Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.
BACKGROUND
Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates.


METHODS
In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments.


RESULTS
The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells.


CONCLUSIONS
Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.
Holmes
E H
EH
Pacific Northwest Cancer Foundation, Department of Cell Surface Biochemistry, Seattle, Washington, USA.
Greene
T G
TG
Tino
W T
WT
Boynton
A L
AL
Aldape
H C
HC
Misrock
S L
SL
Murphy
G P
GP
D000595:Amino Acid Sequence; D002277:Carcinoma / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
D006026:Glycoside Hydrolases; D017430:Prostate-Specific Antigen