Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.
Polysaccharides of the glycosaminoglycan (GAG) type such as hyaluronan (HA) or chondroitin sulfate (CS) are important constituents of the extracellular matrix (ECM) of connective tissues which is omnipresent in all vertebrates [
Motivated by the discovery of the “sulfation code” [
Although the overall degree of sulfation can be easily determined by elementary analysis (i.e., by the determination of the sulfur content of a GAG sample), a more detailed structural analysis of the sulfation pattern is a challenging task. Although 13C nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful methods of structural GAG analysis, this method may easily fail for two reasons. First, the line widths of the 13C NMR resonances of GAG polysaccharides are significant and, thus, prevent the detection of small chemical shift differences [
Even though mass spectrometry (MS) is an additional powerful method of structure elucidation (particularly when combined with MS/MS and/or chromatographic separation) [
Therefore, the most common way of mass spectrometric GAG analysis is based on previous enzymatic digestion of the polysaccharides: using enzymes such as testicular or bacterial hyaluronidases or bacterial chondroitinases (normally of the ABC type which is capable of cleaving CS A, B, and C), the native GAGs can be efficiently converted into characteristic oligosaccharides [
There are two reasons why sulfated oligosaccharides are commercially scarcely available. First, (over)sulfated GAGs (that are available from some animal tissues and/or can be synthesized by chemical sulfation of the native GAG polysaccharides) are less efficiently digested by enzymes such as chondroitinase or hyaluronidase [
We will focus here particularly on the first problem: by using an adapted spectrophotometric assay as well as matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS, we will show that enzymatic digestion of all (over)sulfated GAGs can be basically achieved if the reaction conditions are carefully adjusted. Nevertheless, the yield of digestion products decreases if the degree of sulfation of the polysaccharide educt increases.
Hyaluronan (HA, from
The hyaluronan sulfates (sHA1–sHA3) were synthesized as previously described by Hintze et al. [
The degree of sulfation was determined by estimation of the sulfur content using an automatic elemental analyzer (Euro EA3000 CHNS, EuroVector, Redavalle, Italy). Molecular weight determination was performed by gel permeation chromatography (GPC) with a double detection system consisting of a Postnova Analytics PN 3000 (15°) laser-light scattering (LLS) detector and a Jasco RID-1531 refraction index (RI) detector. Absolute values of number-average (Mn) and weight-average (Mw) molecular weights were determined using the laser-light scattering (LLS) detection system. The calculation of the polydispersity (PD = Mw/Mn) was performed on the basis of Mn and Mw values obtained from RI detection. GPC operating parameters can be found in the work by Hintze et al. [
Selected characteristics of the synthesized HA derivatives.
Sample | sHA1 | sHA2 | sHA3 |
---|---|---|---|
dss | 1.2 | 1.8 | 3.0 |
|
15 910 (39 540) | 16 673 (43 993) | 23 725 (29 275) |
|
26 790 (87 410) | 32 370 (86 150) | 29 525 (48 340) |
PD | 2.2 | 2.0 | 1.7 |
dss, number-average (
Aqueous solutions of (a) native, that is, nonsulfated hyaluronan, and (b) three chemically sulfated hyaluronan derivatives with degrees of sulfation of 1.2, 1.8, and 3.0 were digested with hyaluronidase from
The number of reducing (N-acetylglucosamine) end groups was used as a measure of the extent of the enzymatic digestion: when a GAG is digested, the absolute amount of the GAG remains indeed constant. However, the number of molecules increases. Each of the resulting oligosaccharides possesses a reducing end group and the number of the end groups can be conveniently determined. For the performing of the Reissig method [
All MALDI-TOF mass spectra were acquired on an Autoflex mass spectrometer (Bruker Daltonics, Bremen, Germany) in the linear mode under delayed extraction conditions as previously described [
200 single laser shots were averaged for each mass spectrum. The laser fluence was kept about five percent above threshold to obtain optimum signal to noise ratios.
Saturated 9-aminoacridine (9-AA) [
The GAGs of particular interest for this communication are (a) native (nonsulfated) hyaluronan and (b) chemically sulfated HA derivatives. However, the polymer repeating units remain unchanged upon derivatization: the disaccharide unit of HA is composed of a
Figure
Chemical structure of native (R = H) and chemically sulfated HA (R =
Hyaluronidase from
The Reissig signal [
Figure
Dependence of the Reissig absorption (difference of the absorption at 585 nm without and subsequent to digestion with BTH) on the used hyaluronan samples. All samples were digested with BTH for 20 hours at 37°C at pH 5.7. No salts beside the buffer components were added. For details see text.
The exhaustive enzymatic digestion of (sulfated) HAs leads to tetra-, hexa-, and octasaccharides as the most abundant products [
Mass list of the most abundant digestion products of HA after exhaustive digestion with bovine testicular hyaluronidase.
Hyaluronan oligosaccharide | Monoisotopic molecular masses | |||
---|---|---|---|---|
Number of sulfate residues | ||||
0 | 1 | 2 | 3 | |
Tetrasaccharide (HA-4) | 776.23 | 856.19 | 936.15 | 1016.10 |
Hexasaccharide (HA-6) | 1155.34 | 1235.30 | 1315.26 | 1395.22 |
Octasaccharide (HA-8) | 1534.46 | 1614.41 | 1694.37 | 1774.33 |
The table includes the masses of sulfated derivatives with up to three sulfate residues per oligosaccharide. Please note that all data were calculated by using the monoisotopic masses. Since the data will be used to explain the mass spectra (vide infra), charge compensation by protonation (not by alkali metal ions) is exclusively assumed to occur.
Soft ionization mass spectrometric techniques such as MALDI or ESI are convenient methods to investigate the molecular weights of oligosaccharides [
Negative ion MALDI-TOF mass spectra of differently sulfated HA samples subsequent to digestion with hyaluronidase but without any further purification of the reaction mixture. Trace (a) corresponds to native HA, while (b) and (c) represent HA with dss = 1.2 and 1.8, respectively. All spectra were recorded in the linear mode of the MS device and in the presence of 9-aminoacridine as matrix with a sample to matrix ratio of 2 to 1 (v/v). This corresponds to a 25-fold weight excess of the matrix. Peaks stemming from impurities are marked by asterisks. Please note that the achievable mass accuracy is only of the order of about 200 ppm. This is a rather typical value if the linear (but not the reflector modus) is used and the reason why only one decimal is given.
In the case of the sulfated HA with dss = 1.2 and 1.8, however, only sulfated oligosaccharides are detectable, whereby the tetra-, hexa-, and octasaccharides with one to a maximum of three sulfate residues (cf. Figure
Unfortunately, the digestion of the HA sample with the most significant extent of sulfation (sHA3, dss = 3.0) did not result in an oligosaccharide concentration which could be detected by MALDI MS. However, it should be explicitly noted that oligosaccharide concentrations below the detection limits are only one potential explanation: in addition to this aspect, charged saccharides are always more difficult to detect by means of MS than neutral saccharides. This particularly applies to carbohydrates with strong electrolytes such as sulfate [
In order to improve the extent of the enzymatic degradation of the HA with the most significant extent of sulfation, the digestion conditions were further optimized: the dependence of the Reissig signal (subsequent to BTH digestion) on the used NaCl concentration is shown in Figure
Dependence of the Reissig absorption (as a measure of the reducing end groups, i.e., the extent of the enzymatic digestion) on the NaCl concentration. Since the HA sample with the highest sulfate content (dss = 3) was most refractive to the hyaluronidase digestion, only this sample was investigated. All samples were digested with BTH for 20 hours at 37°C at pH 5.7 prior to measurements.
In addition to the salt concentration, the pH of the reaction mixture was also systematically varied to evaluate the optimum conditions of the enzymatic digestion, whereby the NaCl concentration was kept constant at 0.15 M. The achieved data (given in Figure
Dependence of the Reissig absorption on the pH value of the digestion mixture. Since the HA sample with the highest sulfate content (sHA3) was most refractive to the hyaluronidase digestion, only this sample was investigated. All samples were digested with BTH for 20 hours at 37°C and physiological NaCl concentration (0.15 M).
All data related to the digestion of the HA sample with the highest sulfate content (sHA3) are finally summarized in Figure
Determination of the Reissig absorption under the optimized reaction conditions discussed above. HA with ds = 3 was exclusively used. The applied conditions are indicated directly in the figure. Error bars represent the systematic error of the performed measurements.
The comparison of the Reissig signal intensities after the three different digestions of the highly sulfated HA derivative (Figure
Although this might be considered as a minor progress only, this yield difference is sufficiently pronounced to enable the MALDI MS characterization of the related oligosaccharides: the negative ion MALDI-TOF mass spectrum of the HA polysaccharide with the most pronounced sulfate content (dss = 3.0) subsequent to BTH digestion at the optimized conditions is shown in Figure
Negative ion MALDI-TOF mass spectrum of the sulfated HA with dss = 3.0 subsequent to digestion with BTH at optimized conditions but without further purification of the reaction mixture. All spectra were recorded in the linear mode of the MS device and in the presence of 9-aminoacridine as matrix using a sample to matrix ratio of 2 to 1 (v/v). Sulfated tetrasaccharides are marked by grey and hexasaccharides by white bars, respectively. Obvious impurities are marked by asterisks.
The reader should note that the peak intensities do not necessarily correlate with the concentrations of the individual sulfated oligosaccharides [
Although we are aware of these obvious problems, the present study should be considered as a pioneering work: to our best knowledge it was so far not possible at all to convert highly sulfated HA samples into the corresponding oligosaccharides. Even if further improvements are obviously necessary, this study has provided sufficient evidence that strongly sulfated hyaluronans are basically digestible by bovine testicular hyaluronidase.
We have shown that chemically sulfated hyaluronan derivatives can be digested by bovine testicular hyaluronidase if the conditions of the enzymatic digestion are carefully adjusted, although the oligosaccharide yield is still strongly dependent on the number of sulfate residues per polymer repeating unit.
The molecular weights of the generated oligosaccharides were additionally verified by using MALDI-TOF mass spectrometry. All of the described experiments were exclusively performed on an analytical scale. However, our next step will be the extension of this study to obtain the oligosaccharides in at least mg amounts. If successful, this would be an important progress because the availability of GAG oligosaccharides with defined sulfation patterns is so far extremely limited.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The present research was supported by the German Research Council (Transregio 67, Projects A2 and A8). The authors are indebted to Mrs. Rosmarie Süß for many useful hints regarding the mass spectrometric characterization of sulfated glycosaminoglycans.