Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells

Abstract

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galβ1-3GalNAc (core 1) and mono-and disialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1-and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.

The ST6GalNAc-I Sialyltransferase Localizes throughout the Golgi and Is Responsible for the Synthesis of the Tumor-associated Sialyl-Tn O-Glycan in Human Breast Cancer

“…The myelogenous leukemia cell line, K562, was cultured in RPMI, 10% FCS. CHO MUC1 cells were developed by transfection of CHO cells with a MUC1-murine IgG2a fusion cDNA construct containing 16 tandem repeats (36) and were initially cultured in Iscove's modified Dulbecco's medium, 10% FCS with 600 g/ml G418. For large scale production, they were adapted to serum-free medium (see below).…”

“…Thus, hST6GalNAc-I was able to compete very effectively with the core 1 enzyme to produce STn. (36). The presence of hST6GalNAc-I significantly reduced this number to an average occupancy of 3.8, with only 25% of the tandem repeats being fully glycosylated, with the repeats having four O-glycans being the most common (39%) (Fig.…”

“…cans from 20 g of purified MUC1-IgG were released by reductive ␤-elimination and analyzed by LC-ESI MS (36). The relative amounts of the different oligosaccharides were obtained from the integrated peak areas in the chromatogram corresponding to each of the molecular ions (Neu5Ac␣2-6)GalNAcol (m/z 513), Neu5Ac␣2-3Gal␤1-3GalNAcol and Gal␤1-3(Neu5Ac␣2-6)GalNAcol (m/z 675), and Neu5Ac␣2-3Gal␤1-3(Neu5Ac␣2-6)GalNAcol (m/z 966 and 483).…”

“…The relative amounts of the different oligosaccharides were obtained from the integrated peak areas in the chromatogram corresponding to each of the molecular ions (Neu5Ac␣2-6)GalNAcol (m/z 513), Neu5Ac␣2-3Gal␤1-3GalNAcol and Gal␤1-3(Neu5Ac␣2-6)GalNAcol (m/z 675), and Neu5Ac␣2-3Gal␤1-3(Neu5Ac␣2-6)GalNAcol (m/z 966 and 483). The occupancy of the O-glycosylation sites in MUC1 was determined as described previously (36), except that the treatment with Vibrio cholerae neuraminidase was performed at pH 4.5.-5.0 and the glycopeptides generated by Clostripain digestion were analyzed by nano-LC-ESI MS using a Finnegan LTQ mass spectrometer (Thermo Electron, Waltham, MA). Integration of peaks in mass chromatograms was performed using XCalibur software.…”

“…Instead we turned to the production of a secreted MUC1 protein in CHO cells. The construct that was transfected into the CHO cells contains most of the extracellular domain of MUC1, including 16 tandem repeats, fused to mouse IgG2a Fc to aid secretion (36,37) (Fig. 7a).…”