Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method
Summary
In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen.
Abstract
Glycogen particles are branched polysaccharides composed of linear chains of glucosyl units linked by α-1,4 glucoside bonds. The latter are attached to each other by α-1,6 glucoside linkages, referred to as branch points. Among the different forms of carbon storage (i.e., starch, β-glucan), glycogen is probably one of the oldest and most successful storage polysaccharides found across the living world. Glucan chains are organized so that a large amount of glucose can quickly be stored or fueled in a cell when needed. Numerous complementary techniques have been developed over the last decades to solve the fine structure of glycogen particles. This article describes Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). This method quantifies the population of glucan chains that compose a glycogen particle. Also known as chain length distribution (CLD), this parameter mirrors the particle size and the percentage of branching. It is also an essential requirement for the mathematical modeling of glycogen biosynthesis.
Introduction
Glycogen, used as carbon and energy storage, is a homopolymer of glucose consisting of linear chains of glucosyl units linked by (1 → 4)-α bonds and attached through (1 → 6)-α glycosidic bonds or branching points. They appear as β- and α-particles in the cytosol of a wide range of organisms. β-particles are tiny water-soluble particles mainly observed in prokaryotes. Their diameter ranges from 20-40 nm, likely dictated by the glycogen metabolizing enzymes and steric hindrance1,2.
First described in animal cells, the larger α-particles display up to 300 nm in diameter with a cauliflower-like shape. This particular organization may originate from the aggregation of several β-particles or may arise by budding out of a single β-particle3. Interestingly, a recent study has reported the presence of α-particles in Escherichia coli4. However, unlike α-particles from animal cells, the latter falls apart quickly during the extraction process, which may explain the lack of data in the literature4. The appearance of α-particles in eukaryotes and prokaryotes involves phylogenetically unrelated glycogen metabolizing enzymes5. This raises questions regarding the function of such particles and the nature of potential cross-linker agents between β-particles5.
Although two opposing mathematical models were proposed for glycogen molecule formation6,7,8,9, it is generally accepted that β-particles have evolved in response to their metabolic function as a highly efficient fuel reserve for the rapid release of large amounts of glucose. A large body of evidence indicates that glycogen properties such as digestibility and solubility in water are correlated with the average chain length (ACL), which will then dictate the percentage of branching points and the particle size2,6,7,8,10,11. ACL is defined by the ratio between the total number of glucose residues and the number of branching points. Typically, the ACL values vary from 11-14 and 7-23 glucose residues in eukaryotes and prokaryotes, respectively10. In humans, several glycogen disorder diseases are due to abnormal glycogen accumulation. For instance, Andersen's disease is associated with the deficient activity of a glycogen branching enzyme, resulting in the accumulation of abnormal glycogen11. In prokaryotes, cumulative studies suggest that ACL is a critical factor impacting the degradation rate of glycogen and bacterial survival ability12,13. It has been reported that bacteria synthesizing β-particles with a low ACL value degrade more slowly and therefore withstand starvation conditions longer. Thus, knowledge of the architecture of β-particles is essential for understanding the formation of abnormal glycogen particles in human glycogen storage diseases and prokaryotes survival in a nutrient-deficient environment.
Since the first isolation of glycogen from dog liver by the French physiologist Claude Bernard in the late nineteenth century14, many techniques were developed to characterize glycogen particles in detail. For instance, transmission electron microscopy for glycogen morphology (α- or β-particles)15, proton-NMR spectrometry for determining the percentage of α-1,6 linkages16, size exclusion chromatography with multi-detectors for inferring the molecular weight, Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)17 or High-Performance Anion exchange chromatography with Pulsed Amperometric Detection (HPAEC-PAD) for both chain length distribution (CLD) and ACL determination18.
This work focuses on the fluorophore-assisted carbohydrate electrophoresis method, which relies on the reductive amination of the hemiacetal group by primary amine function. Historically, 8-amino-1,3,6-naphthalene trisulfonic acid (ANTS) was first used for labeling. Later, it was replaced by the more sensitive fluorophore, 8-amino 1,3,6 pyrene trisulfonic acid (APTS)19.
Figure 1: The reductive amination reaction with 8-amino 1,3,6 pyrene trisulfonic acid (APTS). Reductive amination reaction of the hemiacetal group by primary amine function of 8-amino 1,3,6 pyrene trisulfonic acid (APTS) under reductive conditions Please click here to view a larger version of this figure.
As depicted in Figure 1, the hemiacetal function of the reducing end of a glucan chain interacts with the primary amine of APTS under reducing conditions. The sulfonic groups of APTS carry negative charges that enable the separation of glucan chains according to their degree of polymerization (DP). The reductive amine reaction is highly reproducible and efficient. An average efficiency labeling of 80% is obtained for DP3 to DP135 and up to 88% and 97% for maltose (DP2) and glucose, respectively17,20. Because one molecule of APTS reacts with the reducing end of each glucan chain, individual chains could be quantified and compared to each other on a molar basis.
Protocol
1. Incubation with debranching enzymes
- Mix 200 µL of purified glycogen at 0.5-2 mg/mL with 200 µL of 100 mM of acetate buffer (pH 4.8). Add 2 µL of isoamylase (180 U/mg of protein) and 1.5 µL of pullulanase (30 U/mg of protein) (see Table of Materials), mix gently by pipetting up and down, and incubate at 42 °C for 16 h in a 1.5 mL tube.
NOTE: Degradation or amylase contamination might occur during the glycogen purification process. To appreciate the presence of free malto-oligosaccharides, samples can be incubated without the debranching enzyme cocktail in parallel. A standard (e.g., maltoheptaose) is included in the analysis to determine the relationship between the elution time and the degree of polymerization. - Stop the reactions by incubating at 95 °C for 5 min.
- Centrifuge at 16,100 x g for 5 min at room temperature to pellet and remove any insoluble material.
- Remove the supernatants using a pipette and transfer them to new annotated tubes. Desalt the supernatants by adding the equivalent of 100 µL of anion/cation exchange resin beads (AG-501-X8, see Table of Materials) and agitate.
- Regularly agitate the beads for 5 min. Collect the samples by pipetting and place them in new annotated tubes.
- Freeze-dry or use a vacuum evaporator set up (see Table of Materials) at 30 °C to dry the samples.
- Store dried samples at room temperature or at -20 °C.
NOTE: The samples can be stored for 1 month.
2. Reductive amination
- Mix the dried samples with 2 µL of 1 M sodium cyanoborohydride in tetrahydrofuran (THF) and 2 µL of APTS (5 mg of APTS resuspended in 48 µL of 15% acetic acid) (see Table of Materials).
- Incubate at 42 °C for 16 h in the dark.
CAUTION: Sodium cyanoborohydride is handled with adapted personal protection equipment and under a chemical hood. Inhalation and contact with skin are highly toxic and can be fatal, causing severe skin burns and eye damage. Highly toxic gases can be generated when mixing sodium cyanoborohydride with acetic acid. In contact with water, sodium cyanoborohydride releases flammable gases, which may ignite spontaneously. Concentrated samples are handled under a chemical hood and using adapted personal protection equipment starting from this step.
3. FACE analysis
- Add 46 µL of ultrapure water to each sample.
- Dilute the samples directly to 1/50 in micro vials of 100 µL by adding 1 µL of the sample to 49 µL of ultrapure water. Keep the samples in the dark while setting the FACE (5-10 min).
- Perform reverse polarity electrophoresis with a capillary electrophoresis instrument with a laser-induced fluorescence (LIF) detector (see Table of Materials). Set the polarity to "reverse mode" for separation, set the LIF at 488 nm emission wavelength and the detector at 512 nm.
- Set the Injection time for 10 s and the injection pressure at 0.5 psi.
- Carry out the APTS-labelled-glucans separation at 30 kV in a bare fused silica capillary of 60.2 cm in length with an inner diameter of 50 µm (375 µm outer diameter) in the N-linked carbohydrate separation buffer diluted to 1/3 in ultrapure water (see Table of Materials).
NOTE: The N-linked carbohydrate separation buffer is replaced every 20 runs.
4. DATA processing
- Export the ".ASC "and ".CDF "files containing the electropherogram profile and integration data, respectively.
- Open the .ASC file and draw the relative fluorescence unit according to the time chart.
- Open the .CDF file, proceed with a first automatic integration and adjust the following parameters: width; valley to valley integration; minimum area.
- Check and correct any improper integration event manually.
Representative Results
Determination of the average chain length of glycogen
Figure 2 represents the workflow required to infer the chain length distribution and the average chain length (ACL) of glycogen.
Figure 2: Workflow to determine the chain length distribution (CLD) and average chain length. Please click here to view a larger version of this figure.
Figure 3 displays the electropherograms of commercial maltohexaose and debranched bovine liver glycogen. The fluorescence signals observed between 4.13-4.67 min in all experiments originated from the unreacted APTS. The elution time of labeled maltohexaose (DP6) was estimated at 8.49 min (Figure 3A). The APTS-labelled glucans of bovine glycogen were identified based on the elution time of DP6 (Figure 3B). No trace of free malto-oligosaccharide was detected in the control sample (glycogen not incubated with debranching enzymes) (Figure 3C).
Figure 3: Electropherograms of a standard and bovine liver glycogen. (A) The time elution (8.49 min) of a glucan standard, maltohexaose (DP6), was used as a reference to determine the degree of polymerization (DP) of APTS-labelled glucans released from bovine liver glycogen after the action of debranching enzyme activities (B). The inset panel shows a separation of glucan chains up to 44 DP. In parallel, untreated bovine glycogen was labeled with APTS to detect possible traces of free malto-oligosaccharides in sample (C). Please click here to view a larger version of this figure.
It can be concluded that the release of APTS-labelled glucan is due to the cleavage of branching points by both isoamylase and pullulanase activities. It should be noticed that the capillary electrophoresis profile can be redrawn in a more appropriate format to create a mosaic figure containing several profiles. To do this, DATA files containing fluorescence values are generated with "asc" extension and opened in the spreadsheet program by choosing CSV (comma-separated value) format. Unfortunately, the exported fluorescence values are not associated with the corresponding elution time. Consequently, they must be manually added according to the acquisition frequency setup on the FACE apparatus (4 Hz means one value acquisition every 0.25 s).
Peak areas were then inferred using the FACE instrument's native application or exported as a DATA file with a "cdf" extension to use another application. The area values are exported in a spreadsheet program and normalized by expressing the DP as a percentage of the total surface area (Figure 4).
Figure 4: Data normalization, chain length distribution, and average chain length value. Fluorescence peak areas were imported and normalized in a spreadsheet. The chain length distribution is shown as the percentage of DP for each DP. The average chain length (ACL) is calculated by summing each percentage chain times the corresponding degree of polymerization. Please click here to view a larger version of this figure.
Finally, the average chain length (ACL) is inferred by calculating the sum of each percentage chain times the corresponding degree of polymerization. Similar experiments were carried out in triplicate on rabbit liver glycogen (Figure 5A), on bovine liver glycogen (Figure 5B), and oyster glycogen (Figure 5C).
Figure 5: Chain length distribution of commercial glycogen. The rabbit liver (A), bovine liver (B), and oyster glycogen (C) were incubated in the presence of debranching enzymes (isoamylase and pullulanase). The APTS-labelled glucans were then separated according to their degree of polymerization (DP) using FACE analysis. Maltose (DP2), maltohexaose (DP6) maltoheptaose (DP7) represent the most abundant glucans in rabbit liver, oyster, and bovine liver glycogen, respectively. The Standard Error of Mean (SEM) was inferred from three independent experiments. Please click here to view a larger version of this figure.
The chain length distribution of rabbit liver glycogen clearly showed a higher content of short malto-oligosaccharide (DP2) than bovine liver glycogen (DP 7) or oyster glycogen (DP6). As a result, the rabbit liver glycogen possesses the lowest average chain length (ACL = 9.8) compared to bovine liver glycogen (ACL = 11.9) and oyster glycogen (ACL = 12.6). It is to be noted that those commercial glycogens are usually used for assaying the glycogen phosphorylase or glycogen synthase activity. This suggests that the determination of kinetic parameters (Vmax and Km) of glycogen metabolizing enzyme activities will vary according to the source of glycogen.
Subtractive analyses
The subtractive analysis is a simple method to compare the glucan chains distribution of two samples. For example, CLDs of glycogen produced by the wildtype (WT) Synechocystis PCC6803 strain and single isogenic glgA1 and glgA2 mutant strains were determined (Figure 6A).
Figure 6: Comparison of chain length distributions using subtractive analysis. (A) The chain length distributions of glycogen purified from cyanobacterial strains: wildtype (WT) Synechocystis PCC6803 and single isogenic glgA1 and glgA2 mutant strains were determined using FACE analysis. The Standard Error of Mean (SEM) was inferred from three independent experiments. (B) Subtractive analyses were performed by subtracting the % of each DP of WT to the % of each DP of ΔglgA1 and subtracting the % of each DP of WT to the % of each DP of DP ΔglgA2. This straightforward mathematical manipulation displays the alteration of glucan chains in mutant strains (black lines). (C) The average chain length distributions of glycogen from wildtype and mutants of Synechocystis were normalized according to the maximum peak observed for each CLD (DP6 for all samples). Two components are evidenced by plotting the normalized CLD on a logarithmic scale (Nde (DP)). Each component indicates a different mechanism of growth stoppage (for more information, see Reference2). Please click here to view a larger version of this figure.
To recall, most cyanobacteria strains possess two genes encoding glycogen synthase activities: GlgA1 and GlgA221. Both enzymes transfer the residue glucose of ADP-glucose onto the non-reducing ends of linear glucan chains. As depicted in Figure 6A, it is challenging to compare samples by only looking at the chain length distribution profiles. The subtractive analysis consists of subtracting the percentage of each DP between samples (Figure 6B). The subtractive analysis of %DP of WT minus % of DP ΔglgA1 mutant reveals an excess of DP3, 4, and 5 (negative values) and a decreased content of DP 10-20. In contrast, the subtractive analysis between %DP of WT and % of DP ΔglgA2 mutant indicates an opposite effect. Because the subtractive analysis profiles are different between GlgA1 and GlgA2, this suggests a specific function of each glycogen synthase isoform in glycogen biosynthesis21.
It is important to note that subtractive analysis is only applicable to experiments that involve a reference sample performed in parallel with the samples. Otherwise, subtractive analysis can be empirical since it lies on the normalized CLD of the reference. In 2015, Deng and collaborators, who investigated stopping mechanisms of glycogen growth in mice and humans, proposed an alternate plotting and interpreting glycogen CLDs to address this issue. This plot uses the maximum peak area to normalize each CLD. The data are then plotted on a logarithmic scale highlighting two components. The latter illustrate two different mechanisms for chain elongation stopping2. By drawing lines fitting to the higher DP component, absolute parameters (i.e., slopes and intercepts of the lines) can be used for CLD comparison without normalization to a reference profile. CLDs of the wildtype (WT) Synechocystis PCC6803 strain and the single isogenic glgA1 and glgA2 mutants were plotted on a log scale, and fitting lines were determined for each profile (Figure 6C). The first component was highly similar between samples, peaking with a maximum at DP 6. This illustrates that the branching enzyme explicitly produces a maximum of such chains. The second component appeared as a broad shoulder, which was already described for mice and human glycogens2. The inclination of the second component arose at a higher DP in ΔglgA1 and the slope of the corresponding fitting line (red line) was lower than the wildtype profile. Thus, the lack of GlgA1 slows down the arrest of chain elongation during biosynthesis that was proposed to occur by steric hindrance for mice and human glycogen2. These data suggest that the remaining elongating enzyme (i.e., GlgA2) produces longer chains before chain crowding. In ΔglgA2, the opposite effect was observed with a more dramatic drop of the fitting line, corroborating that the chains produced by the remaining GlgA1 are overall shorter than those synthesized by GlgA2 alone before steric hindrance. This analysis suggests that both isoforms possess distinct kinetics and/or that their respective concertation with branching enzyme activity differ.
Discussion
The physicochemical properties of glycogen particles (e.g., size, morphology, solubility) are directly associated with the length of glucans composing the particles. Any imbalance between biosynthetic and catabolic enzymes results in the alteration of the chain length distribution and, per se, the accumulation of abnormal glycogen that can be hazardous for the cell11. The FACE analysis is a method of choice to determine glycogen's chain length distribution (CLD). As depicted in Figure 2, the determination of CLD allows the inference of the average chain length value of glycogen (ACL), which mirrors the structure of glycogen particles. Animal glycogens with high ACL values are associated with the appearance of abnormal particles. The subtractive analysis is a helpful method for comparing two glycogen samples from different genetic backgrounds (mutant versus wildtype). By plotting on a logarithmical scale CLDs normalized to the maximum peak, on the other hand, has the advantage of comparing CLD independently of a reference and gave us information on the growing glycogen mechanism.
In addition, FACE analysis is a powerful technique for characterizing the catalytic properties of glycogen metabolizing enzymes. For instance, all glycogen branching enzymes cleave (1 → 4)-α linkages and transfer oligomaltosyl groups onto a (1 → 6)-α position or branching points. Branching enzymes are distinguishable due to their affinity for polysaccharides (e.g., amylopectin, glycogen) and the length of transferred glucans despite the similar catalytic mechanism22. Thus, various sources of branching enzymes (human, plant, bacteria) can be characterized and classified through a series of incubation experiments and CLD comparisons using FACE analysis23.
As mentioned in the introduction, HPAEC-PAD is also an alternative method for determining the chain length distribution18. Both techniques require the complete hydrolysis of (1 → 6)-α linkages or branching points by isoamylase-type debranching enzymes before separating the pool of linear glucans according to their degree of polymerization (DP). However, the HPAEC-PAD method harbors two disadvantages compared to FACE: (1) the amperometric pulse response decreases as the glucan chains increase, which does not provide quantitative information. This mass bias issue can be circumvented using HPAEC-ENZ-PAD that involves a post-column enzyme reactor between the anion exchange column and PAD24. The column enzyme reactor hydrolyzes malto-oligosaccharides into glucose residues allowing a constant pulse amperometric response. (2) The HPAEC-PAD allows the separation of glucan chains with a degree of polymerization up to 70. Although the resolution is enough for determining the CLD of glycogen samples, FACE separates chains with DPs up to 150, suitable for starch samples17. It is essential to keep in mind that both HPAEC-PAD and FACE analysis have pros and cons. For instance, the amination reaction requires a free hemiacetal group to react with the primary amine function of the APTS. This implies that APTS labeling cannot be used for glucan chains lacking reducing ends (e.g., inulin). The HPAEC-PAD method does not require the presence of reducing ends. A second interesting aspect of the HPAEC-PAD method is that the anion exchange column can be loaded with a few milligrams of linear glucans, allowing the purification of malto-oligosaccharide with a specific DP or 14C-radiolabeled malto-oligosaccharide for enzymatic assay18,25. Finally, although mass spectrometry (e.g., MALDI-TOF) is a fast and sensitive technic to determine chain length distribution, this technic appears to be less reproducible. It overestimates the amount of long glucan chains26. Nevertheless, the latter can be used for a specific application such as MALDI imaging to map the presence of glycogen across cellular tissue27.
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the CNRS, the Université de Lille CNRS, and the ANR grants "MathTest" (ANR-18-CE13-0027).
Materials
| AG-501-X8 Resin | BioRad | #1436424 | Storage Room temperature |
| 100 mM sodium acetate buffer, pH 4.8 | Dissolve 0.82 g of sodium acetate in 80 mL of water. Adjust pH to 4.8 with acetic acid and complete the volume to 100 mL with water—storage at room temperature. | ||
| APTS stock solution | Merck | 09341-5MG | Dissolve 5 mg of APT in 48 mL acetic acid 0.2 M. Storage at -20 °C. |
| Capillary | Sciex Separations, Les Ulis, France | ||
| Chromeleon 6.80 SR8 Build 2623 | Thermofisher | select :File>import/restore>ANDI/chromatography in the open window: select "add" select cdf file > import > next Choose the folder where your file will downloaded in Chromeleon software> finish click on QNT-Editor> parameter "Min Area" select "Range" 0.05 [Signal]*min. |
|
| Excel | Microsoft | Open Excel> New file> save the file > File menu click on Import > In the open window choose "csv." as type file > select your asc file > a new window appears Step 1: choose Macintosh or Window and then used default setting for the steps 2 and 3. > Y values appear in column A> Manually add a Time column by incrementing 0.25 second to each cell that corresponds to the frequency (4Hz) for acquisition data . Then plot the graph. | |
| Free-Dry apparatus | Christ | alpha 2-4 LO plus | before the freezing-drying process, samples are stored at -80 °C for 1 h. |
| Isoamylase | Megazyme | E-ISAMY | 180 U/mg of protein |
| Maltoheptaose | Merck | M7753 | |
| N-Linked carbohydrate separation buffer | Sciex Separations, Les Ulis, France | 477623 | Storage at 4 °C |
| Pullulanase | Megazyme | E-PULKP | 30 U/mg of protein |
| Sodium cyanoborohydride | Sigma-Aldrich | 296813-100ML | 1 M Sodium cyanoborohydride in THF |
| Vaccum-evaporator | Eppendorf | Concentrator 5301 | Set the temperature at 30 °C. Centrifuge until the samples are dried |
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