Oligosaccharide Profiles of the Prostate Specific Antigen in Free and Complexed Forms from the Prostate Cancer Patient Serum and in Seminal Plasma: a Glycopeptide Approach

Glycobiology vol. 18 no. 1 pp. 2–8, 2008 doi:10.1093/glycob/cwm117 Advance Access publication on October 23, 2007 Oligosaccharide profiles of the prostate specific antigen in free and complexed forms from the prostate cancer patient serum and in seminal plasma: a glycopeptide approach Michiko Tajiri2,3 , Chikara Ohyama4 , and Yoshinao Wada1,2 2 Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho Izumi, Osaka 594-1101, Japan; 3 CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi. Saitama 332-0012, Japan; and 4 Department of Urology, Hirosaki University School of Medicine, Hirosaki, Aomori 036-8563, Japan Received on July 18, 2007; revised on October 16, 2007; accepted on October 16, 2007 The oligosaccharide structures of prostate specific antigen (PSA) are expected to be useful in discriminating prostate cancer from benign conditions both accompanied by increased serum PSA levels. A large proportion of PSA forms a covalent complex with a glycoprotein, α 1 -antichymotrypsin, in human blood. In the present study, the glycan profiles of free and complexed forms of PSA from cancer patient serum and of seminal plasma PSA were compared by analyzing the glycopeptides obtained by lysylendopeptidase digestion of the electrophoretically separated PSA with mass spectrometry. The profiles of the PSA N-glycans from the free and complexed molecules were quite similar to each other and consisted of fucosylated biantennary oligosaccharides as the major class. They were mostly sialylated, and a considerable sialic acid fraction was α2,3-linked as determined by Streptococcus pneumoniae neuraminidase digestion of the glycopeptides. In the seminal plasma PSA, high-mannose and hybrid types of oligosaccharides were predominant, and the sialic acids attached to the latter as well as to biantennary oligosaccahrides were exclusively α2,6-linked because they were removed by Arthrobacter ureafaciens neuraminidase but resistant to S. pneumoniae neuraminidase. Complextype oligosaccharides from other sources were found in the seminal plasma sample, indicating that analysis of released glycans carries a risk of being misleading. The results suggest that identification of α2,3-linked sialic acids on PSA potentially discriminates malignant from benign conditions, if the analysis is applied to oligosaccharides specifically attached to the N-glycosylation site of PSA in either a free or a complexed form in the serum. Keywords: N-glycans/prostate specific antigen (PSA)/ prostate cancer/sialic acid Introduction Prostate cancer is one of the common cancers, and the incidence continues to rise in most regions of the world. The measurement whom correspondence should be addressed: Tel: +81-725-56-1220; Fax: +81-725-57-3021; e-mail: 1 To of prostate specific antigen (PSA), which has been available for 20 years and led to the early detection, management and followup of patients with prostate cancer (Stamey et al. 1987), is considered to be one of the best biochemical markers currently available in the field of oncology. PSA is a serine protease belonging to the kallikrein multigene family and consists of 237 amino acids and one N-linked oligosaccharide chain at Asn45 (van Halbeek et al. 1985; Bélanger et al. 1995). The majority of PSA produced by the prostate is excreted in semen but a small proportion leaks into the systemic circulation. PSA testing is based on the fact that prostate cancer tissues release 30 times more PSA into the circulation than the normal prostate tissue, perhaps due to the loss of normal tissue architecture (Stamey et al. 1987). The PSA that reaches the serum is found either in free form or bound with plasma proteins. The major binding proteins are α1 -antichymotrypsin (ACT) (Christensson et al. 1990) and α2 -macroglobulin, both of which are extracellular protease inhibitors abundantly present in the serum. Complex formation with α1 -antichymotrypsin–prostate specific antigen (ACT–PSA) results in the exposure of a limited number of the antigenic epitopes of PSA, whereas α2 -macroglobulin encapsulates the currently identifiable antigenic epitopes of PSA (Christensson et al. 1990). ACT–PSA is, therefore, the predominant immunoreactive form in the serum, whereas free PSA accounts for 5–40% of total immunoreactive PSA (Jain et al. 2002). Measuring free and complexed PSA and determining their ratio improve the diagnostic specificity of PSA testing (Lilja et al. 1991; Stenman et al. 1991) and can decrease the number of negative prostatic biopsies by 20–25% (Catalona et al. 1998). However, the incidence of prostate cancer has been shown to be as high as 22% in patients with a normal PSA range of 2.6–4.0 ng/mL (Catalona et al. 1997). Furthermore, while the PSA test is essentially organ specific, it is not cancer specific with elevated serum concentrations found in noncancerous diseases such as benign prostatic hypertrophy and prostatitis. There has, therefore, been an increasing emphasis on the need for novel serum markers for use in the diagnosis of prostate cancer. The carbohydrate structure of cancer cells is well known to differ considerably from that of nonmalignant cells (Fukuda 1996; Kim and Varki 1997), and a number of carbohydrate tumor markers have been used in clinical situations. Accordingly, the carbohydrate structures of PSA potentially discriminate cancer from benign diseases despite both showing serum PSA elevations. The structure of PSA carbohydrate is thought to be a biantennary complex-type oligosaccharide (Bélanger et al. 1995; Sumi et al. 1999; Prakash and Robbins 2000; Okada et al. 2001; Peracaula et al. 2003; Ohyama et al. 2004). PSA from prostate cancer tissues and a prostate cancer cell line was reported to contain the complex-type oligosaccharides with more antennas than the PSA from benign prostatic hypertrophy
C The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 2 N-glycans of prostate specific antigen tissues and seminal fluid (Sumi et al. 1999; Prakash and Robbins 2000). Although an obvious extension of these studies is to characterize the oligosaccharides of the serum PSA, the low PSA content of the human serum makes this difficult, and only a few reports are available on the structural details of the PSA glycans from cancer patient sera (Ohyama et al. 2004; Tabarés et al. 2006). To date, most of the studies on PSA glycans have been carried out on the oligosaccharides released from PSA samples, and they are not inherently free of contaminating glycans from other sources. In the present study, on the other hand, glycopeptides were analyzed by mass spectrometry (MS). MS and tandem MS of glycopeptides allow characterization of the site-specific glycans of glycoproteins in an efficient and quite reliable manner (Wada et al. 2004; Tajiri et al. 2005). The glycan profiles specific to PSA were analyzed for the free PSA (...truncated)