Isolation and Analysis of Urinary Glycosaminoglycans

Materials and Methods

Preparation of urinary polyanionic macromolecules

Reagents
0.025M sodium acetate buffer
Propan-1-ol
5%cetylpyridinium chloride
Sodium acetate-saturated ethanol

1. A simple of urine(~50ml) was obtained from one of the classmate. The simple was centrifuged at 3000rpm, 5min to remove the dead cell.
2. 23ml of supernatant was obtained from decantation. 30ml of 0.025M sodium acetate buffer, pH 5.8 was added to dilute the simple. 4ml of 5% cetylpyridinium chloride was added. The mixture was mixed well and allowed to precipitate at 4°C for 1.5 hr.
3. The tube was centrifuged at 4500rpm for 10min at 4°C. (About 3ml of white precipitate was formed at the bottom of the yellow solution.) The supernatant was decanted off until 5ml of solution was remaining. The solution was centrifuged at 4500rpm, 5min again.(A white pellet of volume 1 ml was formed at the bottom.) All the supernatant was discarded.
4. 2ml of propan-1-ol was added to dissolve the pellet. 2ml of the resulting solution was transferred to a 15ml centrifuge tube. 8 ml of sodium acetate saturated ethanol was added to the centrifuge tube. The solution was mixed well and allowed to precipitate at 4°C for 50 minutes.(A very pale yellow pellet was formed from clear supernatant)
5. The tube was centrifuged at 5min at 4°C. (Size of pellet reduced and became brown). The supernatant was discarded and 1 ml of 100% ethanol was added as to wash the pellet. (The pellet was firmly attached on the centrifuge tube.) The tube was centrifuged again at 4500rpm, 5min, 4°C.(A small brown pellet was formed with clear supernatant.) The supernatant was discarded and remaining ethanol was drained off. The pellet was dried by sucking.

Preparation of extracts for fluorescent derivatization

Reagents
100mM ammonium acetate, pH7.0
Chondroitinase ABC(10U/ml, stock)

1. The extract was dissolved in 100μl ammonium acetate buffer. The resulting solution was aliquoted 40μl each into a separate 1.5ml tube. The remaining 20μl was kept at -20°C until analysis with agarose gel electrophoresis on day 2.
2. One of the 40μl-aliquots with 10μl chondroitinase ABC(100mU/ml) for 2 hour, 37°C; 10μl buffer was added to the other aliquot and treated as control.
3. The resistant material was precipitated in 4 volumes of NaAc/EtOH(4°C, 1hr) at the end of the incubation. The precipitate which contains chondroitinase and chondroitinase-resistant materials was spun down. The supernatant was transferred to a separate 1.5ml tube and the EtOH was removed by spinning in a Speedvac.

Derivatization of oligosaccharides components of UPM with AMAC

Reagents
AMAC, 12.5mM in acetic acid-DMSO(3:17, v/v). This solution may be stored at -70°C for 2 weeks. Sodium cyanoborohydride, 1.25M in solution in ultrapure water. This solution must be made fresh and used within a few minutes.

1. 10μl of AMAC(500nmoles) was added to each of the disaccharide products to be derivatized. 10μl of sodium cyanoborohydride (50,000 nmoles) was added. The solution was incubated for 16hr at 37°C in the dark.
2. 10μl of glycerol(30%) was added to the reaction mixture. The solution was stored at -70°C in dark until FACE on day2.

Polyacrylamide gel electrophoresis (FACE)

Reagents
Stock acrylamide solution: 30% (w/v) acrylamide, 0.8% (w/v) N, N’-methylenebisacrylamide
10%(w/v) Ammonium persulfate, freshly prepared
N,N,N’,N’-tetramethylenediamine(TEMED)
Electrophroetic buffer: 0.1M tris-borate, pH8.3
Stock gel buffer: same as above
Bromophenol blue and phenol red as marker dyes, in DMSA-glycerol-water, 2:1:7(v/v)

1. The gel-casting apparatus was assembled according to instructions. 1.5mm spacers were used. The apparatus was connected to the cooling system.
2. For each gel, 15ml of resolving gel solution was made by mixing 5.0ml of stock acrylamide solution, 2.5ml of water, 7.5ml of stock gel buffer, 100μl of ammonium persulfate solution, and 15 μl of TEMED. The gel solution was poured into the gel mode without delay after adding the TEMED. We have ensured there were no bubbles in the gel solution before it sets. A comb was inserted into the gel solution to make sample wells. 50min was allowed for the gel to polymerize.
3. The comb was removed with care. Electrophoretic buffer was placed in the anode compartment of the apparatus and the wells.  5μl of each sample was loaded by layering underneath the buffer in a well. A tip with a flat end was used for application. The marker dye (1μl) was applied in lane 1 and two reference CS disaccharides respectively in lanes 2 and 5.
   
   • Composition of marker in lane 2: ∆DiHA, ∆Di2S, ∆Di6S, ∆DiS_P(∆Di2,6diS)
   • Composition of marker in lane 5: ∆Di0S, ∆Di4S, ∆DiS_B(∆Di2,4diS), ∆DiS_E(∆Di4,6diS)
4. Electrophoresis was performed at constant voltage(200V) with cooling for 30min. The apparatus was covered with aluminum foil. The gel was loosen from the glass plate, placed on a piece of transparency. It was than placed in a box covered with aluminum foil. The pal pattern was captured under gel documentation system.

Agarose electrophoresis

Reagents
Electrophoretic buffer, 0.05M barium acetate/acetic acid, pH5.8
0.2% Alcian blue in solvent of 0.05M MgCl26H2O, 0.025M sodium acetate/acetic acid, pH5.8 in 50% EtOH/H2O
0.2% CPC
1% agarose solution in electrophoretic buffer

1. Electrophoretic tank was filled with buffer. The buffer level was similar in two compartments. 2 pieces of filter paper was soaked with buffer and used as wick by hanging each on a baffle at either side of the tank.
2. 1μl of each of the following was applied to the designated well of the supplied agarose gel.
   
   1. Well 1, reference standard
   2. Well 2, test sample after chondroitinase treatment;
   3. Well 3, test sample after control treatment.
3. The slide was placed gel-side down with ends resting on the paper wicks. The electrophoresis was performed at 60mA(since two slide were undergoing electrophoresis at the same time), room temperature for 30 min.
4. The gel-slide was soaked at 0.2% CPC for 30 min. The gel was dried under hot air. The fixed gel was stained in Alcian blue for 10 minutes and the background was rinsed off in the solvent.

Results

FACE Analysis: In this section, FACE was carried out by labeling chondroitinase-treated UPM and control-treated UPM with AMAC. Two markers (composition stated in procedure D.3) were added to analysis the composition of sample. Bromophenol blue and phenol red was used as marker dyes to trace the electrophoresis progress.

Figure 1, Photo taken of the FACE gel (Lane 1: Dye, Lane2: Marker1, Lane 4: Maker 2, Lane3,6: Chondroitinase treated, Lane4,7:Control)

Agarose electrophoresis: In this section, the chondroitinase-treated UPM precipitate and control-treated UPM precipitate was added to agarose gel. A standard containing chondroitin sulphate, Dermatan Sulphate and Heparin was also added as to analyze the component of the samples. The slide was processed with CPC and stained with Alcian blue afterwards.

Figure 2a, Agarose gel result

Figure 2b, Agarose gel result of classmate

Discussion

The interpretation of FACE gel electrophoresis results.

We have used two standard markers with different composition to trace the composition of disaccharide in chondroitinase-treated and control-treated UPM. First, disaccharides with more sulphate groups are more negatively charged, hence move faster in the electrophoresis towards the positive pole. As a result, lowest spots (fastest) should represent disulphated disaccharides. Spots in the middle should represent single sulphated disaccharides and spots in the top (lowest spots) should represent non-sulphated disaccharides. Secondly, due to the characteristic of tris-borate buffer used, 2-sulphated disaccharide should move faster than 4-sulphated one and 6-sulphated disaccharide move slowest. Thirdly, zero-sulphated Chondroitin Sulphate moves faster than non-sulphated hyaluronan. Hence, we can identify marker spots as the disaccharide species stated in figure 1.

Various factors affect the result of polyacrylamide gel electrophoresis. First, diffusion of molecules makes the bend became blur. In addition, the duration of electrophoresis may not be enough to separate disaccharide of small charge difference, this may be the reason that the bend of ∆Di2S/∆Di6S is indistinguishable. Furthermore, the cooling system is not effective enough. The heat generated during electrophoresis cannot be effectively carried out by the cooling water, resulting in local heating and hence distortion in speed of electrophoresis in different area. Hence, the bends seems not moving in vertical direction and do not show a horizontal shape.

Despite the imperfect standard marker pattern, we can conclude spot one to be ∆DiHA or ∆Di0S as spot one is significantly nearer to ∆DiHA/∆Di0S than other spots. Spot1 seems to be nearer to ∆DiHA than ∆Di0S, but as the difference is very small and hence inappropriate to conclude spot1 represent hyaluronan.

Spot2 is near to the standard spots of mono-sulphated Chondroitin Sulphate. As the spots of ∆Di2S/∆Di6S fused together and shape is so distorted, it do not give enough information in identifying spot2.

No spots is observed near the di-sulphated Chondroitin Sulphate bends, we can conclude the GAG sample do not contain di-sulphated CS.

To further identify spot2, we can make use of 4-sulphatase and 6-sulphatase. 4-sulphatase will remove the sulphate group of CS at 4’ position, and hence the digested product will give a bend at ∆Di0S position and no bend at mono-sulphated CS region. Similarly, 6-sulphatase removes sulphate of CS at 6’ position. If spot2 isolate still giving a bend in mono-sulphated CS region after digestion by 4-sulphatase and 6-sulphatase, we can conclude that some mono-sulphated CS is not digested by sulphatase and that should be ∆Di2S.

For controls (lane 4 and 7), because the pellet was not digested by Chondroitinase ABC, there was no free disaccharide in the supernatant (Procedure B3). Hence they did not give out florescence.

To improve FACE analysis, we should use a better cooling system so that the heating effect is not so big. We can use ice-water instead of tap water as the temperate of tap water is quite high. Also, florescence intensity of the spots of chondroitin-treated is high, indicating high concentration of disaccharide present. This will increase the concentration gradient between spots and the surrounding gel, making the bend less clear. Hence fewer samples should be used for FACE next time.

Analysis of agarose gel electrophoresis result

Agarose gel result of our group is shown in figure 2a, there were only spots produced by the standard. There was no spot in Chondroitinase-treated and control-sample. We expect no spot in Chondroitinase-treated lane as all proteoglycans have been digested by chondroitinase and no GAGs were present. Hence the sample contains no polyanionic macromolecule for Alcian Blue to attach. Experimental result confirmed our expectation.

We expect spot in control-treated lane as proteoglycans were not digested by enzyme and GAGs should be attached to the core protein, hence large negative charge was present and let the Alcian blue to attach on. However, the experimental result is contradictory to our expectation with no spot in the control. This may due to many reasons. First, the concentration of GAGs may be too slow so that they are not observable after staining with Alcian blue. Second, the GAGs were lost by diffusion during the processing of agarose gel (soaking in CPC/staining by Alcian blue).

A better experimental result was obtained by a group of bioinformatics students and shown as figure 2b. They are able to obtain a large linear spot for the standard and a small spot for the control. Standard is consisting of Chondroitin Sulphate, Dermatan Sulphate and Heparin. Heparin carries largest negative charge, followed by Dermatan Sulphate and Chondroitin Sulphate is the least negatively charged. Because of using barium acetate as buffer, the positively charged barium ion will bind to the disaccharides and reducing their negative charge. Disaccharides that have higher density of negatively charged sulphate group should now move slowest. Also, barium binds differently to Heparin and Dermatan Sulphate, bind to Heparin more effectively than Dermatan Sulphate. As a result, Chondroitin Sulphate move fastest towards positive pole, Dermatan sulphate come second with Heparin come lowest. However, time taken for electrophoresis is not long enough so that three chemical species can be separated effectively. In addition, diffusion occurs such that the spots merged together and become indistinguishable.

Yet, the spot for control is in the position resemble the tip of the standard spot, which should be contributed by Chondroitin Sulphate which moves fastest. Hence, we can conclude chondroitin sulphate is present in the UPM sample.
To improve the agarose gel electrophoresis, I will let the electrophoresis to run for longer time, so that distance between spots is large enough to separate them. Also, more samples should be added to intensify the spots as to improve visibility of spots.

Extending the experiment to give quantitative determination of GAGs.

The experiment can be extended to give quantitative determination of the amount of each disaccharide present. The intensity of florescence is proportional to the concentration of the disaccharide present. We can first run a gel of FACE using disaccharides of different known concentration. The degree of florescence can be detected by a photometer, and hence a florescence-concentration curve can be obtained. Then we run a gel of FACE with unknown disaccharide concentration. The degree of florescence of each spots can be used to determine the concentration with the help of florescence-concentration curve.

Purity of separation

The separation of proteoglycans using cetylpyridinium chloride, based on its positive-charged detergent property, is not pure. Cetylpyridinium Chloride will bind to negative-charged molecules, including proteoglycans, other proteins and other negatively-charged molecules. Hence urinary polyanionic macromolecules (UPM) are prepared instead of pure proteoglycans.

However, when UPM are treated with chondroitinase, the specificity of chondroitinase will only cut chondroitin sulphate in GAGs into unsaturated disaccharides.  As the macromolecules were spun down, the supernatant should only contain GAGs derivatives. Hence, even though AMAC can tag any reducing sugar, there were only GAG components visualized in FACE.

Yet, for the agarose gel electrophoresis, we analyze the precipitate generated in procedure B-3, which contains UPM other than GAGs like negative charged protein, etc. These impurities will remain in agarose gel and stained by Alcian Blue because of its negative charge (Alcian Blue is a cation, attach on anion because of electrostatic attraction) and may influence the electrophoresis pattern.