The metastasis-inducing protein AGR2 is O-glycosylated upon secretion from mammary epithelial cells

Abstract

AGR2 is overexpressed in multiple cancers, particularly those arising from breast and prostate tissues, and higher levels of AGR2 are associated with earlier patient death. Although AGR2 is normally resident within the endoplasmic reticulum, the protein has been found in the extracellular space in several model systems. However, it has never been expressly demonstrated that this extracellular form of the protein is secreted and does not just accumulate in the extracellular space as a result of cell lysis. We show in this paper that AGR2 protein is secreted by both human and rat mammary epithelial cells in culture. Furthermore, this secreted form of AGR2 becomes O-glycosylated, with no detectable presence of N-glycosylation. Importantly, this post-translationally modified AGR2 is only detected in the conditioned medium from non-leaky cells, suggesting that membrane integrity must be maintained to allow AGR2 glycosylation. The results suggest a possible role for O-glycosylation in modulating the extracellular functions of AGR2.

Keywords: AGR2; Adhesion; Glycosylation; Secretion.

Fig. 1

A higher molecular weight form of AGR2 is released into the medium of AGR2-expressing rat mammary epithelial tumour cells. Conditioned medium was collected from vector only-expressing or WT AGR2-expressing Rama 37 cells and analysed for the presence of AGR2 and lactate dehydrogenase (LDHA) by Western blot. The presence of LDHA in the conditioned medium was used as an indication of the contamination of the secreted protein pool by intracellular proteins [64]. Extracellular AGR2 was 0.90 (SD ± 0.12) kDa larger than intracellular AGR2 on average (n = 3)

Fig. 2

In human mammary epithelial cells, higher molecular weight AGR2 is only released from non-leaky cells. MCF7A cells were incubated for 24 h in serum-free DMEM or serum-free Opti-MEM medium, with and without non-essential amino acids (NEAA). Conditioned medium was collected and probed for AGR2 and LDHA by Western blot. The presence of LDHA in the conditioned medium was used as an indication of the contamination of the secreted protein pool by intracellular proteins under each serum-free condition. Extracellular AGR2 was 0.93 (SD ± 0.09) kDa larger than intracellular AGR2 on average (n = 3)

Fig. 3

A highly secreted mutant form of AGR2 is also released in a high molecular weight form from rat mammary tumour cells. Rama 37 cells were engineered to express AGR2 devoid of the C-terminal KTEL ER-retention sequence in order to increase its secretion. Note that, as ΔKTEL AGR2 is highly secreted, intracellular levels are almost undetectable and thus expression of intracellular WT AGR2 is shown for size comparison. Extracellular AGR2 was 0.94 (SD ± 0.09) kDa larger than intracellular AGR2 on average (n = 3)

Fig. 4

Treatment with O-glycosidase reduces the molecular weight of secreted AGR2. a Conditioned medium from ΔKTEL AGR2-expressing cells (secreted AGR2) and whole cell lysate from WT AGR2-expressing cells (intracellular AGR2) were treated with the indicated deglycosylation enzymes for 4 h at 37 °C, as per the manufacturer’s instructions. Treated samples were subjected to Western blot and probed for AGR2. PNGase F is an N-glycosidase. O-glycosidase-treated samples were simultaneously treated with neuraminidase, as the presence of terminal sialic acid residues blocks the activity of O-glycosidase [65]. Deglycosylation mix consists of PNGase F, O-glycosidase, neuraminidase, β1-4 galactosidase and β-N-acetylglucosaminidase. Untreated extracellular AGR2 was 0.80 (SD ± 0.04) kDa larger than intracellular AGR2 on average (n = 3), and extracellular AGR2 treated with O-glycosidase or deglycosylation mix was 0.27 (SD ± 0.04) kDa larger than intracellular AGR2 on average (n = 3). b To ensure that PNGase F was active in the presence of culture medium components, α-acid glycoprotein (α-AG) was added to conditioned medium and treated with PNGase as in a. Reaction mixtures were run on an SDS-PAGE gel and stained with Coomassie blue

Fig. 5

Predicted O-glycosylation sites on secreted AGR2. Amino acids predicted as possible sites of O-glycosylation by the NetOGlyc 4.0 server [52] are shaded in grey boxes. Secondary structure elements are shown above the primary sequence. α:α-helix. β:β-sheet. Figure adapted from [43]