Shifted Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65 results in formation of high mannose N-glycans in aggressive prostate cancer cells

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 11; pp. 2891 - 2901
• Main Authors: Bhat, Ganapati, Hothpet, Vishwanath-Reddy, Lin, Ming-Fong, Cheng, Pi-Wan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2017
• Subjects: alpha-mannosidase; alpha-Mannosidase - chemistry; alpha-Mannosidase - metabolism; Autoantigens - genetics; Autoantigens - metabolism; biomarkers; Biomarkers, Tumor - biosynthesis; Biomarkers, Tumor - chemistry; Cell Line, Tumor; confocal microscopy; Giantin; glycoproteins; glycosyltransferases; Glycosyltransferases - chemistry; Glycosyltransferases - metabolism; GM130-GRASP65; GNL; Golgi Apparatus - enzymology; Golgi Apparatus - metabolism; Golgi Apparatus - pathology; Golgi α-mannosidase IA; High mannose N-GLYCANS; Humans; In situ proximity ligation assay; Male; mannose; Mannose - metabolism; mass spectrometry; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membrane Proteins - antagonists & inhibitors; Membrane Proteins - genetics; Membrane Proteins - metabolism; neoplasm cells; patients; Polysaccharides - biosynthesis; Polysaccharides - chemistry; precipitin tests; prostatic neoplasms; Prostatic Neoplasms - enzymology; Prostatic Neoplasms - metabolism; Prostatic Neoplasms - pathology; Protein Binding; Protein Transport - genetics; trans-Golgi glycosyltransferases; GM130; PLA; GNL; Golgi α-mannosidase IA; Man II; Mgat2; Mgat1; Co-IP; NMIIA; Man IA; GRASP65; β4GalT1; C1GalT1; GM130-GRASP65; ST3GalT1; In situ proximity ligation assay; C2GnT; Maldi-Tof-MS; trans-Golgi glycosyltransferases; Giantin; High mannose N-GLYCANS
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2017.08.006

There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man8GlcNAc2 down to Man5GlcNAc2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells. •Golgi targeting sites of two glycosyltransferases and α-mannosidase are identified.•How high mannose N-glycans are generated in advanced cancer cells is described.•Shifted Golgi targeting of glycozymes causes formation of high mannose N-glycans.•Proximity ligation assays for aggressive prostate cancer cells are developed.•The results of this study can aid treatment decision on prostate cancer.