- Authors: Ehrencrona E. et al.
- Year: 2022
- Journal: J Biol Chem 297 100871
- Applications: in vitro / DNA / FectoCHO Expression System, FectoPRO
- Cell type: CHO-S
Description: Chinese hamster ovary cells
Plasmids were transformed into competent Escherichia coli XL1-Blue (Agilent), and the DNA was purified using Qiagen plasmid Mini or Maxi kits (QIAGEN) according to manufacturer’s instructions. The truncated proteins were transiently expressed using FectoPRO (Polyplus) in suspension-growing CHO-S cells and prepared from 300 ml (FCGBP-ND2 GDPH and FCGBP-ND2 GAPH mutant) and 1000 ml (FCGBP-N and D13) of FectoCHO (Polyplus) cell medium. Full-length mouse Fcgbp was expressed in CHO-S by transient transfections of the Fcgbp-Myc-DDK plasmid using the FectoPRO transfection reagent (Polyplus)
Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.