Different glycoforms of prostate-specific membrane antigen are intracellularly transported through their association with distinct detergent-resistant membranes

Abstract

Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-transmembrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant membranes) at different stages of the protein maturation pathway in human prostate carcinoma LNCaP cells. At least three PSMA glycoforms were biochemically identified based on their extractability behaviour in different non-ionic detergents. In particular, one precursor glycoform of PSMA is associated with Tween 20-insoluble DRMs, whereas the complex glycosylated protein segregates into membrane structures that are insoluble in Lubrol WX and display a different lipid composition. Association of PSMA with these membranes occurs in the Golgi compartment together with the acquisition of a native conformation. PSMA homodimers reach the plasma membrane of LNCaP cells in Lubrol WX-insoluble lipid/protein complexes. At the steady state, the majority of PSMA remains within these membrane microdomains at the cell surface. We conclude that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.

Case study on the pathophysiology of Fabry disease: abnormalities of cellular membranes can be reversed by substrate reduction in vitro

“…Equal volume from each fraction for LRs analysis or protein amounts from the total lysates were resolved by SDS/PAGE and transferred to PVDF membranes (Roche Diagnostics). Immunoblotting was performed as described before using primary antibodies against flotillin-2 (Santa Cruz Biotechnology, Germany), RhoA (Santa Cruz Biotechnology, Germany) and α-galactosidase A (Sigma, Germany) and then HRP–conjugated secondary antibodies [15]. Protein bands were visualized using ECL with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc system (Bio–Rad, Germany).…”

Cloning and characterization of canine prostate‐specific membrane antigen

“…We could show before that human PSMA is transported via different microdomains along the secretory pathway [14]. These rafts are described to be involved in signaling processes, trafficking and endocytosis.…”

Protocadherin of the Liver, Kidney, and Colon Associates with Detergent-resistant Membranes during Cellular Differentiation

“…Extraction-Association of PLKC with DRMs was investigated by a discontinuous sucrose-density gradient as reported previously (28). In brief, cells at a subconfluent stage of differentiation (around 50% optical confluence) and 2 days after confluence were lysed with 1% (w/v) of Triton X-100 in PBS.…”