrates.

<p>Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the MC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His_<b>6</b>-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 Bam HI and one SalI site, which are indicated in the bottom of panel A. Panels C-G show the cleavage of a number of substrates by rMCP-2. 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 (Panel B).</p

The major Fc receptor gene loci in a panel of different vertebrates.

<p>Each horizontal line corresponds to a chromosome on which different Fc receptor genes are located. Genes are color coded. The Fc receptor-like (FcRL) genes are shown in yellow except the FcRLA and B that are in light green, classical IgG receptors in red (pseudogenes in striped red), IgE receptor in dark green, the IgM receptor in orange, the poly-Ig receptor (PIGR) and the IgA/IgM receptor in blue. The figure contains the genes identified for the following animal species: Zebrafish (<i>Danio rerio</i>), Western clawed frog (<i>Xenopus silunarana</i> and <i>Xenopus tropicalis</i>), Chicken (<i>Gallus gallus</i>), Turkey (<i>Meleagris gallopovo)</i>, Zebra Finch (<i>Taeniopygia guttata</i>), Green anole (<i>Anolis carolinennsis</i>), Platypus (<i>Ornithorhynchus anatinus</i>), Opossum (<i>Monodelphis domestica</i>), Rat (<i>Ratuus norvegius</i>), Mouse (<i>Mus musculus</i>), Horse (<i>Equus caballus</i>), Rabbit (<i>Oryctologus cunicullus</i>), Pig (<i>Sus scrofa</i>), Dog (<i>Canis lupus familaris</i>), Cattle (<i>Bos taurus</i>), Orangutan (<i>Pongo abelli</i>), Chimpanzee (<i>Pan troglodytes</i>), Rhesus macaque (<i>Macaca mulatta</i>), Human (<i>Homo sapiens</i>).</p

The immunoglobulin heavy chain locus of a panel of selected vertebrates from fish to humans.

<p>The locus depicts every single gene as a block and without individual exons. The figure is not to scale and the genes have been color coded; IgM in black, IgD in dark green, IgA and IgX in light green, IgY in magenta, IgG in blue, IgE in purple, IgO in red, IgZ in yellow, IgF in orange and pseudogenes in shaded grey or purple.</p

Identification of potential in vivo targets for rMCP-2.

<p>By screening of the entire rat proteome with a 4 amino acid peptide sequence having the following sequence: P2 (L/V), P1 (F/Y), P1' (S/A/V/R), P2' (A/S/V) resulted in 4825 rat specific hits. This sequence was derived from the rMCP-2 cleavage consensus sequence. Nuclear and cytoplasmic proteins were removed from the list, as they most likely do not represent likely <i>in vivo</i> targets. Among these originally 4825 hits, 78 were considered to be particularly interesting and could be considered as potential substrates for rMCP2 and these are listed in this table.</p><p>Identification of potential in vivo targets for rMCP-2.</p