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Establishment of an HPLC-based method to identify key proteases of proteins in vitro
Accepted Manuscript Establishment of an HPLC-based method to identify key proteases of proteins in vitro Qingqing Wei, Hong Tian, Fan Zhang, Wenbo Sai, Yang Ge, Xiangdong Gao, Wenbing Yao PII:
S0003-2697(19)30022-3
DOI:
https://doi.org/10.1016/j.ab.2019.02.030
Reference:
YABIO 13264
To appear in:
Analytical Biochemistry
Received Date: 6 January 2019 Revised Date:
27 February 2019
Accepted Date: 27 February 2019
Please cite this article as: Q. Wei, H. Tian, F. Zhang, W. Sai, Y. Ge, X. Gao, W. Yao, Establishment of an HPLC-based method to identify key proteases of proteins in vitro, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2019.02.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Establishment of an HPLC-based method to identify key proteases of proteins in vitro
Qingqing Wei, Hong Tian, Fan Zhang, Wenbo Sai, Yang Ge, Xiangdong Gao* and Wenbing Yao*.
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Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China
[email protected] (Wb.S.),
authors. (H.T.),
E-mail
addresses:
[email protected]
[email protected]
(Y.G.),
(F.Z.),
(Qq.W.),
[email protected]
[email protected]
(Xd.G.),
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[email protected] (Wb.Y.).
[email protected]
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*Corresponding
Abstract
Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases
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that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and
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exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone
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was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability. 1
ACCEPTED MANUSCRIPT Keywords: Protease, Identify, Protein, Degradation, Competition, HPLC 1. Introduction Due to the rapid development of recombinant technology and biotechnology, numerous protein and peptide drugs have been studied and emerged in the market.
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However, the majority of these drugs with outstanding pharmacological activities have failed to show satisfactory effects, owing to low stability, immunogenicity or toxicity [1]. The main possible reason is that some protein and peptide drugs exhibit a short plasma half-life in the range of only several minutes to a few hours [2-4], which
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is not effective in delivering a sufficient amount of drug to target tissue. This short half-life is often associated with enzymatic degradation in the blood, liver, and kidney.
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Considering that the majority of protein drugs are administered via parenteral routes, degradation by proteases in blood is the most direct way to influence the effect of drugs on the human body.
The degradation of proteins results in protein fragments and terminally truncated proteins, thus leading to changes in drug efficacy. This degradation is caused by
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endopeptidases and exopeptidases, which cleave off peptides and proteins on internal and one or several terminal amino acids. The degradation of proteins may cause the biological functions of proteins to be impaired or even eliminated [5-7]. For instance,
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the activities of NADP-dependent isocitrate dehydrogenase [8], varicella-zoster virus gpI (gE) candidate subunit vaccine [9] and acetylcholine receptor [10] were lost when the N-terminus was truncated. Thus, methods to identify proteases that degrade
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proteins in systemic circulation are urgently needed. Early efforts to determine the degradation of proteins were based on
endopeptidases, mainly through incubation of proteins with proteases at 37°C and then detection through SDS-PAGE or Western blot; subsequently, the cleavage sites were identified by Edman degradation or mass spectrometry [11-13]. Although this approach can be used, it suffers from a fundamental problem: applications of these methods are limited to endopeptidases, with enzymatic products sharing different molecular weights or other properties. However, traditional ultraviolet- or fluorescence-based protein quantification methods are unsatisfactory considering the 2
ACCEPTED MANUSCRIPT similar spectral characteristics between the terminally truncated proteins and their complete forms. In addition, liquid chromatography or electrophoresis cannot meet the demand to separate the two forms owing to slight differences in protein sequences. Regardless of the fact that the two forms can be quantitatively distinguished by mass
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spectrometry, the protein standards required are difficult to obtain and cost more, especially those for the truncated forms [14]. Therefore, it is critical to develop an efficient and convenient method to identify potential proteases that degrade proteins, including endopeptidases and exopeptidases, during systemic circulation.
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In this work, we describe a method to identify key proteases that hydrolyze proteins during systemic circulation and that can be implemented for endopeptidases
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and exopeptidases. Here, degradation of the target protein by proteases was detected using HPLC by competition with the corresponding standard substrates. An increase in the residual rate of the standard substrates indicates degradation of the target protein by the corresponding proteases, offering a way to identify key proteases of the target protein. Based on this strategy, several protein drugs were then evaluated by
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this method, and the results were confirmed by microscale thermophoresis (MST) and activity detection. Evidence has proven that our method can be used to identify potential proteases that degrade proteins during systemic circulation and provide
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guidance for researchers to modify cleavage sites to improve the pharmacokinetics
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properties of protein drugs in the future.
2. Materials and methods 2.1 Materials
A high-performance liquid chromatography system was purchased from Agilent
(US). The chromatographic column used was an XBridge C18, with a size of 2.1 mm×5.0 mm and a particle size of 3.5 µm. A Monolith NT.115 was purchased from NanoTemper (Germany). A coagulation analyzer was purchased from Steellex (Beijing, China). Eight proteases (see Table 1) were purchased from R&D Systems (US). The standard substrates (see Table 1) were synthesized by GenScript (Nanjing, China). 3
ACCEPTED MANUSCRIPT Pertuzumab and factor VII were provided by Jiangsu Chia Tai Tianqing Pharmaceutical (Nanjing, China). Growth hormone was purchased from GenScript (Nanjing, China). Fibrinogen was purchased from Sigma (US). GLP-1 and BNP-32 were synthesized by GL Biochem (Shanghai, China). The PT kit was purchased from
commercially available and of analytical grade.
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Xinfan Biotechnology (Shanghai, China). All other reagents and solvents were
2.2 Degradation of proteins detected by competition with standard substrates of proteases by using HPLC
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The degradation of proteins was detected by incubating proteins with each protease and the corresponding standard substrate in a final volume of 200 µL at 37°C
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for 1 h (each mixture contained 100 µM standard substrate and 10 µM protein). After terminating the reaction with 200 µL 1% (v/v) TFA, the samples were centrifuged at 12000 rpm at 4°C for 5 min. Analysis was performed by using an Agilent 1260 HPLC system with an XBridge C18 (Agilent) employing a linear acetonitrile gradient at a flow rate of 1.0 mL/min. Absorbance was monitored at 214 nm. The concentrations of
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each protease and assay buffer are listed in Supplemental Table S1. 2.3 Detection of the interactions between proteins and proteases through MST Proteins were labeled with FITC according to the product manual and incubated
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with unlabeled proteases at a range of concentrations at 37°C for 10 min in the dark. Then, samples were loaded into MST NT.115 standard glass capillaries and analyzed. Measurements were performed with blue filters at 37°C using 20% MST power with a
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laser off/on time of 5 s and 20 s, respectively. Data were calculated using NanoTemper software. All experiments were repeated three times and are shown as the mean±SEM.
2.4 Activity detection of factor VII after incubation with proteases Factor VII was added to human factor VII-deficient plasma at a series of concentrations. The control group was normal human plasma treated with assay buffer (20 mM Hepes, 140 mM NaCl, 2% w/v BSA, pH of 7.4). The clotting time was measured after mixing 50 µL sample with 100 µL PT reagent (all preheated at 37°C for 3 min) on a coagulation analyzer. All experiments were repeated three times and 4
ACCEPTED MANUSCRIPT are shown as the mean±SEM.
3. Results 3.1 Study design
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Proteases and the corresponding standard substrates are shown in Table 1. Proteases in blood exhibit a wide range of enzymatic hydrolysis properties, indicating that the standard substrates are highly hydrolyzed by each protease (according to statistics
on
MEROPS:
https://www.ebi.ac.uk/merops/,
see
Table
1).
The
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concentration of each protease used followed the product manual, while the assay buffer was adjusted into a unified system under the premise of ensuring enzymatic
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activity, as shown in Supplemental Table S1. The sequences and residue rates of the standard substrates are shown in Supplementary Table S2.
The degradation of proteins by proteases was detected through competition with standard substrates. The procedures of our method are shown in Scheme 1. In the process, three groups of samples were analyzed: (I) standard substrate in PBS; (II)
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standard substrate with protease; and (III) a mixture of standard substrate, protease and protein. The detection of the relative residue rate of proteins included five steps in the following order: the three groups of samples mentioned above were mixed (A),
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incubated at 37°C for 1 h (B), terminated (C), and centrifuged and subjected to HPLC (D), and the relative residue rate was analyzed (E). An increase in the residual rate of the standard substrates indicates degradation of the target proteins by the proteases,
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offering a way to identify key proteases of the target protein. Here, the relative residue rate of the target protein was introduced and calculated as the peak area of the standard substrate of group II/group III×100%. Degradation of the target protein can be evaluated by the relative residue rate, as it decreases with increasing protein degradation. Otherwise, the aim of group I was to confirm the degradation of the standard substrate in group II by comparing the peak area between the two groups.
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Table 1 Eight proteases with a wide range of enzymatic hydrolysis properties and the corresponding standard substrates. Proteases
Abbreviation
3.4.11.2 3.4.14.5 3.4.17.2 3.4.15.1 3.4.24.1 3.4.24.86 S01.017 S01.132
Aminopeptidase N Dipeptidyl Peptidase-4 Carboxypeptidase B Peptidyl Dipeptidase A Neprilysin Tumor Necrosis Factor α-Convertase Kallikrein-Related Peptidase 5 Mannan-binding Lectin-associated Serine Peptidase-3
AP-N DPP4 CPB PDA/ACE NEP TNFC/ADAM17 KR-5 MLS-3
Type of hydrolysis
Substrates
exopeptidases exopeptidases exopeptidases exopeptidases Endopeptidases Endopeptidases Endopeptidases Endopeptidases
[Met5]-Enkephalin [15] Inducible Cytokine A22 [16] Cholecystokinin 8 [17] Angiotensin 1 [18] Substance P [19] Synthetic Peptide [20] Kallikrein-10 Precursor [21] Synthetic Peptide [22]
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EC
6
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Scheme 1. Overview of the process used to detect the degradation of proteins. In the process, three groups of samples were analyzed: (I) standard substrate in PBS; (II) standard substrate with protease; and (III) a mixture of standard substrate, protease and protein. The process involved five steps: the three groups of samples mentioned above were mixed (A), incubated at 37°C for 1 h (B), terminated (C), and centrifuged and subjected to HPLC (D), and the relative residue rate of the protein was analyzed (E). The relative residue rate was calculated as the peak area of the standard
the mean±SEM.
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substrate of group II/group III×100%. All experiments were repeated three times and are shown as
3.2 Detection of the relative residue rate of proteins by competition with
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standard substrates of proteases
To test our hypothesis that the degradation of proteins can be detected by
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competition with standard substrates of proteases, we tested the method with several proteins. Previous studies have shown that growth hormone and fibrinogen are degraded by KR-5 [11-12], GLP-1 is degraded by DPP4 on the first two amino acids at its N-terminus [23], and BNP-32 is degraded by NEP [24]. Thus, these four drugs were used to further test our method. By competition with the standard substrate of KR-5, the hydrolysis of growth hormone was observed. The peak area of the standard substrate of KR-5 increased with the constant addition of growth hormone; thus, the relative residue rate decreased gradually (Fig. 1A), indicating that growth hormone was degraded by KR-5. This 7
ACCEPTED MANUSCRIPT pattern was also consistent with the results of fibrinogen (Fig. 1B), GLP-1 (Fig. 1C) and BNP-32 (Fig. 1D), suggesting that fibrinogen was degraded by KR-5, GLP-1 was degraded by DPP4, and BNP-32 was degraded by NEP. All these results are consistent with information reported in earlier works, indicating that competition with standard
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substrates can be used to detect the degradation of proteins.
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Fig. 1. Degradation of growth hormone and fibrinogen by KR-5, GLP-1 by DPP4, and BNP-32 by NEP detected by a range of protein concentrations competing with standard substrates of the corresponding proteases. All experiments were repeated three times and are shown as the mean±SEM.
3.3 Validation of the analytical methodology
Validation was performed to prove that this method meets the demands of detecting the content of samples by HPLC with considerable reliability. In this method, only the peak area of the standard substrates was detected and calculated to obtain the relative residue rate; therefore, the standard substrate of AP-N, [Met5]-enkephalin modified by biotin on the C-terminus (CB-Met5), was used to 8
ACCEPTED MANUSCRIPT prove that detection by HPLC was reliable. The linearity, accuracy, precision, and durability of this method were validated. The calibration curve for CB-Met5 was obtained by plotting the peak area against the concentration of CB-Met5 over a concentration range of 40~160 µM with three
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sequential runs under the same conditions. The results exhibited good linearity, with correlation coefficients (r) higher than 0.999 (Fig. 2). CB-Met5, at concentrations of 60 µM, 100 µM and 140 µM, was prepared and analyzed on the same day and on three consecutive days to determine the intra- and interday precision and accuracy.
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The results are shown in Table 2. The intra- and interday precision were less than 3% relative standard deviation (RSD) (n=3). The accuracy was between 95% and 101%,
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as calculated by the measured concentration/theoretical concentration. The measured concentration was calculated by substituting the peak area into the regression curve. The durability of CB-Met5 was measured by slightly changing the column temperature (28°C, 30°C, and 32°C) and flow rate (0.9 mL/min, 1.0 mL/min, and 1.1 mL/min) (Table 3). The durability of the retention time was less than 4% relative
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standard deviation (RSD) (n=3).
Overall, our method demonstrates fairly good linearity, accuracy, precision and
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durability, suggesting that our detection by HPLC is accurate and reliable.
Fig. 2. Calibration curve of CB-MET5. CB-MET5 (40 µM, 60 µM, 80 µM, 100 µM, 120 µM, 140 µM and 160 µM) was analyzed by HPLC, and a calibration curve was obtained by plotting the peak area against the concentration of the samples. All experiments were repeated three times, and the results are shown as the mean±SEM. 9
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Determining accuracy and precision by using CB-MET5 (n=3). x ± s 57.5 ± 0.2 100.7 ± 0.4 138.8 ± 0.5
Accuracy (%) 95.8 100.7 99.2
RSD: relative standard deviation.
Table 3
Intraday precision
Interday precision
x ± s 57.5 ± 0.5 96.9 ± 1.2 135.0 ± 1.6
x ± s 57.2 ± 0.3 99.2 ± 1.2 137.0 ± 1.1
RSD (%) 1.60 2.22 1.99
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60 100 140
Accuracy
RSD (%) 0.86 2.04 1.41
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Concentration (µM)
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Table 2
Determining the durability of the column temperature and flow rate by using CB-MET5 (n=3).
100
11.19 11.22 11.16
28
11.19
30
11.22
32 140
28 30 32
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Temperature (°C) 28 30 32
Retention time (min)
Durability of flow rate
RSD (%)
Flow rate (mL/min)
Retention time (min)
RSD (%)
0.30
0.9 1.0 1.1
11.61 11.21 10.81
3.58
0.9
11.60
1.0
11.20
11.14
1.1
10.80
11.18 11.21 11.13
0.9 1.0 1.1
11.58 11.19 10.79
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60
Durability of column temperature
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Concentration (µM)
0.36
0.34
3.58
3.55
RSD: relative standard deviation.
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ACCEPTED MANUSCRIPT 3.4 Identification of key proteases of protein drugs Four protein drugs, namely, growth hormone, pertuzumab, factor VII and fibrinogen, were subjected to this method to assess their degradation by proteases (shown in Table 4). Here, the key protease of the target protein was defined as the
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protease with a relative residue rate of the target protein higher than 95%. The degradation of growth hormone and fibrinogen by KR-5 was observed, consistent with previous studies [11-12]. Moreover, other key proteases of the protein drugs were found.
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The degradation of growth hormone by KR-5 was reported previously [11], while the degradation of AP-N has not yet been reported, suggesting that the degradation of
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AP-N and KR-5 may be one of the causes of its decreased half-life in vivo. Pertuzumab was hardly degraded by the eight proteases, suggesting that enzymatic hydrolysis may not be the main mechanism affecting the half-life of pertuzumab. The key proteases of factor VII were CPB, NEP, DPP4 and PDA, suggesting that the degradation of factor VII by the four proteases may lead to a shortened half-life in
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vivo. Fibrinogen, as reported, can be digested by KR-5 into 4 fragments [12], while its degradation by CPB has not been reported, indicating that this may be one of the factors that reduces its half-life in vivo.
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Table 4
Relative residue rates of growth hormone, pertuzumab, factor VII and fibrinogen by proteases.
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Protein (10 µM) was incubated with eight proteases and 100 µM of the corresponding standard substrate. The proteases with a relative residue rate less than 95% were identified as key proteases. All experiments were repeated three times, and the results are shown as the mean±SEM. Proteases NEP MLS-3 TNFC DPP4 CPB KR-5 PDA
Relative residue rates of proteins (%) Growth hormone
Pertuzumab
Factor VII
Fibrinogen
100.8 ± 0.7 98.7 ± 0.9 97.7 ± 1.3 103.2 ± 0.8 98.1 ± 2.3 92.3 ± 1.3 98.1 ± 0.5
103.2 ± 1.3 107.0 ± 0.9 100.5 ± 0.3 96.4 ± 0.5 98.5 ± 0.5 108.7 ± 1.6 99.4 ± 0.2
81.2 ± 1.1 99.0 ± 2.4 97.6 ± 0.1 88.0 ± 0.8 71.8 ± 0.1 97.9 ± 0.3 89.7 ± 0.7
112.0 ± 6.1 116.8 ± 4.3 107.1 ± 1.5 104.0 ± 8.0 81.9 ± 4.1 56.1 ± 1.2 96.0 ± 1.7 11
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88.5 ± 1.1
104.5 ± 1.1
103.6± 0.1
98.0 ± 1.0
3.5 Examination of the results of this method by MST In this method, the degradation of proteins was indirectly detected by incubating the proteins with proteases and the corresponding standard substrates, which differs
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from in vivo conditions. Therefore, the binding phenomenon between the proteins and proteases that occurred in the system was further examined through MST, which yielded an equilibrium dissociation constant (Kd), to confirm the results (Table 4).
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Each molecule has unique thermophoretic properties that are determined by its size, charge, and hydration shell. The binding of ligands typically changes at least one
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of these parameters, resulting in changes in the thermophoretic movement of the molecule. A change in thermophoresis can be used to derive the Kd within minutes by sequentially scanning capillaries with varying ligand concentrations [25]. A lower Kd indicates a stronger interaction.
The interactions of growth hormone, pertuzumab, factor VII and fibrinogen with
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proteases are shown in Table 5. The MST results and relative residue rates shown in Table 4 were consistent, since the Kd of the key proteases was smaller than that of the others. However, the Kd of the other proteases was also detected, indicating that MST was more sensitive. Compared with the other proteases, growth hormone showed
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higher affinity to KR-5 and AP-N. Additionally, pertuzumab was hardly bound to the proteases; factor VII strongly combined with CPB, NEP, DPP4 and PDA; and
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fibrinogen was bound to CPB, KR-5 and DPP4. These results agree with the information presented in Table 4 and thus upheld the results of our method.
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Table 5 Equilibrium dissociation constants of growth hormone, pertuzumab, factor VII and fibrinogen with proteases detected by MST. Proteins were labeled with FITC, incubated with proteases and detected on an NT.115 instrument. Data were calculated using NanoTemper software. Growth hormone showed high affinity to KR-5
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and AP-N. Additionally, pertuzumab was hardly bound to the proteases; factor VII strongly combined with CPB, NEP, DPP4 and PDA; and fibrinogen was firmly bound to CPB and KR-5. All experiments were repeated three times, and the results are shown as the mean±SEM.
(2.3 ± 0.1) ×103 (3.2 ± 0.3) ×103 (96 ± 0.0) ×103 (3.5 ± 0.8) ×102 (3.0 ± 0.6) ×102 55 ± 19 (7.0 ± 1.0) ×102 26 ± 5.0
(8.2 ± 2.0) ×102 (1.6 ± 0.0) ×105 (2.0 ± 0.0) ×103 (2.7 ± 0.1)×102 (2.2 ± 0.3) ×102 (2.9 ± 0.1) ×104 (3.8 ± 0.4) ×102 (8.9 ± 0.7) ×102
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Pertuzumab
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Growth hormone
Factor VII
Fibrinogen
3.6 ± 0.7 (2.2 ± 0.2) ×103 (1.4 ± 0.1) ×102 4.5 ± 1.0 6.5 ± 4.7 (3.5 ± 0.6) ×102 31 ± 4.8 (5.9 ± 0.4) ×102
(6.2 ± 0.8) ×102 (1.9 ± 0.2) ×103 (1.3 ± 0.2) ×104 49 ± 1.5 65 ± 19 (1.1 ± 0.2) ×102 (1.4 ± 0.2) ×103 (3.0 ± 0.3) ×103
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NEP MLS-3 TNFC DPP4 CPB KR-5 PDA AP-N
Equilibrium dissociation constant of proteins (Kd, nM)
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Proteases
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ACCEPTED MANUSCRIPT 3.6 Substantiation of results by evaluating the biological activity of factor VII after incubation with its key proteases To further substantiate our method, the activity of the degradation of factor VII by its key proteases was detected, as shown by prothrombin time (PT) in human factor
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VII-deficient plasma with recalcification as the trigger. Under conditions mimicking hemophilia A, factor VII incubated with its key proteases (CPB, DPP4, NEP and PDA) at 37°C for 4 h showed significantly decreased activity, with a prolonged PT in the range of 17.9 s to 28.0 s, while similar activity with normal human plasma was
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observed in the groups with four other proteases (Table 6). The concentration of factor VII (0.5 nM) was used to meet the same level as that of normal plasma (Supplemental
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Table S3). The decreased activity of factor VII incubated with key proteases supported the results as well as the importance of our method. Table 6
Prothrombin time of factor VII in factor VII-deficient plasma. Factor VII incubated with its key proteases (CPB, DPP4, NEP and PDA) showed decreased activity, with a PT in the range of 17.9 s
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to 28.0 s, while factor VII incubated with the other four proteases showed comparable activity with normal human plasma. All experiments were repeated three times, and the results are shown as the mean±SEM. Samples
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normal human plasma factor VII deficient plasma 1 (normal factor VII) 2 (CPB) 3 (DPP4) 4 (NEP) 5 (PDA) 6 (KR-5) 7 (TNFC) 8 (MLS-3) 9 (AP-N)
PT (s) 12.7 ± 0.5 38.1 ± 0.1 12.5 ± 0.2 19.1 ± 0.7b 21.2 ± 1.5b 26.4 ± 0.9b 19.9 ± 1.1a 13.9 ± 0.8 13.3 ± 0.6 13.3 ± 0.2 13.4 ± 0.4
1: Factor VII-deficient plasma with 0.5 nM factor VII; 2~9: factor VII-deficient plasma with 0.5 nM factor VII incubated with CPB, DPP4, NEP, PDA, KR-5, TNFC, MLS-3 and AP-N, respectively. a P<0.01 and b P<0.001, compared with group 1, using Student’s t-test. 14
ACCEPTED MANUSCRIPT 4. Discussion In this work, we developed a method to identify key proteases of proteins by competing them with standard substrates of the corresponding proteases and showed that the method can be applied to endopeptidases and exopeptidases. In addition, the
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effectiveness of our method was further proven by MST and activity detection. Moreover, the key proteases of factor VII were newly identified, and we found that degradation by the key proteases would influence the activity of factor VII.
In our method, the target protein was incubated with proteases and the
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corresponding standard substrates. Degradation of the corresponding standard substrates decreased when the target protein was being degraded, thus proving that
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our idea of competition can be used to detect the degradation of proteins. Here, the relative residue rate was introduced to describe the degree of degradation, and key proteases of proteins were obtained, which were defined as those with a relative residue rate higher than 95%. The benefit of this method is that it can be used to identify
endopeptidases
and
exopeptidases,
while
SDS-PAGE
and
liquid
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chromatography can be applied only to endopeptidases. In addition, samples were analyzed by HPLC, which is more convenient and less expensive than mass spectrometry.
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First, two protein drugs (growth hormone and fibrinogen) and two peptide drugs (GLP-1 and BNP-32) were used to prove the feasibility of our method. Encouragingly, the results were consistent with information reported earlier, supporting the feasibility
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of our method. With validation of our method, we firmly believe that our method exhibits good linearity, accuracy, precision and durability. Subsequently, four protein drugs, namely, growth hormone, pertuzumab, factor
VII and fibrinogen, were further tested. Our results suggested that growth hormone was degraded by KR-5 and AP-N, pertuzumab was hardly degraded by the proteases, factor VII was degraded by CPB, NEP, DPP4 and PDA, and fibrinogen was degraded by KR-5 and CPB. To the best of our knowledge, these results are reasonable. Growth hormone plays an important role in human body growth, with a T1/2 of only 2.3 h (Mr≈22 kDa); thus, our results indicate that the degradation of AP-N and KR-5 may 15
ACCEPTED MANUSCRIPT be one of the reasons for its short half-life in vivo. Pertuzumab is an antibody used in breast cancer treatment (Mr≈148 kDa), with a quite long T1/2 of 18 days [26-27], and the glycosylation-enhanced complexity of its structure would reduce its degradation by proteases [28-29]. Therefore, the results for pertuzumab were expected. Factor VII
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is a type of vitamin K-dependent single-chain glycoprotein (Mr≈50 kDa) that is secreted into blood, where it circulates in its zymogen form [30-31]. The plasma half-life of factor VII is 2.5 h, which is the shortest among all coagulation factors [32-33] and can be extended through PEG modification or protein fusion [34-36]. Our
reason for its short plasma half-life in vivo.
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results suggest that the degradation of factor VII by the four proteases may be the
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Furthermore, we examined the binding phenomenon that occurred in the system through MST. Thermophoresis, the motion of molecules in temperature fields, is sensitive to changes in the size, charge, and solvation shell of a molecule and is thus suitable for bioanalytics [37-39]. Detection by MST relies on infrared laser sources for creating localized temperature fields to perform biomolecular interaction studies.
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Compared with our method, MST was more sensitive, but the results of both were consistent, thus confirming the credibility and reliability of our method. In addition, we investigated the activity of factor VII after incubation with its key
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proteases. For the groups incubated with CPB, DPP4, NEP and PDA, the activity decreased to different extents, while those incubated with the other four proteases showed activity similar to that of the control group. Thus, the activity results
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supported the hydrolysis results and illustrated the significance of our method by demonstrating that the degradation of factor VII by proteases influences its effect in vitro.
CPB is characterized as a carboxypeptidase B-like entity that is able to cleave
basic C-terminal amino acids, arginine and lysine [40]; therefore, the site of factor VII cleavage by CPB may lie between L401 and R402. The primary specificity of PDA is the cleavage of C-terminal dipeptides from substrates with a free C-terminus and the absence of a penultimate Pro [41]; thus, the cleavage site of factor VII by PDA may lie between P404 and F405. DPP4 is specific for a proline at the penultimate position 16
ACCEPTED MANUSCRIPT and hydrolyzes the carboxyl side of this residue (Xaa-Pro↓Xbb-) [42], indicating that the cleavage site of factor VII by DPP4 may lie between N2 and A3. It is well documented in the literature that the function specificity of NEP requires a bulky hydrophobic residue in the P1’ position as an endopeptidase [43]; thus, there are
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numerous potential cleavage sites in factor VII for NEP. Further investigations are needed to improve upon this study. Only four protein drugs were tested with this method, which is far from adequate to meet our goal of providing a method that has a wide range of applications, but we fully demonstrated
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that this method can be used to identify key proteases of proteins. In future studies, it will be important to test more protein drugs with our method, including interferon
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gamma-1b, oprelvekin, teceleukin, sargramostim and metreleptin, considering their short plasma half-life of less than two hours (statistics on drug instruction from the FDA, https://www.fda.gov/Drugs/default.htm, and clinical statistics from Integrity, https://integrity.thomson-pharma.com/integrity/xmlxsl/pk_home.util_home).
In
addition, more proteases in the human body will be examined by this method,
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including not only proteases in the blood but also those in the liver, kidney and other organisms. Notably, since this study provides direct evidence that the degradation of factor VII by its key proteases would decrease its activity, its pharmacological effect
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and plasma half-life may be improved by the modification of its cleavage sites in further studies. 5. Conclusion
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In summary, we developed a convenient method to identify key proteases of
proteins by competition with corresponding standard substrates with high accuracy, precision and durability that can be applied to endopeptidases and exopeptidases. This work may provide guidance for the future modification of proteins on cleavage sites to prolong their plasma half-life. 6. Conflicts of interest The authors declared no conflict of interest. 7. Acknowledgments This work was supported by the National Natural Science Foundation of China 17
ACCEPTED MANUSCRIPT [grant number 81430082, 81872850], “Double-First Class” University Project [CPU2018GF08], the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China [No. 111-2-07]. The authors thank Jiangsu Chia Tai Tianqing Pharmaceutical (Nanjing, China) for providing
8. Appendix A. Supplementary data
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pertuzumab and factor VII.
Supporting materials can be seen in “Appendix A Supplemental material” file. 9. References
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Highlights: Establishment of a method to identify key proteases of proteins based on HPLC Rapid identification of endopeptidases and exopeptidases that degrade proteins Factor VII is newly found to be degraded by CPB, NEP, DPP4 and PDA
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Fibrinogen is newly found to be degraded by CPB
S0003-2697(19)30022-3
DOI:
https://doi.org/10.1016/j.ab.2019.02.030
Reference:
YABIO 13264
To appear in:
Analytical Biochemistry
Received Date: 6 January 2019 Revised Date:
27 February 2019
Accepted Date: 27 February 2019
Please cite this article as: Q. Wei, H. Tian, F. Zhang, W. Sai, Y. Ge, X. Gao, W. Yao, Establishment of an HPLC-based method to identify key proteases of proteins in vitro, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2019.02.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Establishment of an HPLC-based method to identify key proteases of proteins in vitro
Qingqing Wei, Hong Tian, Fan Zhang, Wenbo Sai, Yang Ge, Xiangdong Gao* and Wenbing Yao*.
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Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China
[email protected] (Wb.S.),
authors. (H.T.),
addresses:
[email protected]
[email protected]
(Y.G.),
(F.Z.),
(Qq.W.),
[email protected]
[email protected]
(Xd.G.),
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[email protected] (Wb.Y.).
[email protected]
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*Corresponding
Abstract
Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases
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that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and
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exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone
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was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability. 1
ACCEPTED MANUSCRIPT Keywords: Protease, Identify, Protein, Degradation, Competition, HPLC 1. Introduction Due to the rapid development of recombinant technology and biotechnology, numerous protein and peptide drugs have been studied and emerged in the market.
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However, the majority of these drugs with outstanding pharmacological activities have failed to show satisfactory effects, owing to low stability, immunogenicity or toxicity [1]. The main possible reason is that some protein and peptide drugs exhibit a short plasma half-life in the range of only several minutes to a few hours [2-4], which
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is not effective in delivering a sufficient amount of drug to target tissue. This short half-life is often associated with enzymatic degradation in the blood, liver, and kidney.
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Considering that the majority of protein drugs are administered via parenteral routes, degradation by proteases in blood is the most direct way to influence the effect of drugs on the human body.
The degradation of proteins results in protein fragments and terminally truncated proteins, thus leading to changes in drug efficacy. This degradation is caused by
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endopeptidases and exopeptidases, which cleave off peptides and proteins on internal and one or several terminal amino acids. The degradation of proteins may cause the biological functions of proteins to be impaired or even eliminated [5-7]. For instance,
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the activities of NADP-dependent isocitrate dehydrogenase [8], varicella-zoster virus gpI (gE) candidate subunit vaccine [9] and acetylcholine receptor [10] were lost when the N-terminus was truncated. Thus, methods to identify proteases that degrade
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proteins in systemic circulation are urgently needed. Early efforts to determine the degradation of proteins were based on
endopeptidases, mainly through incubation of proteins with proteases at 37°C and then detection through SDS-PAGE or Western blot; subsequently, the cleavage sites were identified by Edman degradation or mass spectrometry [11-13]. Although this approach can be used, it suffers from a fundamental problem: applications of these methods are limited to endopeptidases, with enzymatic products sharing different molecular weights or other properties. However, traditional ultraviolet- or fluorescence-based protein quantification methods are unsatisfactory considering the 2
ACCEPTED MANUSCRIPT similar spectral characteristics between the terminally truncated proteins and their complete forms. In addition, liquid chromatography or electrophoresis cannot meet the demand to separate the two forms owing to slight differences in protein sequences. Regardless of the fact that the two forms can be quantitatively distinguished by mass
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spectrometry, the protein standards required are difficult to obtain and cost more, especially those for the truncated forms [14]. Therefore, it is critical to develop an efficient and convenient method to identify potential proteases that degrade proteins, including endopeptidases and exopeptidases, during systemic circulation.
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In this work, we describe a method to identify key proteases that hydrolyze proteins during systemic circulation and that can be implemented for endopeptidases
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and exopeptidases. Here, degradation of the target protein by proteases was detected using HPLC by competition with the corresponding standard substrates. An increase in the residual rate of the standard substrates indicates degradation of the target protein by the corresponding proteases, offering a way to identify key proteases of the target protein. Based on this strategy, several protein drugs were then evaluated by
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this method, and the results were confirmed by microscale thermophoresis (MST) and activity detection. Evidence has proven that our method can be used to identify potential proteases that degrade proteins during systemic circulation and provide
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guidance for researchers to modify cleavage sites to improve the pharmacokinetics
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properties of protein drugs in the future.
2. Materials and methods 2.1 Materials
A high-performance liquid chromatography system was purchased from Agilent
(US). The chromatographic column used was an XBridge C18, with a size of 2.1 mm×5.0 mm and a particle size of 3.5 µm. A Monolith NT.115 was purchased from NanoTemper (Germany). A coagulation analyzer was purchased from Steellex (Beijing, China). Eight proteases (see Table 1) were purchased from R&D Systems (US). The standard substrates (see Table 1) were synthesized by GenScript (Nanjing, China). 3
ACCEPTED MANUSCRIPT Pertuzumab and factor VII were provided by Jiangsu Chia Tai Tianqing Pharmaceutical (Nanjing, China). Growth hormone was purchased from GenScript (Nanjing, China). Fibrinogen was purchased from Sigma (US). GLP-1 and BNP-32 were synthesized by GL Biochem (Shanghai, China). The PT kit was purchased from
commercially available and of analytical grade.
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Xinfan Biotechnology (Shanghai, China). All other reagents and solvents were
2.2 Degradation of proteins detected by competition with standard substrates of proteases by using HPLC
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The degradation of proteins was detected by incubating proteins with each protease and the corresponding standard substrate in a final volume of 200 µL at 37°C
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for 1 h (each mixture contained 100 µM standard substrate and 10 µM protein). After terminating the reaction with 200 µL 1% (v/v) TFA, the samples were centrifuged at 12000 rpm at 4°C for 5 min. Analysis was performed by using an Agilent 1260 HPLC system with an XBridge C18 (Agilent) employing a linear acetonitrile gradient at a flow rate of 1.0 mL/min. Absorbance was monitored at 214 nm. The concentrations of
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each protease and assay buffer are listed in Supplemental Table S1. 2.3 Detection of the interactions between proteins and proteases through MST Proteins were labeled with FITC according to the product manual and incubated
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with unlabeled proteases at a range of concentrations at 37°C for 10 min in the dark. Then, samples were loaded into MST NT.115 standard glass capillaries and analyzed. Measurements were performed with blue filters at 37°C using 20% MST power with a
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laser off/on time of 5 s and 20 s, respectively. Data were calculated using NanoTemper software. All experiments were repeated three times and are shown as the mean±SEM.
2.4 Activity detection of factor VII after incubation with proteases Factor VII was added to human factor VII-deficient plasma at a series of concentrations. The control group was normal human plasma treated with assay buffer (20 mM Hepes, 140 mM NaCl, 2% w/v BSA, pH of 7.4). The clotting time was measured after mixing 50 µL sample with 100 µL PT reagent (all preheated at 37°C for 3 min) on a coagulation analyzer. All experiments were repeated three times and 4
ACCEPTED MANUSCRIPT are shown as the mean±SEM.
3. Results 3.1 Study design
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Proteases and the corresponding standard substrates are shown in Table 1. Proteases in blood exhibit a wide range of enzymatic hydrolysis properties, indicating that the standard substrates are highly hydrolyzed by each protease (according to statistics
on
MEROPS:
https://www.ebi.ac.uk/merops/,
see
Table
1).
The
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concentration of each protease used followed the product manual, while the assay buffer was adjusted into a unified system under the premise of ensuring enzymatic
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activity, as shown in Supplemental Table S1. The sequences and residue rates of the standard substrates are shown in Supplementary Table S2.
The degradation of proteins by proteases was detected through competition with standard substrates. The procedures of our method are shown in Scheme 1. In the process, three groups of samples were analyzed: (I) standard substrate in PBS; (II)
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standard substrate with protease; and (III) a mixture of standard substrate, protease and protein. The detection of the relative residue rate of proteins included five steps in the following order: the three groups of samples mentioned above were mixed (A),
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incubated at 37°C for 1 h (B), terminated (C), and centrifuged and subjected to HPLC (D), and the relative residue rate was analyzed (E). An increase in the residual rate of the standard substrates indicates degradation of the target proteins by the proteases,
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offering a way to identify key proteases of the target protein. Here, the relative residue rate of the target protein was introduced and calculated as the peak area of the standard substrate of group II/group III×100%. Degradation of the target protein can be evaluated by the relative residue rate, as it decreases with increasing protein degradation. Otherwise, the aim of group I was to confirm the degradation of the standard substrate in group II by comparing the peak area between the two groups.
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Table 1 Eight proteases with a wide range of enzymatic hydrolysis properties and the corresponding standard substrates. Proteases
Abbreviation
3.4.11.2 3.4.14.5 3.4.17.2 3.4.15.1 3.4.24.1 3.4.24.86 S01.017 S01.132
Aminopeptidase N Dipeptidyl Peptidase-4 Carboxypeptidase B Peptidyl Dipeptidase A Neprilysin Tumor Necrosis Factor α-Convertase Kallikrein-Related Peptidase 5 Mannan-binding Lectin-associated Serine Peptidase-3
AP-N DPP4 CPB PDA/ACE NEP TNFC/ADAM17 KR-5 MLS-3
Type of hydrolysis
Substrates
exopeptidases exopeptidases exopeptidases exopeptidases Endopeptidases Endopeptidases Endopeptidases Endopeptidases
[Met5]-Enkephalin [15] Inducible Cytokine A22 [16] Cholecystokinin 8 [17] Angiotensin 1 [18] Substance P [19] Synthetic Peptide [20] Kallikrein-10 Precursor [21] Synthetic Peptide [22]
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Scheme 1. Overview of the process used to detect the degradation of proteins. In the process, three groups of samples were analyzed: (I) standard substrate in PBS; (II) standard substrate with protease; and (III) a mixture of standard substrate, protease and protein. The process involved five steps: the three groups of samples mentioned above were mixed (A), incubated at 37°C for 1 h (B), terminated (C), and centrifuged and subjected to HPLC (D), and the relative residue rate of the protein was analyzed (E). The relative residue rate was calculated as the peak area of the standard
the mean±SEM.
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substrate of group II/group III×100%. All experiments were repeated three times and are shown as
3.2 Detection of the relative residue rate of proteins by competition with
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standard substrates of proteases
To test our hypothesis that the degradation of proteins can be detected by
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competition with standard substrates of proteases, we tested the method with several proteins. Previous studies have shown that growth hormone and fibrinogen are degraded by KR-5 [11-12], GLP-1 is degraded by DPP4 on the first two amino acids at its N-terminus [23], and BNP-32 is degraded by NEP [24]. Thus, these four drugs were used to further test our method. By competition with the standard substrate of KR-5, the hydrolysis of growth hormone was observed. The peak area of the standard substrate of KR-5 increased with the constant addition of growth hormone; thus, the relative residue rate decreased gradually (Fig. 1A), indicating that growth hormone was degraded by KR-5. This 7
ACCEPTED MANUSCRIPT pattern was also consistent with the results of fibrinogen (Fig. 1B), GLP-1 (Fig. 1C) and BNP-32 (Fig. 1D), suggesting that fibrinogen was degraded by KR-5, GLP-1 was degraded by DPP4, and BNP-32 was degraded by NEP. All these results are consistent with information reported in earlier works, indicating that competition with standard
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substrates can be used to detect the degradation of proteins.
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Fig. 1. Degradation of growth hormone and fibrinogen by KR-5, GLP-1 by DPP4, and BNP-32 by NEP detected by a range of protein concentrations competing with standard substrates of the corresponding proteases. All experiments were repeated three times and are shown as the mean±SEM.
3.3 Validation of the analytical methodology
Validation was performed to prove that this method meets the demands of detecting the content of samples by HPLC with considerable reliability. In this method, only the peak area of the standard substrates was detected and calculated to obtain the relative residue rate; therefore, the standard substrate of AP-N, [Met5]-enkephalin modified by biotin on the C-terminus (CB-Met5), was used to 8
ACCEPTED MANUSCRIPT prove that detection by HPLC was reliable. The linearity, accuracy, precision, and durability of this method were validated. The calibration curve for CB-Met5 was obtained by plotting the peak area against the concentration of CB-Met5 over a concentration range of 40~160 µM with three
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sequential runs under the same conditions. The results exhibited good linearity, with correlation coefficients (r) higher than 0.999 (Fig. 2). CB-Met5, at concentrations of 60 µM, 100 µM and 140 µM, was prepared and analyzed on the same day and on three consecutive days to determine the intra- and interday precision and accuracy.
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The results are shown in Table 2. The intra- and interday precision were less than 3% relative standard deviation (RSD) (n=3). The accuracy was between 95% and 101%,
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as calculated by the measured concentration/theoretical concentration. The measured concentration was calculated by substituting the peak area into the regression curve. The durability of CB-Met5 was measured by slightly changing the column temperature (28°C, 30°C, and 32°C) and flow rate (0.9 mL/min, 1.0 mL/min, and 1.1 mL/min) (Table 3). The durability of the retention time was less than 4% relative
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standard deviation (RSD) (n=3).
Overall, our method demonstrates fairly good linearity, accuracy, precision and
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durability, suggesting that our detection by HPLC is accurate and reliable.
Fig. 2. Calibration curve of CB-MET5. CB-MET5 (40 µM, 60 µM, 80 µM, 100 µM, 120 µM, 140 µM and 160 µM) was analyzed by HPLC, and a calibration curve was obtained by plotting the peak area against the concentration of the samples. All experiments were repeated three times, and the results are shown as the mean±SEM. 9
ACCEPTED MANUSCRIPT
Determining accuracy and precision by using CB-MET5 (n=3). x ± s 57.5 ± 0.2 100.7 ± 0.4 138.8 ± 0.5
Accuracy (%) 95.8 100.7 99.2
RSD: relative standard deviation.
Table 3
Intraday precision
Interday precision
x ± s 57.5 ± 0.5 96.9 ± 1.2 135.0 ± 1.6
x ± s 57.2 ± 0.3 99.2 ± 1.2 137.0 ± 1.1
RSD (%) 1.60 2.22 1.99
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60 100 140
Accuracy
RSD (%) 0.86 2.04 1.41
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Concentration (µM)
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Table 2
Determining the durability of the column temperature and flow rate by using CB-MET5 (n=3).
100
11.19 11.22 11.16
28
11.19
30
11.22
32 140
28 30 32
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Temperature (°C) 28 30 32
Retention time (min)
Durability of flow rate
RSD (%)
Flow rate (mL/min)
Retention time (min)
RSD (%)
0.30
0.9 1.0 1.1
11.61 11.21 10.81
3.58
0.9
11.60
1.0
11.20
11.14
1.1
10.80
11.18 11.21 11.13
0.9 1.0 1.1
11.58 11.19 10.79
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60
Durability of column temperature
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Concentration (µM)
0.36
0.34
3.58
3.55
RSD: relative standard deviation.
10
ACCEPTED MANUSCRIPT 3.4 Identification of key proteases of protein drugs Four protein drugs, namely, growth hormone, pertuzumab, factor VII and fibrinogen, were subjected to this method to assess their degradation by proteases (shown in Table 4). Here, the key protease of the target protein was defined as the
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protease with a relative residue rate of the target protein higher than 95%. The degradation of growth hormone and fibrinogen by KR-5 was observed, consistent with previous studies [11-12]. Moreover, other key proteases of the protein drugs were found.
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The degradation of growth hormone by KR-5 was reported previously [11], while the degradation of AP-N has not yet been reported, suggesting that the degradation of
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AP-N and KR-5 may be one of the causes of its decreased half-life in vivo. Pertuzumab was hardly degraded by the eight proteases, suggesting that enzymatic hydrolysis may not be the main mechanism affecting the half-life of pertuzumab. The key proteases of factor VII were CPB, NEP, DPP4 and PDA, suggesting that the degradation of factor VII by the four proteases may lead to a shortened half-life in
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vivo. Fibrinogen, as reported, can be digested by KR-5 into 4 fragments [12], while its degradation by CPB has not been reported, indicating that this may be one of the factors that reduces its half-life in vivo.
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Table 4
Relative residue rates of growth hormone, pertuzumab, factor VII and fibrinogen by proteases.
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Protein (10 µM) was incubated with eight proteases and 100 µM of the corresponding standard substrate. The proteases with a relative residue rate less than 95% were identified as key proteases. All experiments were repeated three times, and the results are shown as the mean±SEM. Proteases NEP MLS-3 TNFC DPP4 CPB KR-5 PDA
Relative residue rates of proteins (%) Growth hormone
Pertuzumab
Factor VII
Fibrinogen
100.8 ± 0.7 98.7 ± 0.9 97.7 ± 1.3 103.2 ± 0.8 98.1 ± 2.3 92.3 ± 1.3 98.1 ± 0.5
103.2 ± 1.3 107.0 ± 0.9 100.5 ± 0.3 96.4 ± 0.5 98.5 ± 0.5 108.7 ± 1.6 99.4 ± 0.2
81.2 ± 1.1 99.0 ± 2.4 97.6 ± 0.1 88.0 ± 0.8 71.8 ± 0.1 97.9 ± 0.3 89.7 ± 0.7
112.0 ± 6.1 116.8 ± 4.3 107.1 ± 1.5 104.0 ± 8.0 81.9 ± 4.1 56.1 ± 1.2 96.0 ± 1.7 11
ACCEPTED MANUSCRIPT AP-N
88.5 ± 1.1
104.5 ± 1.1
103.6± 0.1
98.0 ± 1.0
3.5 Examination of the results of this method by MST In this method, the degradation of proteins was indirectly detected by incubating the proteins with proteases and the corresponding standard substrates, which differs
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from in vivo conditions. Therefore, the binding phenomenon between the proteins and proteases that occurred in the system was further examined through MST, which yielded an equilibrium dissociation constant (Kd), to confirm the results (Table 4).
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Each molecule has unique thermophoretic properties that are determined by its size, charge, and hydration shell. The binding of ligands typically changes at least one
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of these parameters, resulting in changes in the thermophoretic movement of the molecule. A change in thermophoresis can be used to derive the Kd within minutes by sequentially scanning capillaries with varying ligand concentrations [25]. A lower Kd indicates a stronger interaction.
The interactions of growth hormone, pertuzumab, factor VII and fibrinogen with
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proteases are shown in Table 5. The MST results and relative residue rates shown in Table 4 were consistent, since the Kd of the key proteases was smaller than that of the others. However, the Kd of the other proteases was also detected, indicating that MST was more sensitive. Compared with the other proteases, growth hormone showed
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higher affinity to KR-5 and AP-N. Additionally, pertuzumab was hardly bound to the proteases; factor VII strongly combined with CPB, NEP, DPP4 and PDA; and
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fibrinogen was bound to CPB, KR-5 and DPP4. These results agree with the information presented in Table 4 and thus upheld the results of our method.
12
ACCEPTED MANUSCRIPT
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Table 5 Equilibrium dissociation constants of growth hormone, pertuzumab, factor VII and fibrinogen with proteases detected by MST. Proteins were labeled with FITC, incubated with proteases and detected on an NT.115 instrument. Data were calculated using NanoTemper software. Growth hormone showed high affinity to KR-5
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and AP-N. Additionally, pertuzumab was hardly bound to the proteases; factor VII strongly combined with CPB, NEP, DPP4 and PDA; and fibrinogen was firmly bound to CPB and KR-5. All experiments were repeated three times, and the results are shown as the mean±SEM.
(2.3 ± 0.1) ×103 (3.2 ± 0.3) ×103 (96 ± 0.0) ×103 (3.5 ± 0.8) ×102 (3.0 ± 0.6) ×102 55 ± 19 (7.0 ± 1.0) ×102 26 ± 5.0
(8.2 ± 2.0) ×102 (1.6 ± 0.0) ×105 (2.0 ± 0.0) ×103 (2.7 ± 0.1)×102 (2.2 ± 0.3) ×102 (2.9 ± 0.1) ×104 (3.8 ± 0.4) ×102 (8.9 ± 0.7) ×102
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Pertuzumab
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Growth hormone
Factor VII
Fibrinogen
3.6 ± 0.7 (2.2 ± 0.2) ×103 (1.4 ± 0.1) ×102 4.5 ± 1.0 6.5 ± 4.7 (3.5 ± 0.6) ×102 31 ± 4.8 (5.9 ± 0.4) ×102
(6.2 ± 0.8) ×102 (1.9 ± 0.2) ×103 (1.3 ± 0.2) ×104 49 ± 1.5 65 ± 19 (1.1 ± 0.2) ×102 (1.4 ± 0.2) ×103 (3.0 ± 0.3) ×103
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NEP MLS-3 TNFC DPP4 CPB KR-5 PDA AP-N
Equilibrium dissociation constant of proteins (Kd, nM)
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Proteases
13
ACCEPTED MANUSCRIPT 3.6 Substantiation of results by evaluating the biological activity of factor VII after incubation with its key proteases To further substantiate our method, the activity of the degradation of factor VII by its key proteases was detected, as shown by prothrombin time (PT) in human factor
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VII-deficient plasma with recalcification as the trigger. Under conditions mimicking hemophilia A, factor VII incubated with its key proteases (CPB, DPP4, NEP and PDA) at 37°C for 4 h showed significantly decreased activity, with a prolonged PT in the range of 17.9 s to 28.0 s, while similar activity with normal human plasma was
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observed in the groups with four other proteases (Table 6). The concentration of factor VII (0.5 nM) was used to meet the same level as that of normal plasma (Supplemental
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Table S3). The decreased activity of factor VII incubated with key proteases supported the results as well as the importance of our method. Table 6
Prothrombin time of factor VII in factor VII-deficient plasma. Factor VII incubated with its key proteases (CPB, DPP4, NEP and PDA) showed decreased activity, with a PT in the range of 17.9 s
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to 28.0 s, while factor VII incubated with the other four proteases showed comparable activity with normal human plasma. All experiments were repeated three times, and the results are shown as the mean±SEM. Samples
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normal human plasma factor VII deficient plasma 1 (normal factor VII) 2 (CPB) 3 (DPP4) 4 (NEP) 5 (PDA) 6 (KR-5) 7 (TNFC) 8 (MLS-3) 9 (AP-N)
PT (s) 12.7 ± 0.5 38.1 ± 0.1 12.5 ± 0.2 19.1 ± 0.7b 21.2 ± 1.5b 26.4 ± 0.9b 19.9 ± 1.1a 13.9 ± 0.8 13.3 ± 0.6 13.3 ± 0.2 13.4 ± 0.4
1: Factor VII-deficient plasma with 0.5 nM factor VII; 2~9: factor VII-deficient plasma with 0.5 nM factor VII incubated with CPB, DPP4, NEP, PDA, KR-5, TNFC, MLS-3 and AP-N, respectively. a P<0.01 and b P<0.001, compared with group 1, using Student’s t-test. 14
ACCEPTED MANUSCRIPT 4. Discussion In this work, we developed a method to identify key proteases of proteins by competing them with standard substrates of the corresponding proteases and showed that the method can be applied to endopeptidases and exopeptidases. In addition, the
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effectiveness of our method was further proven by MST and activity detection. Moreover, the key proteases of factor VII were newly identified, and we found that degradation by the key proteases would influence the activity of factor VII.
In our method, the target protein was incubated with proteases and the
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corresponding standard substrates. Degradation of the corresponding standard substrates decreased when the target protein was being degraded, thus proving that
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our idea of competition can be used to detect the degradation of proteins. Here, the relative residue rate was introduced to describe the degree of degradation, and key proteases of proteins were obtained, which were defined as those with a relative residue rate higher than 95%. The benefit of this method is that it can be used to identify
endopeptidases
and
exopeptidases,
while
SDS-PAGE
and
liquid
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chromatography can be applied only to endopeptidases. In addition, samples were analyzed by HPLC, which is more convenient and less expensive than mass spectrometry.
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First, two protein drugs (growth hormone and fibrinogen) and two peptide drugs (GLP-1 and BNP-32) were used to prove the feasibility of our method. Encouragingly, the results were consistent with information reported earlier, supporting the feasibility
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of our method. With validation of our method, we firmly believe that our method exhibits good linearity, accuracy, precision and durability. Subsequently, four protein drugs, namely, growth hormone, pertuzumab, factor
VII and fibrinogen, were further tested. Our results suggested that growth hormone was degraded by KR-5 and AP-N, pertuzumab was hardly degraded by the proteases, factor VII was degraded by CPB, NEP, DPP4 and PDA, and fibrinogen was degraded by KR-5 and CPB. To the best of our knowledge, these results are reasonable. Growth hormone plays an important role in human body growth, with a T1/2 of only 2.3 h (Mr≈22 kDa); thus, our results indicate that the degradation of AP-N and KR-5 may 15
ACCEPTED MANUSCRIPT be one of the reasons for its short half-life in vivo. Pertuzumab is an antibody used in breast cancer treatment (Mr≈148 kDa), with a quite long T1/2 of 18 days [26-27], and the glycosylation-enhanced complexity of its structure would reduce its degradation by proteases [28-29]. Therefore, the results for pertuzumab were expected. Factor VII
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is a type of vitamin K-dependent single-chain glycoprotein (Mr≈50 kDa) that is secreted into blood, where it circulates in its zymogen form [30-31]. The plasma half-life of factor VII is 2.5 h, which is the shortest among all coagulation factors [32-33] and can be extended through PEG modification or protein fusion [34-36]. Our
reason for its short plasma half-life in vivo.
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results suggest that the degradation of factor VII by the four proteases may be the
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Furthermore, we examined the binding phenomenon that occurred in the system through MST. Thermophoresis, the motion of molecules in temperature fields, is sensitive to changes in the size, charge, and solvation shell of a molecule and is thus suitable for bioanalytics [37-39]. Detection by MST relies on infrared laser sources for creating localized temperature fields to perform biomolecular interaction studies.
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Compared with our method, MST was more sensitive, but the results of both were consistent, thus confirming the credibility and reliability of our method. In addition, we investigated the activity of factor VII after incubation with its key
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proteases. For the groups incubated with CPB, DPP4, NEP and PDA, the activity decreased to different extents, while those incubated with the other four proteases showed activity similar to that of the control group. Thus, the activity results
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supported the hydrolysis results and illustrated the significance of our method by demonstrating that the degradation of factor VII by proteases influences its effect in vitro.
CPB is characterized as a carboxypeptidase B-like entity that is able to cleave
basic C-terminal amino acids, arginine and lysine [40]; therefore, the site of factor VII cleavage by CPB may lie between L401 and R402. The primary specificity of PDA is the cleavage of C-terminal dipeptides from substrates with a free C-terminus and the absence of a penultimate Pro [41]; thus, the cleavage site of factor VII by PDA may lie between P404 and F405. DPP4 is specific for a proline at the penultimate position 16
ACCEPTED MANUSCRIPT and hydrolyzes the carboxyl side of this residue (Xaa-Pro↓Xbb-) [42], indicating that the cleavage site of factor VII by DPP4 may lie between N2 and A3. It is well documented in the literature that the function specificity of NEP requires a bulky hydrophobic residue in the P1’ position as an endopeptidase [43]; thus, there are
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numerous potential cleavage sites in factor VII for NEP. Further investigations are needed to improve upon this study. Only four protein drugs were tested with this method, which is far from adequate to meet our goal of providing a method that has a wide range of applications, but we fully demonstrated
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that this method can be used to identify key proteases of proteins. In future studies, it will be important to test more protein drugs with our method, including interferon
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gamma-1b, oprelvekin, teceleukin, sargramostim and metreleptin, considering their short plasma half-life of less than two hours (statistics on drug instruction from the FDA, https://www.fda.gov/Drugs/default.htm, and clinical statistics from Integrity, https://integrity.thomson-pharma.com/integrity/xmlxsl/pk_home.util_home).
In
addition, more proteases in the human body will be examined by this method,
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including not only proteases in the blood but also those in the liver, kidney and other organisms. Notably, since this study provides direct evidence that the degradation of factor VII by its key proteases would decrease its activity, its pharmacological effect
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and plasma half-life may be improved by the modification of its cleavage sites in further studies. 5. Conclusion
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In summary, we developed a convenient method to identify key proteases of
proteins by competition with corresponding standard substrates with high accuracy, precision and durability that can be applied to endopeptidases and exopeptidases. This work may provide guidance for the future modification of proteins on cleavage sites to prolong their plasma half-life. 6. Conflicts of interest The authors declared no conflict of interest. 7. Acknowledgments This work was supported by the National Natural Science Foundation of China 17
ACCEPTED MANUSCRIPT [grant number 81430082, 81872850], “Double-First Class” University Project [CPU2018GF08], the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China [No. 111-2-07]. The authors thank Jiangsu Chia Tai Tianqing Pharmaceutical (Nanjing, China) for providing
8. Appendix A. Supplementary data
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pertuzumab and factor VII.
Supporting materials can be seen in “Appendix A Supplemental material” file. 9. References
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Highlights: Establishment of a method to identify key proteases of proteins based on HPLC Rapid identification of endopeptidases and exopeptidases that degrade proteins Factor VII is newly found to be degraded by CPB, NEP, DPP4 and PDA
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Fibrinogen is newly found to be degraded by CPB