Team:Stockholm/Measurement

Best Innovation in Measurement Special Prize

Mucus is a multifunctional network of heavily glycosylated proteins, whose composition and properties can vary during diseases. Our project aims to degrade thickened mucus using glycosidases such as sialidase and beta-endo-galactosidase. With the help of our advisors, we developed a strategy to measure the changes in mucus composition and physical properties caused by these enzymes. The analyses employed High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) to detect compositional changes as well as rheology measurements to assess the visco-elastic properties of degraded mucus.

The HPAEC-PAD technique can provide a precise analysis of oligosaccharides such as sialic acid. This allowed us to quantify the sialic acid released from bovine submaxillary mucin (BSM) after digestion with sialidase, both from commercial sources and expressed by our engineered bacterial system.

As a consequence of the sialidase activity, terminal residues of sialic acid were cleaved off from the mucin structure and were subsequently quantified through a customized HPAEC-PAD protocol. Signal peaks for sialic acid were registered at an approximate retention time of 5 minutes and translated into sialic acid concentrations employing a previously made calibration curve of sialic acid.

Sulfuric acid treated BSM was employed as a positive control, considering that the highly acidic conditions result in near to total removal of sialic acid. An additional calibration curve, using sulfuric acid with varying sialic acid concentrations, was used for quantification of the positive control digestion. This was performed for an optimal measurement, as HPAEC-PAD signal strength varies with pH. Untreated BSM was employed as a negative control and showed no sign of sialic acid residues.

By using this technique, we were able to confirm and demonstrate the activity of sialidase, thus, displaying its potential to alter mucus composition and properties. However, the test delivered inconclusive results on the correlation between enzyme concentration and sialic acid degraded (figure 1).

Figure 1. Concentration of sialic acid from BSM after digestion with sialidase.

Once we confirmed the enzymatic activity of our expressed sialidase, we proceeded to demonstrate the effect of glycan removal on viscoelastic properties of mucus.

To be more specific, mucus is a non-Newtonian, thixotropic gel which displays an elastic solid-like behavior under low shear stress and viscous liquid-like behavior under high shear stress (Lai et al. 2009). Viscosity is a way to express the fluidity of a given substance and can be calculated by the parameters stress and shear rate.

Depending on the site of mucus in the body and the disease state, the mucus composition changes with the physiological need or the pathological conditions. Several studies have shown that small differences in the mucin concentration can affect the mucus viscoelasticity tremendously (e.g. 2-4 fold increase in mucin concentration can result in 100-fold higher viscosity) (Lai et al., 2009). In cystic fibrosis for instance, a disease with an abnormally high mucin concentration, the viscosity at low shear rates (close to standstill) can be compared to that of rubber (Lai et al., 2009) (Mestecky et al., 2015).

Thus, rheology testing is a suitable measurement method enabling us to gain information about how the glycosylation state of mucins changes the viscoelastic properties of mucus. To perform rheology testing, pig gastric mucins (PGM) samples were treated with a deglycosylation reagent which cleaves O-linked glycans in a non-reducing manner with minimal protein or glycan destruction. Thereby, glycan mass equal to 39% of the initial PGM mass was removed (glycans constitute up to 80% of the dry mucus mass). Rheology measurement showed decreased viscosity with increasing shear rate in deglycosylated compared to native PGM (figure 2).

Figure 2. Rheology measurement displaying viscosity and stress of untreated mucus (native PGM) versus treated mucus (deglycosylated PGM) .

Taken together, with this dual measurement we showed degradation, not only by enzymatic cleavage of sialic acids, but also by a reduction in viscosity. The combination of these methods tied the approach of enzymatically degrading mucus and the potential impact of mucus degrading enzymes as a therapeutic approach together. We have therefore shown ability to adapt measurement methods, common for other areas, to confirm our hypothesis.

References

  • S. K. Lai, Y.-Y. Wang, D. Wirtz, J. Hanes (2009). Micro- and macrorheology of mucus. Adv Drug Deliv Rev. Doi:10.1016/j
  • Cone R. Mucus. In: Michael WS, Lamm E, McGhee Jerry R, Mayer Lloyd, Mestecky Jiri, Bienenstock John, editors. Mucosal Immunlogy. San Diego: Academic Press; 1999. pp. 43–64.

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