Waikato Medical Research Foundation
The aim of this proposal was to explore the hypothesis that milk-derived QSOX
enzyme plays a role in intestinal barrier function through generating disulphide bonds
that cross-link mucin in the mucus layer. This function may be important to the
immature intestine of the neonate, which has only low levels of QSOX compared to the
adult (Isaacs et al. 1984). If QSOX does play a role in this function then its inclusion in
infant formula could provide a benefit to the bottle-fed infant. In addition, QSOX could
be used for treating patients with inflammatory bowel diseases that exhibit a bacterially
damaged mucous layer.
We explored the effect of QSOX on mucin integrity through examining the chemical,
physical and functional properties of mucin and QSOX-treated mucin. In all
experiments, mucin was from pig gastric mucus layer and either type II (crude) or type
III (purified). In order to break existing disulphide bonds, mucin was treated with beta
mercaptoethanol and urea according to Fogg et al. (1996), and then exhaustively
diafiltered over a 50 Kda ultrafilter against degassed water to remove any reducing
agent. Reduced mucin was then freeze-dried and stored between 4 and 8 °C. QSOX
was obtained as an enriched fraction from pasteurised bovine milk using cation
exchange and heparin affinity chromatography, and ultrafiltration. The final product
had an estimated purity of between 12 and 15% by SDS-PAGE and was stored as a
freeze-dried powder between 4 and 8 °C. The sulphydryl oxidase activity of this
product was confirmed by measuring its ability to oxidise the free thiol DDT according
Jaje et al. (2007).
2.1 Chemical properties of QSOX-treated mucin
The ability of QSOX to create disulphide bonds in mucin was determined by measuring
free thiol content according to Mantle et al.(1990). Reduced and non-reduced mucin
was dissolved in buffer at 0.625 mg mucin per mL and allowed to hydrate overnight.
This was then treated with 0.1 mg/mL QSOX fraction, or with buffer alone, for 2 h at
room temperature. Free thiol content was measured by adding 200 uM aldrithiol and
measuring the absorbance at 324 nm.
2.2 Physical properties of QSOX-treated mucin
The ability of QSOX to cross-link mucin molecules was determined by examining the
molecular size distribution of mucin aggregates using size exclusion chromatography
(SEC). Reduced and non-reduced mucin was treated with QSOX as described above.
Samples were subjected to SEC using a Sephacryl S300 HR chromatography column, which can separate globular proteins in the molecular weight range 10 kDa to 1500
kDa, and polymeric open structures (e.g. Dextran) in the range 2 to 400 kDa.
Chromatography was performed in 10 mM phosphate buffer pH 7.2 containing 150
mM NaCl, and absorbance measured at 280 nm.
2.3 Permeability of QSOX-treated mucin
The effect of QSOX on the permeability of mucin was determined by measuring the
effective diffusion of fluorescently labelled polystyrene microspheres through a
hydrated mucin hydrogel. Reduced and non-reduced mucin was treated with QSOX as
described above, except that the concentration of mucin was increased to 25 mg/mL to
represent levels similar to those observed in the intestine. Fluorescently labelled, biotin
tagged microspheres were added to the mucins and mixed in a tube. A proportion was
added to the well of a 96-well streptavidin coated fluorescent microplate and left
overnight. The degree of diffusion of microspheres through the mucin hydrogel was
determined by measuring the level of fluorescent biotin beads captured on the surface
of the streptavidin microplate after the mucin had been removed and the well washed
with buffer. As control, the effect of QSOX on the capture of fluorescent beads alone