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Measurement of Dipeptidylpeptidase Activity in vitro and in vivo
体外和体内二肽基肽酶活性测定   

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Abstract

Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and non-hematopoietic cells and is also present in many biological fluids in a bioactive soluble form. DPP4 expression is modulated by inflammation, and measurements of its activity have been used as biomarker for disease. Here, we describe a method to evaluate the enzymatic activity of DPP4 in vitro and in vivo.

Keywords: Dipeptidylpeptidase 4(二肽基肽酶4), Enzyme(酶), Tumor(肿瘤), Plasma(血浆), DPP4 inhibitor(DPP4抑制剂)

Background

The magnitude of the enzymatic activity of DPPs can be an indicator of inflammation; and an important pharmacodynamics parameter for usage of DPP4 inhibitors. Here, we report details for the usage of a commercially available kit to evaluate DPP-enzymatic activity in vitro; and provide details on the generation of biological samples tested. Furthermore, we describe how this method can be used for evaluation of DPP activity in vivo.

Materials and Reagents

  1. 96 well plates, U bottom (Fisher Scientific, catalog number: 12-565-500 )
  2. Luminometer 96 well plates (Promega, catalog number: Z3291 )
  3. 2 ml Eppendorf tube (Fisher Scientific, catalog number: 05-402-7 )
  4. 1 ml syringe (BD, catalog number: 309659 )
  5. 20 G (0.9 x 25 mm) needle (BD, catalog number: 305175 )
  6. Luciferase-expressing transgenic mouse such as the FVB-Tg(CAG-luc,-GFP)L2G85Chco/J mouse (THE JACKSON LABORATORIES, catalog number: 008450 )
  7. Biologic samples such as mouse plasma/serum or organ homogenates/supernatants (see Notes)
  8. DPPIV-GloTM Protease Assay (Promega, catalog number: G8351 )
  9. 1x PBS (Thermo Fisher Scientific, GibcoTM catalog number: 14190144 )
  10. Recombinant human DPP4 (R&D Systems, catalog number: 1180-SE-010 )
  11. Prionex (Sigma-Aldrich, catalog number: G0411 )
  12. DPP4 inhibitor (such as K579, Sigma-Aldrich, catalog number: D3572 )
  13. Ethanol

Equipment

  1. Luminometer, with plate reader (such as Promega, model: GloMax® 96 Microplate Luminometer )
  2. Multichannel pipet
  3. XENOGEN (PerkinElmer, model: IVIS system )
  4. Precision scale (such as Veritas Analytical Balance, AFFORDABLE SCALES AND BALANCES, model: M124A )
  5. Sterile stainless steel bead 5 mm (QIAGEN, catalog number: 69989 )
  6. TissueLyser II (QIAGEN, catalog number: 85300 )
  7. Tabletop microcentrifuge (such as Eppendorf, model: 5417 R )

Procedure

  1. Measuring DPP4 activity in vitro
    1. The protocol to measure DPP4 enzymatic activity was performed as described by Promega (provider of the DPP4 enzymatic activity kit used in our assays). Briefly, this assay provides a DPP4-sensitive substrate (Gly-Pro-aminoluciferin) that produces a luminescent signal upon reaction with dipeptidylpeptidases (DPP) and luciferase. DPP-mediated truncation of the Gly-Pro dipeptide is necessary for this reaction, and therefore the luminescent signal is indicative of DPP enzymatic activity. Important considerations concern the preparation and dilutions of biologic samples used in these assays:
      1. Mouse serum and plasma samples should be used at 1 to 20 dilution in Prionex solution.
      2. Mouse tumors homogenates should be used at 1 to 2 and 1 to 10 dilution in Prionex solution.
        Note: Please refer to the notes at the end of the protocol for instructions on how to obtain tumor homogenates.
      3. Peritoneal washes (collected with 1 ml of sterile cold PBS) should be used at 1 to 2 dilution in Prionex solution.
      4. A standard curve should be performed using dilutions of recombinant human DPP4 in Prionex solution. Start at 50 ng/ml and perform 2 fold dilutions until 0.01 ng/ml.
      5. Leave 2 wells with Prionex solution only (blank wells) in order to determine the background values of the assay.
      Note: We recommend the dilutions to be done in a 96-well U-bottom plate, before transferring them into the luminometer plate. Prepare the samples in a final volume of at least 60 μl in order to correctly pipet 50 μl of each sample onto the luminometer plate. Transfer of samples should be done with a multichannel pipet, avoiding the generation of bubbles. When all the samples have been transferred, add 50 μl of reaction buffer (provided in the kit) to each well, using a multichannel pipet. Pipetting up and down is not necessary. Before reading the results in the luminometer, incubate plate at room temperature for 15 min, exposed to room light (do not cover the plate with light blocking foil).
    2. Preparing tumor lysates for analysis of DPP4 activity
      1. Collect tumors using sterile dissection tools and place them in cold, sterile 1x PBS.
      2. Carefully dry the tumor with a paper towel and weigh it in a precision scale (such as Veritas Analytical Balance, M124A).
      3. Place tumors in a 2 ml Eppendorf tube. 
        Note: Tumor can be frozen at -20 °C after this step.
      4. To each tube, add a sterile stainless steel bead (5 mm) and 250 μl of cold sterile 1x PBS.
      5. Tumor lysates are generated by high speed shaking, using the TissueLyser II. Speed used is 25 Hz for 2 min. Repeat this step 2 times. The machine should be placed in a cold room. From this moment on, keep the samples on ice at all times.
      6. Centrifuge tubes in an Eppendorf centrifuge at 500 x g for 10 min, at 4 °C.
      7. Collect supernatants containing the tumor lysates into new, sterile tubes.
        Note: It is advisable to make at least 2 aliquots from each supernatant, to avoid multiple freeze/thaw cycles in the future analysis.
      8. Freeze supernatants at -20 °C or use them for evaluation of DPP4 activity.
    3. Collecting peritoneal washes for analysis of DPP4 activity
      1. Sacrifice mice according to the regulations instituted by your working facility. If possible, we recommend the mice to be sacrificed in a CO2 chamber, as cervical dislocation might result in internal organ damage and bleeding into the peritoneal cavity.
      2. Disinfect the skin of the mouse with ethanol and cut it. Expose the entire peritoneal area, without damaging the peritoneal membrane.
      3. Inject 1 ml of cold 1x PBS, into the peritoneal cavity using a 1 ml syringe with a 20 G (0.9 x 25 mm) needle. We recommend the injection to be performed through the abdominal fat pads in order to reduce the probability of damaging organs.
      4. Collect the peritoneal wash: hold up the peritoneal membrane over the intestine (use blunt tweezers) and gently aspirate the liquid with the same syringe used to inject the PBS.
      5. Place each peritoneal wash in a 2 ml Eppendorf tube.
      6. Centrifuge tubes in a tabletop microcentrifuge (such as centrifuge 5417 R, Eppendorf) at 500 x g for 5 min at 4 °C. This step allows elimination of peritoneal cells collected with the washes.
      7. Collect supernatants containing the peritoneal washes proteins into new, Eppendorf tubes.
        Note: It is advisable to make at least 2 aliquots from each supernatant, to avoid multiple freeze/thaw cycles in the future analysis.
      8. Freeze peritoneal washes at -20 °C or use them for evaluation of DPP4 activity.

  2. Measuring DPP activity in vivo
    The luciferin/luciferase system used to measure DPP activity in vitro can also be used to detect DPP activity in vivo, as follows:
    1. Inject 50 μl of 10 nM Gly-Pro-aminoluciferin (provided by the Promega kit) into a luciferase expressing mouse, such as the FVB-Tg(CAG-luc) mice. Injection can be intravenous or intraperitoneal. Intravenous injection results in fast systemic delivery (within 5 min, see Figure 1), while intraperitoneal injection results in luminescence detected in the peritoneal cavity area (Barreira da Silva et al., 2015).


      Figure 1. Example of luminescence image of FVB CAG-luciferase transgenic mice treated with control chow or chow containing DPP4 inhibitor (DPP4i). Mice were fed overnight with respective diets. DPP4i chow was sitagliptin 1.1%. Injection of Gly-Pro-aminoluciferin was done intravenously and images were acquired 3 minutes after injection. Exposition time was 1 sec. Regions of interest (ROI) were selected for quantification of radiance, calculated in photons per second per cm2 (of area selected), per steradian (sec-1 cm-2 sr-1).

    2. Place the mouse in a XENOGEN (IVIS System) and image luminescence 3-5 min after injection. The mouse should be anesthetized during the entire procedure, using an isoflurane-based anesthesia system, as specified for each research institution. For image acquisition, test different exposure times (start with 1 sec, up to 1 min).

Data analysis

  1. It is important to keep in mind that this assay detects the activity DPP4, but other members of the DPP family may also truncate Gly-Pro-aminoluciferin. In mouse serum or plasma samples, at least 95% of the measured DPP activity corresponds to DPP4, as evaluated by comparing samples from wildtype and dpp4-/- mice. Whenever evaluating the specific contribution of DPP4 in other biologic samples (e.g., tumor lysates), we advise assaying the sample in duplicate and adding a DPP4 inhibitor (such as K579) to one of the two samples. The reason behind this additional control is that there are other DPP family members (such as Fibroblast Activating Protein [FAP] and intracellular DPP8 and 9) that might become exposed after tissue lysis, giving positive signals in the assay. K579 inhibits the activity of DPP4 at concentrations of 50 nM. Samples do not need to be incubated at length with K579, as inhibition with these concentrations occurs as soon as the reagents are mixed together. It is recommended to perform the assay using at least 4 biological replicates (e.g., biological samples from 4 mice) and 2 technical replicates per sample.
  2. The representation of results can be done in relative fluorescent units (RFUs) by subtracting the RFU levels of the blank wells to all values. For representation of DPP4-specific activity a subtraction of the activity of samples incubated with K579 can be done. Alternatively, the results can be represented in Units of DPP activity. For this, please refer to the lot number of the recombinant DPP4 used for generation of the standard curve, as there should be stated the Units of activity per weight of enzyme. The values can then be extrapolated from the standard curve of the assay.
  3. Detection of DPP4 activity in the entire body of the mouse can be done following intravenous injection of the Gly-Pro-aminoluciferin. This method can be used to study pharmacodynamics of DPP4 inhibition in vivo, following administration of DPP4 inhibitors, such as sitagliptin (Figure 1). Moreover, it is likely that this technique can be extrapolated to measure DPP4 activity in restricted tissues, such as inoculated luciferase-expressing tumors or transferred immune cells. It is recommended to perform the assay in at least 3 biological replicates.

Recipes

  1. Prionex solution – Prepare fresh for each experiment
    1x PBS supplemented with 0.1% Prionex

Acknowledgments

When using this protocol, please cite Barreira da Silva et al. (2015). Funding was provided by the Pasteur-Roux post-doctoral fellowship (RBdS), the Ligue Contre le Cancer and the Fondation ARC pour la recherche sur le cancer (MLA) and the French government’s Invest in the Future Program, managed by the Agence Nationale de la Recherche (LabEx Immuno-Onco [RBdS, MAI MLA]). We thank M.A. Nicola (Plateforme d’imagerie dynamique, Institut Pasteur, Paris, France) for providing FVB CAG-luciferase transgenic mice. Animal experimental protocols were approved by the comité d’éthique pour l’expérimentation animale (The ethics committee for animal experimentation) Paris.

References

  1. Barreira da Silva, R., Laird, M. E., Yatim, N., Fiette, L., Ingersoll, M. A. and Albert, M. L. (2015). Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy. Nat Immunol 16(8): 850-858.

简介

二肽基肽酶(DPPs)是丝氨酸蛋白酶,其在其N末端的第二位置切割具有脯氨酸或丙氨酸的小蛋白质和肽。在该家族成员中,二肽基肽酶4(DPP4)在细胞外空间中组成型表达。 DPP4存在于许多造血细胞和非造血细胞的表面,并且也以生物活性可溶形式存在于许多生物流体中。 DPP4表达受炎症调节,其活性的测定已被用作疾病的生物标志物。在这里,我们描述了一种评估体外DPP4和/或体内酶活性的方法。

背景 DPPs的酶活性的大小可以是炎症的指标;以及用于DPP4抑制剂的重要药效学参数。在这里,我们报告了市售试剂盒用于评估体外DPP-酶活性的详细信息;并提供测试生物样品生成的细节。此外,我们描述了该方法如何用于体内DPP活性的评估。

关键字:二肽基肽酶4, 酶, 肿瘤, 血浆, DPP4抑制剂

材料和试剂

  1. 96孔板,U底(Fisher Scientific,目录号:12-565-500)
  2. 发光计96孔板(Promega,目录号:Z3291)
  3. 2ml Eppendorf管(Fisher Scientific,目录号:05-402-7)
  4. 1 ml注射器(BD,目录号:309659)
  5. 20 G(0.9 x 25 mm)针(BD,目录号:305175)
  6. 表达荧光素酶的转基因小鼠,例如FVB-Tg(CAG-luc,-GFP)L2G85Chco/J小鼠(THE JACKSON LABORATORIES,目录号:008450)
  7. 生物样品如小鼠血浆/血清或器官匀浆/上清液(见注释)
  8. DPPIV-Glo TM蛋白酶测定(Promega,目录号:G8351)
  9. 1x PBS(Thermo Fisher Scientific,Gibco TM 目录号:14190144)
  10. 重组人DPP4(R& D Systems,目录号:1180-SE-010)
  11. Prionex(Sigma-Aldrich,目录号:G0411)
  12. DPP4抑制剂(例如K579,Sigma-Aldrich,目录号:D3572)
  13. 乙醇

设备

  1. 发光计,带读板器(如Promega,型号:GloMax 96微孔板发光计)
  2. 多通道移液器
  3. XENOGEN(PerkinElmer,型号:IVIS系统)
  4. 精密秤(如Veritas Analytical Balance,AFFORDABLE SCALES AND BALANCES,型号:M124A)
  5. 无菌不锈钢珠5毫米(QIAGEN,目录号:69989)
  6. TissueLyser II(QIAGEN,目录号:85300)
  7. 台式微量离心机(如Eppendorf,型号:5417 R)

程序

  1. 在体外测量DPP4活性
    1. 如Promega(我们的测定中使用的DPP4酶活性试剂盒的提供者)所述进行测量DPP4酶活性的方案。简而言之,该测定提供了与二肽基肽酶(DPP)和荧光素酶反应时产生发光信号的DPP4敏感性底物(Gly-Pro-氨基荧光素)。 DPP介导的Gly-Pro二肽的截短对于该反应是必需的,因此发光信号指示DPP酶活性。重要的考虑事项涉及这些测定中使用的生物样品的制备和稀释:
      1. 应在Prionex溶液中以1〜20稀释使用小鼠血清和血浆样品
      2. 小鼠肿瘤匀浆应在Prionex溶液中以1至2和1至10稀释使用。
        注意:有关如何获得肿瘤匀浆物的说明,请参阅协议末尾的说明。
      3. 应在Prionex溶液中以1-2稀释使用腹膜洗液(用1ml无菌冷PBS收集)。
      4. 应使用Prionex溶液中重组人DPP4的稀释液进行标准曲线。以50ng/ml开始,进行2倍稀释至0.01ng/ml
      5. 仅使用Prionex溶液留出2孔(空白孔),以确定测定的背景值。
      注意:我们建议在96孔U形底板中进行稀释,然后将其转移到发光计板中。准备至少60μl的最终体积的样品,以便将50μl每个样品正确吸取到光度计板上。样品转移应使用多通道移液管进行,避免产生气泡。当所有样品已经转移时,使用多通道移液管将50μl反应缓冲液(试剂盒中提供)加入每个孔中。上下移动是不必要的。在光度计读取结果之前,在室温下孵育板15分钟,暴露于室内光线(不要用遮光箔覆盖板)。
    2. 准备肿瘤裂解物以分析DPP4活性
      1. 使用无菌解剖工具收集肿瘤,并将其置于冷的无菌1x PBS中
      2. 用纸巾小心地干燥肿瘤,并用精密称重称重(如Veritas Analytical Balance,M124A)。
      3. 将肿瘤置于2ml Eppendorf管中。 
        注意:在此步骤后,肿瘤可以在-20°C冷冻。
      4. 向每个管中加入无菌不锈钢珠(5mm)和250μl冷的无菌1x PBS。
      5. 使用TissueLyser II通过高速振动产生肿瘤裂解物。使用的速度为25 Hz,持续2分钟。重复此步骤2次。机器应放在冷藏室内。从此刻起,随时将样品放在冰上。
      6. 离心管在Eppendorf离心机中以500×g×10分钟,在4℃下。
      7. 将含有肿瘤裂解物的上清液收集到新的无菌管中。
        注意:建议从每个上清液中制备至少2个等分试样,以避免将来分析中多次冻融循环。
      8. 将上清液冷冻至-20°C或用于评估DPP4活性。
    3. 收集腹膜洗液用于分析DPP4活性
      1. 根据您的工作设施制定的规定宰杀小鼠。如果可能,我们建议将小鼠在CO 2室中处死,因为颈椎错位可能导致内部器官损伤并渗入腹膜腔。
      2. 用乙醇消毒小鼠的皮肤并切割。暴露整个腹膜区域,而不会损害腹膜。
      3. 使用具有20 G(0.9 x 25 mm)针头的1 ml注射器将1 ml冷1x PBS注入腹腔。我们建议通过腹部脂肪垫进行注射,以减少器官损伤的可能性。
      4. 收集腹膜洗液:将腹膜覆盖在肠上(使用钝镊子),并用与注射PBS相同的注射器轻轻吸出液体。
      5. 将每个腹膜洗液放入2 ml Eppendorf管中
      6. 在4℃下,将500g离心管在台式微量离心机(如离心机5417R,Eppendorf)中以5℃离心5分钟。该步骤可以消除用洗涤液收集的腹膜细胞。
      7. 收集含有腹膜的上清液将蛋白质洗涤到新的Eppendorf管中。
        注意:建议从每个上清液中制备至少2个等分试样,以避免将来分析中多次冻融循环。
      8. -20℃冷冻腹膜洗涤,或用于DPP4活性评估
  2. 在体内测量DPP活动
    用于测量体外DPP活性的荧光素/荧光素酶系统也可用于检测体内DPP活性,如下所示:
    1. 将50μl10nM Gly-Pro-氨基荧光素(由Promega试剂盒提供)注入到表达荧光素酶的小鼠中,例如FVB-Tg(CAG-luc)小鼠。注射可以静脉内或腹膜内。静脉注射导致快速全身递送(5分钟内,参见图1),而腹膜内注射导致在腹膜腔区域中检测到的发光(Barreira da Silva等人,2015)。 >

      图1.用对照食物或含有DPP4抑制剂(DPP4i)的食物处理的FVB CAG-萤光素酶转基因小鼠的发光图像的实施例。小鼠喂食过夜各自的饮食。 DPP4i食物是西他列汀1.1%。静脉内注射Gly-Pro-氨基荧光素,注射后3分钟摄取图像。展览时间为1秒。选择感兴趣区域(ROI)用于定量辐射度,以每秒每光子每平方厘米(选择的面积)计算,每个球状体(秒 -1 cm -2 sr -1 )。

    2. 将鼠标置于XENOGEN(IVIS系统)中,注射后3-5分钟进行成像发光。在整个手术过程中,应按照每个研究机构规定的异氟烷麻醉系统麻醉小鼠。对于图像采集,测试不同的曝光时间(从1秒开始,最长1分钟)。

数据分析

  1. 重要的是要记住,该测定法检测DPP4的活性,但DPP家族的其他成员也可能截短Gly-Pro-氨基荧光素。在小鼠血清或血浆样品中,通过比较来自野生型和dpp4小鼠的样品评估,所测量的DPP活性的至少95%对应于DPP4。每当评估DPP4在其他生物样品(例如肿瘤裂解物)中的具体贡献时,我们建议一式两份测定样品,并将DPP4抑制剂(如K579)添加到两个样品之一中。这种额外控制的原因在于还有其他DPP家族成员(如成纤维细胞激活蛋白[FAP]和细胞内DPP8和9),可能在组织裂解后暴露,在测定中给出阳性信号。 K579以50nM的浓度抑制DPP4的活性。样品不需要长时间与K579一起孵育,因为一旦将试剂混合在一起就会发生这些浓度的抑制。建议使用至少4个生物重复(例如,4只小鼠的生物样品)和每个样品2次技术重复进行测定。
  2. 通过将空白孔的RFU水平减去所有值,可以在相对荧光单位(RFU)中进行结果表示。为了表达DPP4特异性活性,可以减少用K579孵育的样品的活性。或者,结果可以以DPP活性的单位表示。为此,请参考用于产生标准曲线的重组DPP4的批号,因为应该说明每重量酶的活性单位。然后可以从测定的标准曲线推断值。
  3. 在静脉注射Gly-Pro-氨基荧光素后,可以检测小鼠全身中的DPP4活性。在施用DPP4抑制剂如西他列汀后,该方法可用于研究体内DPP4抑制的药效学(图1)。此外,这种技术可能被外推以测量限制性组织中的DPP4活性,例如接种的荧光素酶表达肿瘤或转移的免疫细胞。建议在至少3次生物复制中进行检测。

食谱

  1. Prionex溶液 - 为每个实验准备新鲜
    1x PBS补充0.1%Prionex

致谢

当使用这个协议时,请引用Barreira da Silva等人。 (2015)。资助由巴斯德·鲁夫博士后研究金(RBdS),Ligue Contre le癌症以及法国政府的ARC pour la recherche sur le cancer(MLA)和法国政府的"未来计划投资计划"提供,由国民政府管理la Recherche(LabEx Immuno-Onco [RBdS,MAI MLA])。我们感谢提供FVB CAG荧光素酶转基因小鼠的M.A.Nicola(Plateforme d'imagerie dynamique,Institut Pasteur,Paris,France)。动物实验方案由巴黎的动物实验委员会(动物实验伦理委员会)批准。

参考文献

  1. Barreira da Silva,R.,Laird,ME,Yatim,N.,Fiette,L.,Ingersoll,MA和Albert,ML(2015)。  二肽基肽酶4抑制增强淋巴细胞运输,改善天然存在的肿瘤免疫和免疫治疗。 Nat Immunol 16 (8):850-858。
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引用:Barreira da Silva, R., Ingersoll, M. A. and Albert, M. L. (2017). Measurement of Dipeptidylpeptidase Activity in vitro and in vivo. Bio-protocol 7(6): e2184. DOI: 10.21769/BioProtoc.2184.
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