A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles
Summary
Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.
Abstract
Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.
Introduction
Glycogen is a highly branched polysaccharide that consists of glucosyl residues and also a small but significant amount of proteins, in which all glucosyl residues are linked together via α-1,4-glycosidic bonds in linear chains and α-1,6-glycosidic bonds at branching points1. The structure of glycogen particles is generally divided into three hierarchies: 1) short-chain oligomers, 2) spherical β particles (~20 nm in diameter), and 3) large rosette-shaped α particles aggregated together by β particles, the diameter of which ranges roughly up to 300 nm. Recently, it has been found that glycogen α particles have two structural states in eukaryotes, i.e., a fragile state and a stable state. Here, fragility means the dissociation of larger α particles into smaller β particles in the presence of a chaotropic agent like DMSO2. Further analyses found that glycogen α particles in the diabetic liver are consistently fragile3 and the fragile α particles degrade much faster than stable α particles4. Thus, glycogen structural fragility may exacerbate hyperglycemic conditions in diabetes2,4, which makes fragile α-particle a potential pathological biomarker of diabetes at a molecular level. However, the existence of glycogen α particles in prokaryotes is only sporadically reported5, and there is no report of the two different structural states of glycogen α particles in bacteria.
In order to understand the physiological functions of bacterial glycogen particles, it is essential to determine the fine structure of glycogen molecules, which requires glycogen isolation with maximal yield and minimal degradation1. So far, various techniques have been developed for glycogen extraction, including but not limited to hot water extraction, trichloroacetic acid (TCA) extraction, and hot alkaline (potassium hydroxide, KOH) extraction6. In addition, another method that is commonly used for eukaryotic glycogen isolation, the sugar density gradient ultra-centrifugation (SDGU) method, was also reported for bacterial glycogen isolation in Selenomonas ruminantium and Fibrobacter succinogenes7,8. Although the pros and cons of these methods have been widely discussed in eukaryotic studies9,10, there are rarely comparative studies of glycogen fine structures isolated via different extraction methods in bacteria from the perspective of glycogen particle structures.
In this study, this issue has been addressed by using Escherichia coli BL21(DE3) as the model organism. A total of four glycogen extraction methods were compared, namely, TCA-precipitated hot water extraction (TCA-HW), TCA-precipitated cold-water extraction (TCA-CW), hot 30% KOH solution extraction (KOH-HW), and cold-water extraction using sucrose density gradient ultracentrifugation (SDGU-CW). Glycogen particle size distribution was then measured via size exclusion chromatography (SEC) while chain-length distribution was detected via fluorophore-assisted carbohydrate electrophoresis (FACE), both of which were used for assessing the quality of extraction methods. In addition, the stability and fragility of bacterial glycogen α particles were also compared among the various extraction methods by comparing particle size distribution before and after treating with the commonly used chaotropic agent, dimethyl sulfoxide (DMSO). The detailed procedures for glycogen extraction and structural characterization are presented below. In summary, the SDGU-CW method has the best overall effect in terms of glycogen structural integrity and is, therefore, recommended for bacterial glycogen extraction in future relevant studies.
Protocol
Representative Results
Discussion
Glycogen is an important energy reserve that has been identified in many bacteria16. To dissect the physiological functions of glycogen particles, it is essential to have a better understanding of the fine structure of glycogen molecules. So far, a variety of methods have been developed to extract glycogen from bacterial culture. However, different size distributions of glycogen particles have been observed from different extraction methods, which suggests damaged glycogen structure. Thus, it is n…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We are greatly thankful to Professor Robert G. Gilbert from the University of Queensland and Yangzhou University who provided insights and expertise that greatly assisted the completion of this study. We acknowledge the financial support of the National Natural Science Foundation of China (No. 31900022, No. 32171281), Natural Science Foundation of Jiangsu Province (No. BK20180997), Young Science and Technology Innovation Team of Xuzhou Medical University (No. TD202001), and Jiangsu Qinglan Project (2020).
Materials
| Equipment | |||
| Agilent 1260 infinity SEC system | Agilent | 1260 infinity II | Particle size distribution |
| Analytical column | PSS | 10-1000 | – |
| Centrifuge | Eppendorf | 5420 | – |
| Filter membrane | Cambio | Km-0220 | – |
| Fluorescence-assisted capillary electrophoresis system | Beckman Coulter | – | Chain length distribution |
| Freeze dryer | Xinzhi | SCIENTZ-10N | Lyophilization of bacteria and glycogen |
| Freezer | Thermo Fisher | Forma 900 | Sample storage |
| Guard column | PSS | SUPPERMA | – |
| Incubator | Thermo Fisher | PR505750R-CN | – |
| Low-speed large-capacity centrifuge | Hexi | HR/T20MM | Sample centrifugation |
| Multiskan FC microplate reader | Thermo Fisher | 1410101 | – |
| Optima XPN ultracentrifuge | Beckman | XPN-100/90/80 | For glycogen |
| Oscillator | Xinbao | SHZ-82 | – |
| PA-800 Plus System | Beckman Coulter | A66528 | – |
| pH meter | Mettler Toledo | FE28 -TRIS | – |
| Refractive index detector | Wyatt | Optilab T-rEX | – |
| Refrigerator | Haier | BCD-406WDPD | – |
| Thermomixer | Shanghai Jingxin | JXH-100 | Sample incubation |
| Transmission electron microscope | Hitachi Corporation | H-7000 | Glycogen particle morphology |
| Ultracentrifuge tube | Beckman | 355651 | – |
| Ultrasonic cell crusher | Ningbo Xinzhi | Scientz-IID | Bacteria disruptor |
| Ultrasonic oscillating water bath | Jietuo | JT-1027HTD | – |
| Vortex mixer | Tiangen | OSE-VX-01 | – |
| Water system | Merck Millipore | H2O-MM-UV-T | Deionized water |
| Material | |||
| 8-Aminopyrene-1,3,6-Trisulfonic Acid Trisodium Salt | Sigma-Aldrich | 196504-57-1 | – |
| Absolute ethanol | Guoyao | 10009228 | – |
| Agar powder | Solarbio | A1890 | – |
| Alpha-amylase | Megazyme | E-BLAAM-40ML | – |
| Amyloglucosidase | Megazyme | E-AMGDF-40ML | – |
| cOmplete Mini | Roche | 4693159001 | – |
| D-(+)Glucose | Sigma-Aldrich | G8270-1kg | – |
| D-Glucose Assay Kit (GOPOD Format) | Megazyme | K-GLUC | Glycogen quantification |
| Dimethyl sulfoxide | Vicmed | Vic147 | Chaotropic agent |
| E. coli BL21(DE3) | Tiangen | CB105-02 | – |
| Ethylene diamine tetra-acetic acid | Vicmed | Vic1488 | – |
| Glacial acetic acid | Guoyao | 10000218 | – |
| Glycerol | Guoyao | 10010618 | Bacterial storage |
| Hydrochloric acid | Guoyao | 10011008 | – |
| Hydroxymethyl aminomethane | Sigma-Aldrich | V900483-500g | – |
| Isoamylase | MegaZyme | 9067-73-6 | Glycogen debranch |
| Lithium chloride | Sigma-Aldrich | 62476-100g | – |
| M9, Minimal Salts, 5× | Sigma-Aldrich | M6030-1kg | Bacterial culture |
| Potassium hydroxide | Guoyao | 10017008 | – |
| Pullulan standard | PSS | – | – |
| Sodium acetate trihydrate | Guoyao | 10018718 | – |
| Sodium azide | Sigma-Aldrich | 26628-22-8 | – |
| Sodium chloride | Guoyao | 10019318 | Bacterial culture |
| Sodium cyanoborohydride | Huaweiruike | hws001297 | – |
| Sodium diphosphate | Sigma-Aldrich | 71515-250g | – |
| Sodium Fluoride | Macklin | S817988-250g | – |
| Sodium hydroxide | Guoyao | 10019762 | – |
| Sodium nitrate | Guoyao | 10019928 | – |
| Sodium pyrophosphate | Sigma-Aldrich | V900195-500g | – |
| Sucrose | Guoyao | 10021463 | – |
| Trichloroacetic acid | Guoyao | 40091961 | – |
| Tryptone | Oxoid | LP0042 | Bacterial culture |
| Yeast Extract | Oxoid | LP0021 | Bacterial culture |
Riferimenti
- Wang, L., et al. Molecular structure of glycogen in Escherichia coli. Biomacromolecules. 20 (7), 2821-2829 (2019).
- Deng, B., et al. Molecular structure of glycogen in diabetic liver. Glycoconjugate Journal. 32 (3-4), 113-118 (2015).
- Hu, Z., et al. Diurnal changes of glycogen molecular structure in healthy and diabetic mice. Carbohydrate Polymers. 185, 145-152 (2018).
- Nawaz, A., Zhang, P., Li, E., Gilbert, R. G., Sullivan, M. A. The importance of glycogen molecular structure for blood glucose control. iScience. 24 (1), 101953 (2021).
- Rashid, A. M., et al. Assembly of α-glucan by GlgE and GlgB in mycobacteria and streptomycetes. Biochimica. 55 (23), 3270-3284 (2016).
- Wang, L., et al. Recent progress in the structure of glycogen serving as a durable energy reserve in bacteria. World Journal of Microbiology and Biotechnology. 36 (1), 14 (2020).
- Kamio, Y., Terawaki, Y., Nakajima, T., Matsuda, K. Structure of glycogen produced by Selenomonas ruminantium. Agricultural and Biological Chemistry. 45 (1), 209-216 (1981).
- Gong, J., Forsberg, C. W. Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase. Journal of Bacteriology. 175 (21), 6810-6821 (1993).
- Mojibi, N. Comparison of methods to assay liver glycogen fractions: the effects of starvation. Journal of Clinical and Diagnostic Research. 11 (3), (2017).
- Orrell, S. A., Bueding, E. A comparison of products obtained by various procedures used for the extraction of glycogen. The Journal of Biological Chemistry. 239, 4021-4026 (1964).
- Tan, X., et al. Proteomic investigation of the binding agent between liver glycogen beta particles. ACS Omega. 3 (4), 3640-3645 (2018).
- Hu, Z., et al. Diurnal changes of glycogen molecular structure in healthy and diabetic mice. Carbohydrate Polymers. 185, 145-152 (2018).
- Sullivan, M. A., Harcourt, B. E., Xu, P., Forbes, J. M., Gilbert, R. G. Impairment of liver glycogen storage in the db/db animal model of type 2 diabetes: a potential target for future therapeutics. Current Drug Targets. 16 (10), 1088-1093 (2015).
- Deng, B., et al. Molecular structure of glycogen in diabetic liver. Glycoconjugate Journal. 32 (3-4), 113-118 (2015).
- Sullivan, M. A., et al. Molecular structural differences between type-2-diabetic and healthy glycogen. Biomacromolecules. 12 (6), 1983-1986 (2011).
- Wang, L., et al. Systematic analysis of metabolic pathway distributions of bacterial energy reserves. G3 Genes|Genomes|Genetics. 9 (8), 2489-2496 (2019).
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