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Oct 2020

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Quantitative Measurement of Mucolytic Enzymes in Fecal Samples
粪便粘液溶解酶的定量测定   

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Abstract

The mucus layer in the gastrointestinal tract covers the apical surface of intestinal epithelial cells, protecting the mucosal tissue from enteric pathogen and commensal microorganisms. The mucus is primarily composed of glycosylated protein called mucins, which are produced by goblet cells, a type of columnar epithelial cells in the intestinal tract. Defective mucin barrier facilitates infection caused by enteric pathogen and triggers inflammation due to invasion of commensal or opportunistic pathogens into the intestinal epithelial mucosa. Several bacterial species in the gut produce enzymes that are capable of degradation of the mucus. Defective mucin production or increased abundance of mucolytic bacteria are clinically linked to inflammatory bowel disease. Measurement of mucolytic enzymes in the feces, therefore, can be implicated in clinical and experimental research on intestinal disorders. Here, we describe a step-by-step procedure for the measurement of the mucolytic enzyme activity in fecal samples.

Keywords: Mucus (粘液), Mucin (粘蛋白), Mucolytic enzymes (粘液溶解酶), Mucus degrading bacteria (粘液降解菌)

Background

The gastrointestinal tract (GI) is home for trillions of microorganisms which play diverse functions in the physiological processes (Sommer and Backhed, 2011). Commensal gut microbiota process undigested food, provide energy, nutrients and vitamins, activate the immune system, and prevent pathogens from infecting the intestinal mucosal tissue (Round and Mazmanian, 2009; Pickard et al., 2017). Despite these beneficial roles, gut commensal microorganisms may act as opportunistic pathogens when they get the opportunity to colonize intestinal epithelial barrier and invade into the mucosal tissue. However, a gel like mucus layer above the apical surface of the epithelial cells throughout the intestinal tract ensures physical separation of commensal microbes from the intestinal mucosal tissue and helps maintain intestinal homeostasis (Pullan et al., 1994; Linden et al., 2008; Atuma et al., 2011; Johansson et al., 2011; Juge, 2012). In the large intestine, mucus barrier is very thick, about 700 nm, and can be divided into two distinct layers – a thick outer layer and a thin inner layer (Johansson et al., 2008 and 2011). While the outer layer is nutrient rich, easy to be dislodged, and often colonized with anaerobic bacteria, the inner layer is firmly attached to the epithelial layer and is mostly sterile (Johansson et al., 2008 and 2011).


The mucus is primarily composed of glycoprotein called mucin, produced by goblet cells which are a type of columnar epithelial cells in the intestinal tract. Upon synthesis, mucin proteins are O-glycosylated or N-glycosylated with oligosaccharides and transported to the cell surface or secreted outside (McGuckin et al., 2011). Secretory mucins are heavily O-glycosylated and are homo-oligomerized via inter-molecular disulphide bond formed between the cysteine-rich D domain at the C and N terminus (Thornton et al., 2008). The major mucins in the outer layer that oligomerize to form the matrix are MUC2, MUC5AC, MUC5B, MUC6, and MUC19 (Thornton et al., 2008; McGuckin et al., 2011). The mucus is embedded with many antimicrobial peptides and immunoglobulins, which also keep the inner mucus layer sterile (McGuckin et al., 2011). On the other hand, oligosaccharides of the mucin serve as ligands and a source of nutrients for many anaerobic bacteria. Thus, several intestinal commensals as well as pathogens produce mucolytic enzymes, such as sulphatase, proteases, neuraminidases, α-glycosidase, β-glycosidase, β-galactosidase, fucosidase, β-N-acetylglucosaminidase, α-N-acetylgalactosaminidase, etc., to degrade mucins (Corfield et al., 1992; Linden et al., 2008; Johansson et al., 2011; Desai et al., 2013). Based on the diversity and complexity of mucin oligomers, cooperative actions are required from a number of enzymes as mentioned above for the degradation of mucins (Lombard et al., 2014). The major mucosa-associated bacteria belong to the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Verrucomicrobia (Derrien et al., 2010; Tailford et al., 2015).


Enzymatic degradation of the mucus layer allows gut commensal bacteria or pathogen to breach the mucus barrier (Khan et al., 2020). Therefore, increased abundance of mucolytic bacteria facilitates enteric infection and is associated with inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis (Prizont, 1982; Carroll et al., 2010; Png et al., 2010; Hansson, 2012; Sheng et al., 2012). Thus, the level of mucus degrading enzymes in the colon could be a predictive marker for IBD. Measurement of mucolytic enzymes is also very useful in studies aimed at dissecting the mechanism of IBD pathogenesis in experimental or clinical settings.

Materials and Reagents

  1. 1.7 ml Posi-ClickTM Tubes (Denvelle, catalog number: C2170)

  2. 96-well flat bottom plate (Thermo Scientific, catalog number: 12565136)

  3. Aluminium foil (Fisher Brand, catalog number: 01-213-100)

  4. Micropipette barrier tips (from 10 μl to 1,000 μl) (Genesee Scientific)

  5. 8-10 weeks-old C57Bl6/j mice

  6. 4-nitrophenol (Sigma-Aldrich, catalog number: 241326-50G)

  7. 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, catalog number: N9376)

  8. 4-nitrophenyl α-D-galactopyranoside (Sigma-Aldrich, catalog number: N0877)

  9. 4-nitrophenyl β-D-glucopyranoside (Sigma-Aldrich, catalog number: N7006)

  10. Fresh or -80 °C stored feces pellets

  11. Acetone (EM Science, catalog number: AX0120-8)

  12. DNases (Sigma-Aldrich, catalog number: 11284932001)

  13. Lysozyme (Fisher BioReagentsTM, catalog number: BP535-1)

  14. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M-0250)

  15. Methanol (Fisher Chemical, catalog number: A433P-4)

  16. p-nitrophenyl α-L-fucopyranoside (Sigma-Aldrich, catalog number: N3628)

  17. p-nitrophenyl β-D-xylopyranoside (Sigma-Aldrich, catalog number: N2132)

  18. PierceTM BCA protein assay kit (Thermo Scientific, catalog number: 23227)

  19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541-500G)

  20. Protease inhibitor cocktail tablet (Roche, catalog number: 26733200)

  21. Triton X-100 (Sigma-Aldrich, catalog number: T-9284)

  22. Trizma® hydrochloride (Sigma-Aldrich, catalog number: S8045-1KG)

  23. 4-Nitrophenyl (4NP) standard curve (see Recipes)

  24. Mucolytic enzyme buffer (see Recipes)

  25. Nitrophenyl-linked substrates and their corresponding mucolytic enzymes (see Recipes)

Equipment

  1. A pair of sterile forceps

  2. -80 °C freezer (Thermo Scientific)

  3. Centrifuge (Thermo Scientific, Legend Micro 21R)

  4. Ice making machine (Hoshizaki American Inc.)

  5. Micropipette (from 10 μl to 1,000 μl) (Labnette)

  6. Multi-channel pipette (300 μl) (Fisher Brand)

  7. Sonicator with 3 mm tapered microtip (Branson Digital Sonifier, Model: 102C)

  8. Spectrophotometer (TECAN, SPARK 10M)

  9. Vortex Genie 2 (VWR Scientific Products)

  10. Weighing balance (ADAM Equipment, PW124)

Procedure

  1. Collect 2-3 fecal pellets (approximately 50 mg) from each mouse into a tube.

    Note: Feces can be stored at -80 °C until measurement.

  2. Add 0.5 ml of ice-cold mucolytic enzyme buffer (Recipe 1) into the tube containing fecal pellets and gently vortex.

    Note: Vertexing is not necessary if feces are not solid.

  3. Sonicate samples using ultrasonic processor for 5 s with 35% amplitude and 3 mm tapered microtip on ice. Repeat ultrasonication following a 10 s interval for a total of 9 cycles or 45 s.

  4. Centrifuge sonicated samples at 10,000 × g for 10 min at 4 °C.

  5. Transfer ~400 μl supernatant into fresh 1.5 ml tubes and measure protein concentration.

    Note: Supernatant can be stored at -80 °C, until measurement.

  6. Adjust the protein concentration to 1 mg/ml by adding ice-cold mucolytic enzyme buffer.

  7. Transfer 5 μl (5 μg protein) supernatant of a particular fecal sample into three wells (5 μl each) of a 96-well flat-bottom plate. Add 150 μl of a specific 10 mM nitrophenyl-based substrate (Recipe 2) prepared in ice-cold mucolytic enzyme buffer. For each enzyme tested, corresponding substrate is added into triplicate wells containing the same sample. These procedures are repeated when multiple samples are used.

  8. Measure the absorbance at 405 nm in a plate reader at 37 °C at every 30 min interval.

  9. Using a known concentration of 4-nitrophenol standard curve (Recipe 3), determine the individual mucolytic enzyme activity (Figure 1).



    Figure 1. Time-dependent mucolytic enzymatic activity in mouse fecal samples. Fecal samples collected from healthy mouse were homogenized and processed as described in the protocol. The activity of indicated enzymes was measured in the fecal lysate following the procedure described in the protocol.

Recipes

  1. Mucolytic enzyme buffer (pH 7.25)

    Stock solution Working solution For 100 ml

    1 M Tris            50 mM 

    1 M KCl            100 mM 

    1 M MgCl2       10 mM 

    Lysozyme                                        5-10 mg

    12% Triton X-100                           100 μl

    DNases                                            5-10 mg

    Protease inhibitor                         One tablet

  2. Nitrophenyl-linked substrates and their corresponding mucolytic enzymes

    Nitrophenyl-linked substrates          Mucolytic enzymes
    4-nitrophenyl α-D-galactopyranoside          α-galactosidase                        Plant glycans
    4-nitrophenyl N-acetyl-β-D-glucosaminide           β-N-acetylglucosaminidas  Mucin
    4-nitrophenyl β-D-glucopyranoside                     β-glucosidase Plant glycans
    p-nitrophenyl α-L-fucopyranoside          α-fucosidase Mucin
    p-nitrophenyl β-D-xylopyranoside          β-xylosidase Plant glycans

    Note: We mentioned above nitrophenyl-linked substrates, which we use in the Figure 1.

  3. 4-Nitrophenol (4NP) standard curve

    4-Nitrophenol (4NP) is an enzymatic product of p-nitrophenyl-linked substrates. The amount of 4NP produced during reaction of mucolytic enzymes with its substrate corresponds to the mucolytic enzyme activity (Recipe 2). 4NP provides yellow color and can be measured spectrophotometrically at 405 nm. Thus, a standard curve of 4NP can be used to measure mucolytic enzyme activity in a reaction mixture of mucolytic enzymes and its p-nitrophenyl-linked substrates.

    1. Prepare a 100 mM 4NP (MW = 139.11) stock solution by dissolving 0.0139 g (13.9 mg) of 4NP in 1 ml of methanol.

    2. Prepare a working stock solution of 1 mM 4NP (from 100 mM 4NP) for standard curve with mucin enzyme assay buffer.

    3. Prepare a series of 4NP concentration ranging from 0 to 1,000 µM (or 0 to 1 mM) from the working stock (1 mM) prepared in mucin enzyme assay buffer.

    4. Add 150 µl of each standard solution into a 96-well plate including blank and read the absorbance with a spectrophotometer at 405 nm at 37 °C.

    5. Plot the standard curve with known standard concentration on the X-axis and absorbance on the Y-axis.

Acknowledgments

This protocol was adapted with minor modification from previous study published by Desai et al. (2016) and Khan et al. (2020). We would like to thank the UT Southwestern Animal Resource Center (ARC) for maintenance and care of our mouse colony. This work was supported by The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institute of Health (NIH) under Award Number R01DK125352, and Cancer Prevention and Research Institute of Texas (CPRIT) Individual Investigator Awards (RP200184), and UT Southwestern funding given to H.Z.

Competing interests

The authors declare no competing interests.

Ethics

This study was approved by the Institutional Animal Care and Use Committee (IACUC; approval No. 2016-101683), and was conducted in accordance with the IACUC guidelines.

References

  1. Atuma, C., Strugala, V., Allen, A. and Holm, L. (2001). The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am J Physiol Gastrointest Liver Physiol 280(5): G922-929.
  2. Carroll, I. M., Chang, Y. H., Park, J., Sartor, R. B. and Ringel, Y. (2010). Luminal and mucosal-associated intestinal microbiota in patients with diarrhea-predominant irritable bowel syndrome. Gut Pathog 2(1): 19.
  3. Corfield, A. P., Wagner, S. A., Clamp, J. R., Kriaris, M. S. andHoskins, L. C. (1992). Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria. Infect Immun 60(10): 3971-3978.
  4. Desai, M. S., Seekatz, A. M., Koropatkin, N. M., Kamada, N., Hickey, C. A., Wolter, M., Pudlo, N. A., Kitamoto, S., Terrapon, N., Muller, A., Young, V. B., Henrissat, B., Wilmes, P., Stappenbeck, T. S., Nunez, G. and Martens, E. C. (2016). A dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility. Cell 167(5): 1339-1353 e1321.
  5. Derrien, M., van Passel, M. W., van de Bovenkamp, J. H., Schipper, R. G., de Vos, W. M. and Dekker, J. (2010). Mucin-bacterial interactions in the human oral cavity and digestive tract. Gut Microbes 1(4): 254-268.
  6. Hansson, G. C. (2012). Role of mucus layers in gut infection and inflammation. Curr Opin Microbiol 15(1): 57-62.
  7. Johansson, M. E., Larsson, J. M. and Hansson, G. C. (2011). The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions. Proc Natl Acad Sci U S A 108 Suppl 1: 4659-4665.
  8. Johansson, M. E., Phillipson, J. M., Petersson, J., Velcich, A., Holm, L. and Hansson, G. C. (2008). The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A 105(39): 15064-15069.
  9. Juge, N. (2012). Microbial adhesins to gastrointestinal mucus. Trends Microbiol 20(1): 30-39.
  10. Khan, S., Waliullah, S., Godfrey, V., Khan, M. A. W., Ramachandran, R. A., Cantarel, B. L., Behrendt, C., Peng, L., Hooper, L. V., Zaki, H. (2020). Dietary simple sugars alter microbial ecology in the gut and promote colitis in mice. Sci Transl Med 12: eaay6218.
  11. Linden, S. K., Sutton, P., Karlsson, N. G., Korolik, V. and McGuckin, M. A. (2008). Mucins in the mucosal barrier to infection. Mucosal Immunol 1(3): 183-197.
  12. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. and Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42: D490-495.
  13. McGuckin, M. A., Linden, S. K., Sutton, P. and Florin, T. H. (2011). Mucin dynamics and enteric pathogens. Nat Rev Microbiol 9(4): 265-278.
  14. Pickard, J. M., Zeng, M. Y., Caruso, R. and Nunez, G. (2017). Gut microbiota: Role in pathogen colonization, immune responses, and inflammatory disease. Immunol Rev 279(1): 70-89.
  15. Png, C. W., Linden, S. K., Gilshenan, K. S., Zoetendal, E. G., McSweeney, C. S., Sly, L. I., McGuckin, M. A. and Florin, T. H. (2010). Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria. Am J Gastroenterol 105(11): 2420-2428.
  16. Prizont, R. (1982). Degradation of intestinal glycoproteins by pathogenic Shigella flexneri. Infect Immun 36(2): 615-620.
  17. Pullan, R. D., Thomas, G. A., Rhodes, M., Newcombe, R. G., Williams, G. T., Allen, A. and Rhodes, J. (1994). Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis. Gut 35(3): 353-359.
  18. Round, J. L. and Mazmanian, S. K. (2009). The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol 9(5): 313-323.
  19. Sheng, Y. H., Hasnain, S. Z., Florin, T. H. and McGuckin, M. A. (2012). Mucins in inflammatory bowel diseases and colorectal cancer. J Gastroenterol Hepatol 27(1): 28-38.
  20. Sommer, F. and Backhed, F. (2013). The gut microbiota--masters of host development and physiology. Nat Rev Microbiol 11(4): 227-238.
  21. Tailford, L. E., Crost, E. H., Kavanaugh, D., Juge, N. (2015). Mucin glycan foraging in the human gut microbiome. Front Genet 6: 81.
  22. Thornton, D. J., Rousseau, K. and McGuckin, M. A. (2008). Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 70: 459-486.

简介

[摘要]胃肠道粘液层覆盖了肠上皮细胞的顶端表面,保护了粘膜组织免受肠道病原体和共生微生物的侵害。粘液主要由称为粘蛋白的糖基化蛋白组成,其由杯状细胞产生,杯状细胞是肠道中的一种柱状上皮细胞。缺陷性粘蛋白屏障促进由肠道病原体引起的感染并由于共生或机会病原体侵入肠道上皮粘膜而引发炎症。在肠道中几种细菌物种产生的酶即能够降解的 黏液 临床上,粘蛋白产生缺陷或粘液溶解细菌丰度增加与炎症性肠病有关。因此,粪便中粘液溶解酶的测定可能与肠道疾病的临床和实验研究有关。在这里,我们描述了粪便样品中粘液分解酶活性的分步测量方法。

[背景]胃肠道(GI)是数万亿个微生物的家园,这些微生物在生理过程中发挥着不同的功能(Sommer和Backhed ,2011年)。共生肠道菌群过程未消化的食物,提供能量,营养物质和维生素,激活的免疫系统,和防止病原体感染肠道粘膜组织(圆形和Mazmanian ,2009;皮卡德等人。,2017)。尽管有这些有益的作用,但是当肠道共生微生物有机会对肠道上皮屏障进行定殖并侵入粘膜组织时,它们仍可能是机会病原体。然而,整个肠道上皮细胞顶表面上方的凝胶状粘液层确保了共生微生物与肠道粘膜组织的物理分离,并有助于维持肠道的稳态(Pullan等,1994;Linden等,2008;Landen等,2008;Landen等,2008)。Atuma等,2011 ;Johansson等,2011;Juge ,2012 )。在大肠中,粘液屏障非常厚,约为700 nm,可以分为两个不同的层-厚的外层和薄的内层(Johansson等人,2008和2011 )。而外层是营养丰富,容易被移去,并经常与厌氧菌定植,内层被牢固地附接到上皮层和主要是无菌的(约翰逊等人,2008和2011 )。

粘液主要由杯状细胞产生,称为糖蛋白,称为糖蛋白,由杯状细胞产生,杯状细胞是肠道中的一类柱状上皮细胞。合成后,粘蛋白蛋白被寡糖O-糖基化或N-糖基化,然后转运到细胞表面或分泌到外部(McGuckin等,2011 )。分泌型粘蛋白被大量O-糖基化,并通过在C和N端富含半胱氨酸的D结构域之间形成的分子间二硫键进行均聚(Thornton等,2008 )。低聚形成基质的外层主要粘蛋白是MUC2,MUC5AC,MUC5B,MUC6和MUC19(Thornton等,2008 ;McGuckin等,2011 )。粘液中嵌入了许多抗菌肽和免疫球蛋白,这也使内部粘液层保持无菌状态(McGuckin等,2011 )。另一方面,粘蛋白的低聚糖充当许多厌氧细菌的配体和营养来源。因此,几个肠共生以及病原体产生粘液溶解酶,如硫酸酯酶,蛋白酶,神经氨酸酶,α糖苷酶,β糖苷酶,β半乳糖苷酶,岩藻糖苷酶,β -N-乙酰氨基,α - N乙酰氨基,等等。,以降解粘蛋白(Corfield等,1992;Linden等,2008;Johansson等,2011;Desai等,2013 )。基于粘蛋白寡聚体的多样性和复杂性,如上所述,多种酶需要协同作用来降解粘蛋白(Lombard等,2014 )。主要的与粘膜相关的细菌分别是门的Proteobacteria,Actinobacteria,Fimicutes,Bacteroidetes和Verrucomicrobia(Derrien等,2010;Tailford等,2015 )。

粘液层的酶促降解使肠道共生细菌或病原体突破了粘液屏障。因此,粘液溶解细菌的丰度增加促进肠道感染和与炎症相关的肠疾病(IBD)如克罗恩'病和溃疡性结肠炎(Prizont ,1982;卡罗尔等人。,2010;巴等人。,2010; Hansson的, 2012;Sheng等,2012 )。因此,结肠中粘液降解酶的水平可能是IBD的预测标志。粘液溶解酶的测量在旨在剖析IBD发病机理的实验或临床环境中的研究中也非常有用。

关键字:粘液, 粘蛋白, 粘液溶解酶, 粘液降解菌

材料和试剂
1.7 ml Posi-Click TM试管(Denvelle,目录号:C2170)
96孔平底板(Thermo Scientific,目录号:12565136)
铝箔(Fisher Brand,目录号:01-213-100)
微量移液器防渗吸头(10个起) μ l至1000 μ升)(杰纳西Scientific)中
8-10周大的C57Bl6 / j小鼠
4-硝基苯酚(Sigma-Aldrich,目录号:241326-50G)
4-硝基苯基N-乙酰基-β-D-氨基葡萄糖苷(Sigma-Aldrich,目录号:N9376)
4-硝基苯基α-D-吡喃半乳糖苷(Sigma-Aldrich,目录号:N0877)
4-硝基苯基β-D-吡喃葡萄糖苷(Sigma-Aldrich,目录号:N7006)
新鲜或-80°C储存的粪便颗粒
丙酮(EM Science,目录号:AX0120-8)
脱氧核糖核酸酶(Sigma-Aldrich,目录号:11284932001)
溶菌酶(Fisher Bi oReagents TM ,目录号:BP535-1)
氯化镁(MgCl 2 )(Sigma-Aldrich,目录号:M-0250)
甲醇(Fisher Chemical,目录号:A433P-4)
对硝基苯基α-L-呋喃果糖苷(Sigma-Aldrich,目录号:N3628)
对硝基苯基β-D-吡喃吡喃糖苷(Sigma-Aldrich,目录号:N2132)
Pierce TM BCA蛋白分析试剂盒(Thermo Scientific,目录号:23227)
氯化钾(KC升)(西格毫安-Aldrich公司,目录号:P9541-500G)
蛋白酶抑制剂鸡尾酒片剂(罗氏,目录号:26733200)
海卫一X-100(Sigma-Aldrich,目录号:T-9284)
盐酸Trizma ((Sigma-Aldrich,目录号:S8045-1KG)
4-硝基苯基(4NP)标准曲线(请参见配方)
粘液溶解酶缓冲液(请参见食谱)
硝基苯基连接的底物及其相应的粘液溶解酶(请参见食谱)


设备


一对无菌钳
-80 °C冰箱(Thermo Scientific)
离心机(Thermo Scientific,Legend Micro 21R)
制冰机(星崎美国公司)
微量(从10微升至1 ,000微升)(Labnette )
多通道移液器(300μl)(Fisher品牌)
带有3毫米锥形微尖端的声波发生器(Branson Digital Sonifier,型号:102C)
分光光度计(TECAN,SPARK 10M)
Vortex Genie 2(VWR科学产品)
称重天平(ADAM设备,PW124)


再修改的è


从每只小鼠收集2-3个粪便沉淀物(约50 mg)到管中。
注意:粪便可以在-80 °C下保存直至测量。


向装有粪便沉淀的试管中加入0.5 ml冰冷的粘液溶解酶缓冲液(配方1 ),并轻轻涡旋。
注意:如果粪便不牢固,则无需进行顶点处理。


使用超声处理器超声处理样品5秒钟,振幅为35%,并在冰上放置3 mm的锥形微尖。间隔10 s后重复超声处理,共9个周期或45 s。
在4 °C下以10,000 × g超声处理样品10分钟。
将〜400μl上清液转移到新鲜的1.5 ml管中,并测量蛋白质浓度。
注意:上清液可以在-80°C下保存,直到测量。


通过添加冰冷的粘液溶解酶缓冲液将蛋白质浓度调节至1 mg / ml。
将特定粪便样品的5μl(5μg蛋白)上清液转移到96孔平底平板的三个孔中(每个5μl)。加入150μl在冰冷的粘液溶解酶缓冲液中制备的基于10 mM硝基苯的特定底物(配方2 )。对于每种测试的酶,将相应的底物添加到包含相同样品的一式三份孔中。当使用多个样本时,将重复这些过程。
每隔30分钟在37°C的读板器中测量405 nm处的吸光度。
使用已知浓度的4-硝基苯酚标准曲线(配方3 ),确定各个粘液分解酶的活性(图1)。






图1.小鼠粪便样品中随时间变化的粘液分解酶活性。如协议所述,将从健康小鼠收集的粪便样品均质化并进行处理。指示的酶瓦特的活性如在以下的方案中描述的方法中的粪便裂解物测定。


菜谱


粘液溶解酶缓冲液(pH 7.25)
储备溶液工作溶液100毫升                         

1 M Tris 50毫米                         

100万KC l 100 mM                         

1 M氯化镁2 10 mM                         

溶菌酶5-10毫克                         

12%的Triton X-100 100 μ升                         

脱氧核糖核酸酶5-10毫克                         

蛋白酶抑制剂一粒                         

硝基苯基连接的底物及其相应的粘液溶解酶
硝基苯基连接的底物


粘液溶解酶


4-硝基苯基α-D-吡喃半乳糖苷


α-半乳糖苷酶


植物聚糖


4-硝基苯基N-乙酰基-β-D-氨基葡萄糖


β-N-乙酰氨基葡萄糖苷


粘蛋白


4-硝基苯基β-D-吡喃葡萄糖苷


β-葡萄糖苷酶


植物聚糖


对硝基苯基α-L-呋喃核糖苷


α-岩藻糖苷酶


粘蛋白


对硝基苯基β-D-吡喃吡喃糖苷


β-木糖苷酶


植物聚糖


注意:我们上面提到了硝基苯连接的底物,我们在图1中使用了这种底物。


4- Nitrophen Ö升(4NP)标准曲线
4-硝基苯酚(4NP)是对硝基苯基连接的底物的酶促产物。在粘液溶解酶与其底物反应期间产生的4NP量对应于粘液溶解酶活性(配方2)。4NP提供黄色,可以在405 nm处用分光光度法测量。因此,可以使用4NP的标准曲线来测量粘液溶解酶及其对硝基苯基连接的底物的反应混合物中的粘液溶解酶活性。


一种。通过将0.0139 g(13.9 mg)4NP溶于1 ml甲醇中,制备100 mM 4NP(MW = 139.11)储备液。     

b。准备1 mM 4NP(从100 mM 4NP)的工作储备液,用粘蛋白酶测定缓冲液进行标准曲线分析。     

C。从在粘蛋白酶测定缓冲液中制备的工作储备液(1 mM)中制备一系列4NP浓度,范围从0到1,000 µM(或0到1 mM)。     

d。将150 µl每种标准溶液加到包括空白的96孔板中,并在37°C下用分光光度计在405 nm处读取吸光度。     

e。在X轴上绘制具有已知标准浓度的标准曲线,在Y轴上绘制吸光度。     



致谢


该协议在Desai等人先前发表的研究中进行了较小的修改。(2016)。我们要感谢UT西南动物资源中心(ARC)对我们的小鼠群体的维护和照顾。Ť他的工作是预防癌症和得克萨斯州的研究所(CPRIT)个别研究者奖(RP200184)和UT西南资金给予支持HZ


利益争夺


作者宣称没有利益冲突。


伦理


这项研究已由机构动物护理和使用委员会(IACUC;批准号2016-101683)批准,并根据IACUC指南进行。

参考


Atuma,C. ,Strugala,V. ,Allen,A。和Holm,L。(2001)。粘附的胃肠粘液凝胶层:体内的厚度和物理状态。Am J胃肠胃肠道疾病280 (5):G922-929。
卡罗尔,IM ,长安,Y ^ h ,公园,J. ,萨特,RB和零关系Ringel,Y. (2010)。腹泻型肠易激综合症患者的肠腔内和粘膜相关的肠道菌群。肠病2 (1):19。
Corfield ,AP,Wagner ,SA,Clamp,JR ,Kriaris,MS和Hoskins,LC (1992)。人结肠中的粘蛋白降解:粪便细菌菌株产生唾液酸酶,唾液酸O-乙酰酯酶,N-乙酰神经氨酸裂解酶,芳基酯酶和糖硫酸酯酶活性。感染免疫60 (10):3971-3978 。
德赛,MS ,Seekatz,AM ,Koropatkin,NM ,镰,N. ,希基,C. A. ,沃尔特,M.,Pudlo,NA ,北本,S. ,Terrapon,N. ,穆勒,A. ,杨, VB ,Henrissat,B. ,Wilmes,P. ,Stappenbeck,TS ,Nunez,G.和Martens,EC (2016)。膳食纤维缺乏的肠道菌群会降低结肠粘液屏障并增强病原体敏感性。单元格167 (5):1339-1353 e1321。
Derrien,M. ,van Passel,MW ,van de Bovenkamp,JH ,Schipper,RG ,de Vos,WM和Dekker,J. (2010)。人口腔和消化道中的粘蛋白-细菌相互作用。肠道微生物1 (4):254-268。
Hansson,GC(2012)。粘液层在肠道感染和炎症中的作用。Curr Opin Microbiol 15 (1):57-62 。
约翰逊(ME),拉尔森(JM)和汉森(G.C .)(2011)。结肠的两个粘液层由MUC2粘蛋白组成,而外层是宿主-微生物相互作用的立法者。美国国家科学院院刊108增刊1 :4659-4665。
Ĵ ohansson,ME ,菲利普森,J 。M. ,Petersson,J. ,Velcich,A. ,Holm,L。和Hansson,GC(2008)。结肠中两个Muc2粘蛋白依赖性粘液层的内部不含细菌。美国国家科学院院刊105 (39):15064-15069 。
Juge,N.(2012年)。微生物黏附素对胃肠粘液。趋势微生物学20 (1):30-39 。
Linden,SK ,Sutton,P. ,NG ,Karlsson ,V.Korolik和MA ,McGuckin(2008)。粘蛋白在黏膜屏障中被感染。粘膜免疫1 (3):183-197。
的Lombard,V. ,Golaconda Ramulu,H. ,Drula,E. ,科蒂尼奥,PM和Henrissat,B。(2014)。2013年的碳水化合物活性酶数据库(CAZy)。NucleicAcids Res 42 :D490-495 。
McGuckin,MA ,Linden,SK ,Sutton,P.和Florin,TH(2011)。粘蛋白动力学和肠道病原体。Nat Rev Microbiol 9 (4):265-278。
Pickard,JM ,Zeng,M.Y. ,Caruso,R。和Nunez,G. (2017年)。肠道菌群:在病原体定植,免疫反应和炎症性疾病中的作用。免疫评论279 (1):70-89 。
Png,CW ,Linden,SK ,Gilshenan,KS ,Zoetendal,EG ,McSweeney,CS ,Sly,LI ,McGuckin,MA和Florin,TH(2010)。IBD粘膜患病率增高的粘液溶解细菌会增加其他细菌对粘蛋白的体外利用。美国胃肠病杂志105 (11):2420-2428。
Prizont,R. (1982年)。致病性志贺菌降解肠道糖蛋白的研究。感染免疫36 (2):615-620 。
              Pullan RD ,Thomas,GA ,Rhodes,M. ,Newcombe,RG ,Williams,GT ,Allen,A。和Rhodes,J。(1994)。人结肠黏膜上黏附的黏液凝胶的厚度及其与结肠炎的关系。肠35 (3):353-359
Round,JL和Mazmanian,SK (2009)。肠道菌群会影响健康和疾病期间的肠道免疫反应。Nat Rev Immunol 9 (5):313-323。
Sheng YH ,Hasnain,SZ ,Florin,TH和McGuckin,MA(2012)。黏蛋白在肠炎性疾病和结肠直肠癌中。胃肠肝病学杂志27 (1):28-38 。
Sommer,F.和Backhed,F. (2013年)。肠道菌群-宿主发育和生理学的大师。Nat Rev Microbiol 11 (4):227-238。
Tailford,LE ,Crost,EH ,Kavanaugh,D. ,Juge,N. (2015年)。粘蛋白聚糖在人类肠道微生物组中觅食。前遗传学6 :81。
桑顿,DJ ,卢梭,K和McGuckin ,MA(2008)。气道粘液中聚合粘蛋白的结构和功能。生理学年鉴70 :459-486 。
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  2. Khan, S., Waliullah, S., Godfrey, V., Khan, M. A. W., Ramachandran, R. A., Cantarel, B. L., Behrendt, C., Peng, L., Hooper, L. V., Zaki, H. (2020). Dietary simple sugars alter microbial ecology in the gut and promote colitis in mice. Sci Transl Med 12: eaay6218.
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