μM, for S-2288 hydrolysis by matriptase: 290 ± 10 μM, for S-2288 hydrolysis by thrombin: 2.9 ... nM plasmin; 1 nM matriptase; 0.15 nM thrombin respectively).
1
Supplementary material to Kousted et al. “Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation” (Thromb Haemost 2014; 111.1) SUPPLEMENTARY METHODS DETAILS
Determination of the apparent second order rate constant for PN-1 protease inhibition Variable concentrations of PN-1 were pre-incubated in the absence or presence of 10-fold molar excess of heparin for 30 minutes at room temperature. The appropriate chromogenic substrate was added and the reaction was started by the addition of protease. In all assays, at least 10-fold excess of PN-1 over protease was used in order to maintain pseudo-first-order conditions. The final concentration of serpin was between 5-875 nM, that of protease was 0.05-2 nM and that of substrate was 0.2-3 mM. Substrate turnover was monitored for 60 minutes at 37 °C. For each PN-1 concentration, kobs values were estimated by fitting the progress curve (A405 vs. time) to equation 1, where [P]t and [P]∞ is the product concentration at time t and time infinite respectively.
[ P]t [ P] (1 e kobst )
(Eq. 1)
The estimated kobs values were then plotted against the PN-1, and KM and klim were determined by fitting the data to equation 2: [
- ]
(
[ ]
)
[
- ]
(Eq. 2)
Thus [S]0 is the substrate concentration at time zero and KM is that of protease catalysed hydrolysis of the substrate (see below). [PN-1]0 is the PN-1 concentration at time zero, KD is the dissociation constant for the formation of non-covalent Michaelis complex between serpin and protease and klim is the observed rate constant for formation of covalent serpin-protease complex at saturation. The second order rate constant, k2, is given by k2 = klim / KD. The KM value for S-2444 hydrolysis by uPA was 110 ± 30 M (1), for S-2251 hydrolysis by plasmin: 200 ± 10 M, for S-2288 hydrolysis by matriptase: 290 ± 10 M, for S-2288 hydrolysis by thrombin: 2.9 ± 0.4 M. For KM measurements, the initial rates of substrate hydrolysis were determined at variable concentrations of substrates (0-4 mM S-2251; 0-3 mM S-2288; 0-125 µM S-2288) and constant concentration of protease (1 nM plasmin; 1 nM matriptase; 0.15 nM thrombin respectively). Substrate turnover was monitored for 5 minutes at 37 °C and reaction velocities (V0) were calculated from the progress curves by linear regression.
2
Next, plotting V0 against the substrate concentration [S] allowed determination of KM from the saturation curve assuming Michealis Menten kinetics by fitting to equation 3: [ ]
[ ]
(Eq. 3)
Mass spectroscopy analysis of PN-1 cleavage product by target proteases. The in-gel digestion procedure of proteins separated by SDS-PAGE was performed essentially as described before (2). The samples were digested by sequence grade endoproteinase Lys-C (Promega) or porcine trypsin (Promega), peptides recovered by reverse-phase absorption (C18 Stagetip, Proxeon Biosystems), and the sample eluted directly onto the MALDI target using 1 μl of α-cyano-4-hydroxycinnamic acid in 70% acetonitrile, 0.1% trifluoroacetic acid. Peptides were subsequently analysed by using an Autoflex Smartbeam III instrument (Bruker) operated in positive mode. Prior to analyses, the instrument was calibrated by external calibration using a peptide mix containing 7 calibrants (Bruker). The obtained data were evaluated by using the GPMAW software (gpmaw.com)
SUPPLEMENTARY REFERENCE
1. Petersen HH, Hansen M, Schousboe SL, Andreasen PA. Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme. European journal of biochemistry / FEBS. 2001;268(16):4430-9.
3
SUPPLEMENTARY TABLE
TABLE S1 _______________________________________________________________________________________ Protease
Second order rate constants (M-1 s-1) PN-1 without heparin
PN-1 with heparin
_______________________________________________________________________________________ Thrombin
(2.9 ± 0.3) x 105
(8.8 ± 1.2) x 107
uPA
(4.8 ± 0.7) x 104
(8.0 ± 0.8) x 104
Plasmin
(1.4 ± 0.2) x 105
(2.1 ± 0.1) x 105
Matriptase
(2.3 ± 0.3) x 105
(1.4 ± 0.2) x 105
_______________________________________________________________________________________ Table S1. Second order rate constants for reaction of PN-1 with a variety of serine proteases Second order rate constants, k2 (M-1s-1) describing the reaction between E. coli PN-1 and various serine proteases are shown in the absence or presence of heparin. The data represents mean and standard deviations from at least three independent determinations.
4
SUPPLEMENTARY RESULTS
Mass spectrometric analysis confirms location of the scissile bond in the RCL of PN-1 To determine the site in PN-1, which is cleaved by the target protease in the presence of the antibodies, we performed mass spectrometric analysis of in-gel digests of intact and target protease-cleaved PN-1. We used endoproteinase Lys-C and trypsin for in-gel digestion. Target protease-catalysed cleavage of the reactive centre peptide bond Arg346-Ser347 and subsequent digestion by Lys-C, was expected to produce a Ala335-Arg346 peptide, which should be absent from Lys-C digestion of intact PN-1. This peptide pattern was in fact observed as an ion of m/z 1158.6 corresponding to Ala335-Arg346 (m/zcalc 1158.7) was detected only in the material representing target protease-cleaved PN-1 (Supplementary Figure S4A). Lys-C digestion of intact PN-1 was instead expected to generate a large peptide of m/z 4968.7 representing the C-terminus (Ala335-Pro379). However, this peptide is difficult to detect using in-gel digestion and mass spectrometry, since this approach relies on passive diffusion of peptides out of the gel. With trypsin cleaving after both lysine and arginine residues, we would expect the Ala335-Arg346 peptide to be present following digestion of both intact and target protease-cleaved PN-1. Again, the expected pattern was observed and ions of m/z 1158.8 representing the peptide Ala335-Arg346 peptide (m/zcalc 1158.9) were detected in both intact and target protease-cleaved PN-1 (Supplementary Figure S4B). Theoretically, cleavage of intact PN-1 by trypsin will generate two additional C-terminal peptides Ser347Arg363 and His364-Pro379. When increasing the sensitivity by performing MALDI-MS analysis in linear mode (m/z 1500-1900 range), we were able to detect an ion of m/z 1723.8 following trypsin digestion of intact PN-1 (Supplementary Figure S4C). This ion, which represents the His364-Pro379 peptide (m/zcalc 1723.9), was not detected in the target protease-cleaved PN-1 material consistent with the loss of the C-terminus. An additional His364-Pro379 peptide (m/zobs 1739.8) was detected in intact PN-1, encompassing a methionine sulfoxide (MetO) at position 373 (Supplementary Figure S4C). Taken together, these observations are fully compatible with target protease-cleaved PN-1 being cleaved at the reactive centre peptide bond Arg346-Ser347.
5
SUPPLEMENTARY FIGURES
FIGURE S1 SPR analysis of the specificity of mAbs Displayed is the binding of human E. coli PN-1 (solid black), murine PN-1 (solid red), rat PN-1 (grey dash) and human PAI-1 (blue dash) to mAb33, mAb34 and mAb39 or anti-murine PN-1 mAb16. The monoclonal antibodies were captured at about 430 RU by immobilised anti-mouse IgG and serpins (200 nM) were subsequently injected over mAb33, 34, 39 and 16, respectively. A >10-fold reduced steady-state level was observed for rat PN-1 to mAb34. Shown is the data of a representative single experiment from a series of at least three independent experiments.
6
FIGURE S2 Chromogenic assays of the effect of antibodies on PN-1 inhibitory activity Displayed is the hydrolysis of chromogenic substrate by uPA (A), plasmin (B) or matriptase (C) alone (black circles); in the presence of E. coli PN-1 (grey circles) or in the presence of E. coli PN-1 pre-incubated with mAb33 (squares), mAb34 (triangle down) or mAb39 (triangle up). The following assay conditions were used in A: 1 nM uPA; 10 nM PN-1, 100 nM mAb, 100 M S-2444; B: 2 nM plasmin; 20 nM PN-1, 200 nM mAb, 200 M S-2251; C: 0.2 nM matriptase; 2 nM PN-1, 20 nM mAb, 300 M S-2288. Shown is the data of a representative single experiment from a series of at least three independent experiments for each protease.
7
FIGURE S3 SDS-PAGE analysis of PN-1 activity in the presence of mAbs E. coli PN-1 at 1.6 M (1.5 g) was incubated with 1.5-fold excess of the indicated mAbs for 30 minutes at 22C. Where indicated, thrombin (A) and the serine protease domain (SPD) of matriptase (B) was added in 2-fold molar excess and incubation was then continued for additional 10 minutes at 22C. In the case of thrombin, an additional lane with PN-1 also incubated with 1.5-fold molar excess of heparin was included. All reaction mixtures were subjected to reducing SDS-PAGE. The migration of uPA, PN-1, RCL-cleaved PN-1, uPA-PN-1 complex and antibody heavy and light chain is indicated to the left. Variation in glycosylation state may account for the double band representing the mAb34 heavy chain. The migration of molecular weight markers (Mr) is indicated to the right.
8
FIGURE S4 MALDI MS analysis of in-gel digests Proteins separated by SDS-PAGE were subjected to in-gel digestion using endoproteinase Lys-C (A) or trypsin (B and C). Generated peptides were recovered by reverse-phase extraction and analysed by MALDI MS using reflector mode in the range of m/z 700-2000 (A and B) or linear mode in the m/z 1500-1900 range in order to increase sensitivity (C). Ions are assigned to the corresponding peptide sequences.
9
10
FIGURE S5 SPR analysis of mAbs binding to PN-1:heparin and PN-1:LRP complexes A. Displayed is the binding of mAb33 (short dash), mAb34 (medium dash), mAb39 (long dash), all injected at 10 nM (II), to E. coli PN-1 captured on a heparin coupled sensor chip at 240 RU (I). B. Displayed is the binding of 50 nM E. coli PN-1 alone (solid black line) or incubated with 100 nM mAb33 (solid blue line), mAb34 (solid grey line) or mAb39 (solid red line) to immobilised LRP ligand binding domain cluster IV. As controls, the mAbs alone were injected at 100 nM (broken lines). The curves shown in A and B are representative from a series of at least 3 injections.
A 300
200 RU
II
100
0
Buffer mAb33 mAb34 mAb39
I 0
2
4
6
8
10
Time (min)
B
12
14
16
11
FIGURE S6 SDS-PAGE analysis of the functional integrity of PN-1 variants Wild type or mutant E. coli PN-1 (1.6 M corresponding to 1.5 g) was incubated with or without 3.3 M (3.7 g) uPA for 10 minutes at 22C and the reaction mixtures were subjected to reducing SDS-PAGE. The migration of uPA, PN-1, RCL-cleaved PN-1, and uPA-PN-1 complex is indicated to the left of the gel. The migration of molecular weight markers (Mr) is indicated to the right.
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FIGURE S7 CD analysis of wild type and D153A PN-1 A. Wave length scan reveals identical structure content of wild type (solid grey line) and D153A (solid black line) E. coli PN-1. B. Thermal unfolding of wild type (grey curve, Tm ~57°C) and D153A (black curve, Tm ~53°C) PN-1 determined from the observed decay of structural content at 222 nm. Representative fit of a standard sigmoidal curve equation to the normalised raw data are shown including curve labels.
