Expression of cysteine-rich pig gastric mucin (Muc5AC) domains in Pichia pastoris and their pH-dependent properties
Turner, Bradley Steven
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Gastric mucin glycoproteins provide a viscous protective mucus barrier to the underlying gastric epithelium from the luminal environment of acid, enzymes, bacteria and shear forces. Pig gastric mucin (PGM) forms a viscoelastic gel exhibiting a sol-gel transition at the low pHs found in the stomach. Our laboratory has demonstrated that the non-glycosylated cysteine-rich (Cys-rich) regions of the protein core of PGM play important roles in the hydrophobic, electrostatic and disulfide-bond interactions involved in PGM aggregation and gelation. Here I investigate whether or not recombinant peptides from the Cys-rich regions of PGM expressed in Pichia pastoris exhibit hydrophobic and structural changes as a function of pH. I cloned cDNAs encoding the full-length, Cys-rich C-terminal (CTerm) and inter-repeat (Cys-Rep) domains of the PGM mucin gene Muc5AC. These were cloned into the vector pDEST911 and transformed into the yeast expression host Pichia pastoris. The secreted recombinant peptides were purified from culture supernatant by nickel affinity chromatography yielding 5-15 mg/L. The pH-dependent properties of the peptides were investigated by intrinsic peptide fluorescence of tryptophan (Trp), binding of the fluorescent hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS) and circular dichroism. The peptides induced a concentration-dependent increase in the ANS fluorescence intensity and a concomitant decrease in the ANS peak emission wavelength (blue shift) indicating hydrophobic binding of ANS to the peptides. Similarly, decreasing the pH from 6 to 2 also caused an increase in the ANS fluorescence intensity and blue shift. Conversely, the intrinsic Trp fluorescence exhibited a decrease in intensity and a red shift with decreasing pH, indicating a net movement of the hydrophobic-peptide Trp residues to a more exposed aqueous or polar environment. Circular dichroism at low pH revealed a slight decrease in α-helix content and increased random coil or β-strand for the C-terminal and Cys-repeat peptides respectively. Thus, decreasing pH causes an increase in the exposure of, and binding to, hydrophobic sites of recombinant peptides from the Cys-rich regions of PGM. The increased hydrophobic interactions may well be important in inducing aggregation and gelation of PGM Muc5AC at low pH.
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