MALDI-ToF MS characterization of the substituted peptide after incubation with plasmin. (A) MALDI-ToF MS characterization of the substituted R 1122 to G 1122 P36 peptide after incubation with plasmin (E:S 1:50, 37°C overnight). No cleavage was observed. (B) MALDI-ToF MS characterization of the substituted S 1120 to W 1120 P36 peptide after incubation with plasmin (E:S 1:50, 37°C overnight). Complete digestion was observed. (C) MALDI-ToF MS characterization of the substituted G 1121 to W 1121 P36 peptide after incubation with plasmin (E:S 1:50, 37°C overnight). Complete digestion was observed. (D) MALDI-ToF MS characterization of the substituted C 1123 to W 1123 P36 peptide after incubation with plasmin (E:S 1:50, 37°C overnight). Complete digestion was observed. (E) MALDI-ToF MS characterization of the substituted N 1124 to W 1124 P36 peptide after incubation with plasmin (E:S 1:50, 37°C overnight). Complete digestion was observed. (F) Panel summarizing the cleavage of modified peptides by plasmin. 

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... Mechanisms leading to the cleavage of type XIX collagen and to the release of NC1(XIX) have never been studied. Interestingly, a potential sequence for plasmin cleavage, due to the presence of an arginine residue, is present at the beginning of the NC1(XIX) domain. Plasmin is implicated in the cleavage of many extracellular matrix components, or in the activation of matrix metalloproteinase (MMP) zymogens. It is an important serine proteinase, with a wide range of physiological functions and pathological implications [27]. It hydrolyses Arg-Xaa or Lys-Xaa peptide bonds. The proteolytic cleavage of the inactive plasminogen by tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) generates plasmin [28]. The aim of this study was to investigate if plasmin could release NC1(XIX) from the α1(XIX) collagen chain. For that purpose, we submitted a peptide derived from the α1(XIX) chain to plasmin proteolysis in vitro. We showed that an active anti-tumor fragment of the NC1(XIX) peptide was rapidly released from the α1(XIX) chain. We identified the cleavage sites and sequenced the different proteolytic fragments. By using different amino acid substitutions in the plasmin cleavage site, we demonstrated that the Arg 1122 residue was crucial for plasmin cleavage. Plasmin specifically cleaves extracellular matrix and basement membrane components. To evaluate whether plasmin could cleave the C-terminal domain of type XIX collagen, we used a synthetic peptide (P36) composed of the last 36 residues of the human α1(XIX) chain, homologous to that previously described by Boudko et al [29]. The P36 peptide contained the last 17 amino-acids of the α1(XIX) helical Col1 domain (SPGAPGPQGPPGPSGRC) and the totality of the C-terminal NC1(XIX) domain (NPEDCLYPVSHAHQRTGGN). P36 peptide was submitted to plasmin digestion for 16 h. The molar ratio of plasmin to P36 peptide was 1:50. The digestion products were separated by HPLC using the method 1. The absorbance peak (A 214 nm ) of P36 peptide was detected at 25 min with method 1 (Figure 1A) or at 23.5 min with method 2 (Figure 1B). HPLC analysis of the digestion products showed that plasmin hydrolysis generated 3 main peaks: F1, F2, and Fx (Figure 1A). F1 peak was eluted at 11.5 min, F2 at 21 min and Fx at 25.5 min, respectively. The Fx peak was further fractionated using the method 2. It provided two peaks, F3 and F4, with a retention time 24.5 and 22 min, respectively (Figure 1B). (Figure 1B). The plasmin digestion products were characterized by MALDI-ToF MS analysis. The analysis of intact P36 peptide revealed a 3592.74 Da major peak, which matched the expected P36 Peptide molecular weight (Figure 1C). The major plasmin digestion products (F2, F3, and F4) of the P36 peptide corresponded to 1415.81 Da, 3263.81 Da and 1866.95 Da, respectively (Figure 1D). The 1415.81 Da F2 peak matched the expected mass of the SPGAPGPQGPPGPSGR Col1 domain of P36 peptide. The 1866.95 Da F4 peak matched the expected 16 first amino- acid residue sequence (CNPEDCLYPVSHAHQR) of the NC1(XIX) domain. The 3263.81 Da F3 peak matched the expected entire P36 peptide sequence without the four last C-terminal residues (TGGN) (Figure 1D). The F1 peak seemed to correspond to the C-terminal TGGN residues. MALDI-ToF MS sequence analysis confirmed the in vitro plasmin cleavage of P36 peptide at 2 sites. The first plasmin cleavage occurred at Arg 1138 -Thr 1139 and released the 4 C-terminal amino-acid residues of P36 peptide (peak F1, TGGN). The second cleavage occurred at Arg 1122 -Cys 1123 and released the major part of NC1(XIX) domain (peak F4, CNPEDCLYPVSHAHQR). Amino acid sequences of all the plasmin cleavage products are summarized in Figure 1E. MALDI-ToF MS analyses also demonstrated that the P36 peptide, F3 and the F4 fragment formed an intra-chain disulfide-bond between Cys 1123 and Cys 1128 in solution over the time (Figure S1). The digestion of P36 peptide by plasmin was dose- dependent (Figure 2A). Kinetic analysis of the P36 peptide cleavage by plasmin was performed by incubating P36 with plasmin during 0, 5 min, 30 min or 60 min. HPLC analysis was done with method 1. The digestion of P36 peptide by plasmin was time-dependent (Figure 2B). P36 peptide was cleaved as soon as the first 5 min of incubation whereas plasmin digestion products (Fx) appeared gradually (Figure 2B). (Figure 2B). K M and Kcat were 73.04 nM, and 0.077s –1 respectively and the ratio Kcat/K M was 1506.5 M.s –1 . To confirm plasmin involvement in the P36 peptide cleavage, aprotinin was added to the medium during the incubation of P36 with plasmin. 10 –5 M (pointed grey) and 10 –6 M (red) aprotinin concentrations completely abolished P36 peptide plasmin digestion whereas 10 –7 M aprotinin concentration was not sufficient to prevent it (black) (Figure 2C). Five substituted peptides were synthesized to study the plasmin cleavage site at Arg 1122 and to determine the amino acids crucial for plasmin cleavage (Figure 3). The Arg 1122 –> Gly substituted P36G 1122 peptide was no longer cleaved by plasmin. This confirms that Arg 1122 was crucial for plasmin cleavage (Figure 3A). On the contrary, substitution of the two preceding (P36W 1121 , P36W 1120 ) or the two following residues (P36W 1123 , P36W 1124 ) still allowed P36 peptide cleavage by plasmin (Figures 3B–3E). These results show that only Arg 1122 is necessary for the cleavage of P36 peptide by plasmin, whereas the two preceding and the two following residues are not. SKMEL28 melanoma cells were cultured with or without plasminogen and/or aprotinin. These cells secreted tPA into the extracellular medium (Figure 4A). In the presence of plasminogen, cell-generated-plasmin hydrolyzed the chromogenic substrate (Figure 4B). Aprotinin completely inhibited plasmin activity (Figure 4B). In the absence of plasminogen, no release of F2, F3, and F4 was observed (Figure 4C). When plasminogen was added to the culture medium, cell-generated plasmin released the F2, F3 and F4 fragments (Figure 4D). Aprotinin abolished this release (Figure 4E), as well as the Arg 1122 substitution (Figure 4F). Cell transfection with siRNA specific to human tPA decreased (–60%) tPA mRNA expression (Figure 4G). Zymography (Figure 4H) and immunocytofluorescence (Figure 4I) studies demonstrated that tPA protein expression was also decreased. Under our experimental conditions, siRNA transfection also decreased the proteolysis of P36 peptide by plasmin in cell culture medium (–56%) (Figure 4J). In a scratch wound test F4 peptide (200 μg/mL) decreased wound closure after 24 h (–53%, p < 0.05), 48 h (–39%, p < 0.05) or 72 h (–36%, p < 0.05) of incubation (Figure 5A). Similarly, in an in vivo murine melanoma model, F4 peptide significantly inhibited tumor growth at day 15th (–30%, p < 0.05) (Figure 5B). Visualization of the different trajectories showed that both P19 and F4 peptides, independently of the starting conformation (displayed in Figure 6A), adopted a small amount of hydrogen secondary structure (β-bridge, β-sheet or helix), low number of β-bridge, β-sheet or helix local structure. Globally, the peptides adopted mostly random structures. These observations were confirmed by the analysis of the local secondary structures performed using the DSSP (Define Secondary Structure of Proteins) algorithm as implemented in the GROMACS package [30]. In all simulations, along the 200 ns, the coil local structure proportion was above 40%. The fractions of bends and turns were more important when starting from conformations generated with the Itasser or the Pepfold servers. The presence of the disulfide bridge between the two cystein residues in the F4 peptides also promoted the formation of turns. We also determined the localization and the geometry of the β-turns (Table S1). For the 8 MD simulations, the sequences of type I β-turns occurring more than 5% along the whole simulation (1000 occurrences given the 20001 total snapshots for each simulation) are displayed. A first observation was that the peptides whose starting conformation was not an elongated one showed more sequences of type I turns. Moreover, the type I NPED turn was found in 6 MD simulations out of 8 (75% of the simulations). In the case of F4 peptide with disulfide bridge, this local structure was explored more than 60% of the time. We extracted one snapshot from each of these 6 MDs. We could then highlight the geometry of the NPED type I turn (Figure 6B). In addition, for each type of peptide (P19 or F4), we superimposed the type-I turns (Figure 6C). During tumor invasion, tumor cells invade surrounding normal tissue. In cancer progression, two major classes of proteases play a crucial role, the matrix metalloproteinase (MMP) family and the plasminogen activation system [31]. For migration and invasion, tumor cells degrade the extracellular matrix, and cross vascular epithelial basement membranes. Therefore, matrix metalloproteinases (MMP) and the plasminogen/ plasmin system cleave basement membrane components into multiple fragments released into the tumor microenvironment [32]. Collagen XIX is a minor collagen associated with basement membranes. It can be proteolysed during tumor invasion. We previously demonstrated that its 19 amino acids NC1(XIX) domain exerts anti-tumor and anti- angiogenic properties on melanoma. In the present study, we demonstrated that plasmin may release an anti-tumor fragment of NC1(XIX). Plasmin is largely involved in tumor invasion, particularly in melanoma [33, 34]. Previous studies on other basement membrane collagens have clearly demonstrated that collagen NC1 domains are released during tumor invasion. For instance, endostatin, the NC1 domain of type XVIII collagen, is released by many proteases such as MMP-3, 7, 9, 12, 13, 20 [14, 35, 36], or cathepsins B, L, S, V [37, 38, 39]. Endostatin exerts significant anti-tumor properties in number of experimental cancer models [7, 43] and reduces ...