STAMP2 : subcellular localization and effect on synthesis and sulfation of glycans

Abstract

Six transmembrane protein of prostate 2 (STAMP2) is a six transmembrane domain protein in the STAMP/STEAP family. It is primarily found in the prostate, visceral adipose tissue, and bone marrow, and is overexpressed in prostate cancer cells. STAMP2 has an oxidoreductase activity and is capable of reducing both iron and copper, and is shown to co-localize with the metal transporters DMT1 and CTR1 in endosomes, where it is thought to reduce iron and copper for translocation across the membrane. STAMP2 is also proposed to be involved in the integration of inflammatory and metabolic responses in mice. A similar role is suggested for STAMP2 in humans, although the exact functions of the protein remain to be determined.

The Saatcioglu group had previously expressed a GFP-tagged STAMP2 variant with significant co-localization with Golgi markers. A pilot study conducted by the Prydz and Saatcioglu groups to monitor Golgi functions had indicated that reduction of STAMP2 expression in LNCaP cells had an effect on the incorporation of sulfate into proteoglycans (PGs). Since copper ions previously have been suggested to play a role in PG metabolism, it was of interest to study this possible link further. With the extensive knowledge of PG synthesis and sulfation in the epithelial cell line MDCK, as well as available methodology for transport and subcellular fractionation studies, it would be of interest to study the impact of STAMP2 in transfected MDCK cells, as well as to further study the link between STAMP2 and synthesis and sulfation of PGs in LNCaP cells.

Three STAMP2 variants fused to GFP were transfected into MDCK II cells. One of these was the original construct from the Saatcioglu group with the GFP at the very N-terminus (N- terminal signal sequence for ER import not required). The two other variants had the GFP moved into the N-terminal region of protein, one of these with a mutated ferric-reductase domain. Confocal microscopy and subcellular fractionation studies indicated a difference in the localization of the three variants. Exposure of the N-terminal cytoplasmic tail of STAMP2 caused localization to the plasma membrane and endosome-like strucutres, while blocking the tail with GFP, resulted in significant localization to the Golgi apparatus. Radioactive labeling of control cells and the transfected cell lines with [3H]-glucosamine and [35S]-sulfate gave a significant decrease in the incorporation of glucosamine and sulfate into GAG chains in the cells where STAMP2 had the GFP domain at the very N-terminus and showed Golgi localization.

The stable knockdown of STAMP1 and STAMP2 in LNCaP cells had no effect on the synthesis and sulfation of glycoproteins.