Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing.
Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.
Castelletti
Deborah
D
Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.
Fracasso
Giulio
G
Alfalah
Marwan
M
Cingarlini
Sara
S
Colombatti
Marco
M
Naim
Hassan Y
HY
D020816:Amino Acid Motifs; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000737:chemistry*; D001692:Biological Transport; D019921:Biotinylation; D015153:Blotting, Western; D019556:COS Cells; D037281:Calnexin / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D043425:Glutamate Carboxypeptidase II / Q000096:biosynthesis* / Q000737:chemistry*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008297:Male; D008358:Mannose / Q000737:chemistry; D009362:Neoplasm Metastasis; D011134:Polysaccharides / Q000737:chemistry*; D011471:Prostatic Neoplasms / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms; D014162:Transfection; D014357:Trypsin / Q000737:chemistry
D000954:Antigens, Surface; D018076:DNA, Complementary; D011134:Polysaccharides; D020033:Protein Isoforms; D037281:Calnexin; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D014357:Trypsin; D008358:Mannose