From an increase in the number of tandem repeats through the decrease of sialylation to the downregulation of MUC1 expression level

Abstract

Enhanced glucose uptake by cancer cells was demonstrated in many studies in vitro and in vivo. Glycolysis is one of the main ways of obtaining energy in hypoxia conditions. However, in addition to energy exchange, carbohydrates are also necessary for the posttranslational modification of the protein molecules. Cancer cells are often characterized by an enhanced expression of different glycoproteides.Correct glycosylation defines the structure and activity of such molecules. We demonstrated that under the same cultivation conditions, the intensity of glycosylation does not depend on the total number of potential O-glycosylation sites in one molecule. As a model for the investigation, the tandem repeat region (region with variable number of tandem repeats) of the human mucin MUC1, in which each of the repeats carries four potential O-glycosylation sites, was used. An increase of the tandem repeat number in the recombinant protein did not lead to a proportional increase in the level of sLe a glycosides. A consequence of this was a reduction in the number of recombinant proteins associated with the cytoplasmic membrane at an overall high expression level. Prolongation of the cultivation duration led to a reduction in the expression level of the recombinant proteins by up to 30% of the initial level, and the intensity of this reduction was in a direct ratio to the number of tandem repeats in the protein molecule. K E Y W O R D S cancer, cell metabolism, glycosylation, MUC1, sLe a , tandem repeat J Cell Biochem. 2019;120:4472-4484. wileyonlinelibrary.com/journal/jcb 4472 |

The length of mucin MUC1 extracellular domain affects integrin-mediated cell adhesion to fibronectin and vitronectin

“…These proteins also contain MUC1 transmembrane domain and anchoring fragment (Cys-Gln-Cys). EGFP is located in the C-terminus [28]. The difference in the length of the recombinant proteins was confirmed by immunoblotting.…”

“…HT-29 cells (HT-29_TR-, HT-29_TR1, HT-29_TR4, HT-29_TR8, HT-29_TR12 and HT-29_TR20) expressing proteins with a different number of tandem repeats (0, 1, 4, 8, 12 and 20, respectively) [28] were maintained in DMEM supplemented with 10 % heat-inactivated fetal bovine serum, penicillin/streptomycin and 700 mg/ml G418 in a humidified incubator at 37 °C and 5 % CO 2 .…”

“…At the same time, it is not entirely clear which size of the extracellular domain of the mucin MUC1 is sufficient for this molecule to facilitate integrin-mediated adhesion. To answer this question, we used HT-29 cell lines stably expressing recombinant proteins containing a different number of tandem repeats from the VNTR region of the human mucin MUC1 in their extracellular regions [28].…”