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.
Highlights
Anterior gradient protein 2 (AGR2) is a member of the protein disulphide isomerase family of endoplasmic reticulum (ER) chaperones [1], and its major role to date appears to be in promoting the secretion of several mucin glycoproteins, including MUC1 [2], MUC2 [3, 4], MUC4 [5], MUC5B and MUC5AC [6, 7]
The reduced survival time is thought to be caused by an increase in the rate of metastasis of AGR2-expressing cancers, as a number of studies have shown an increase in AGR2 expression in metastatic cells relative to their primary tumours [16, 25,26,27,28,29,30,31] and importantly, AGR2 was able to induce metastasis of a benign rat mammary cell line when cells overexpressing AGR2 were injected into syngeneic rats [16]
This mass difference in extracellular AGR2 shows that its presence in the culture medium is not due to intracellular AGR2 released from lysed or leaky cells, and this is further confirmed by the lack of detectable levels in the culture medium of AGR2-expressing Rama 37 cells of the intracellular marker protein, lactate dehydrogenase A (LDHA)
Summary
Anterior gradient protein 2 (AGR2) is a member of the protein disulphide isomerase family of endoplasmic reticulum (ER) chaperones [1], and its major role to date appears to be in promoting the secretion of several mucin. Extracellularlyadded recombinant AGR2 has been shown to promote adhesion of rat mammary epithelial cells [16, 43] and the migration and tube formation of human umbilical vein endothelial cells (HUVEC) [38], indicating a possible role for extracellular AGR2 in the promotion of angiogenesis. Changes in both cell adhesion and induction of angiogenesis may be important for a pro-metastatic phenotype. We show here that AGR2 is O-glycosylated upon secretion from human and rat cell lines, and that this form of the protein is released from healthy cells and not as a result of cell lysis
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