2 Department of Urology, Akita University School of Medicine, Akita, Japan; 3 Department of Biochemistry, Cancer Research Institute, Tohoku Pharmaceutical University, Sendai, Japan; 4 Chugai Pharmaceuticals, Bio-Product Technology Laboratory, Gotenba, Japan; 5 Department of Urology, Sendai National Hospital, Sendai, Japan; 6 Department of Urology, Tohoku University School of Medicine, Sendai, Japan; and 7 Glycobiology Program, Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037
Received on January 20, 2004; revised on February 27, 2004; accepted on February 29, 2004
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Abstract |
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Key words:
2,3-linked sialic acid
/
Maackia amurensis agglutinin
/
prostate-specific antigen
/
prostate cancer
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Introduction |
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Because PSA is a serine protease, some PSAs exist in the blood stream in the form of complex with serum protease inhibitors such as 1-antichymotrypsin and
2-macroglobulin, whereas the other PSA components exist in a free form (Christensson et al., 1993
). The total PSA assay detects both free and PSA-
1-antichymotrypsin complex. In spite of the application of free PSA as an additional indicator, there is a considerable overlap in a serum PSA concentration between the patients with prostate cancer and benign prostate hypertrophy patients (Vashi et al., 1997
). PSA is a glycoprotein containing approximately 8% carbohydrates composed of an N-glycan (Bélanger et al., 1995
). The structure of carbohydrate on PSA is thought to be a biantennary N-linked oligosaccharide of the N-acetyllactosamine type (Bélanger et al., 1995
; Okada et al., 2001
; Peracaula et al., 2003
; Prakash and Robbins, 2000
; Sumi et al., 1999
). Furthermore, it was reported that PSA from the prostate cancer tissues and the prostate cancer cell line (LNCaP) contains N-glycans with more antennas than PSA from the benign prostate hypertrophy tissues and the seminal fluid (Prakash and Robbins, 2000
; Sumi et al., 1999
).
Oncogenesis is accompanied by an alteration in the carbohydrate structure on the cell surface (Fukuda, 1996). Carbohydrates are expressed on the tumor cell surface and involved in the malignant potentials represented by invasion and metastasis (Fukuda, 1996
; Ohyama et al., 1999
), and a number of carbohydrate tumor markers have been used in the clinical situations. Some glycoprotein tumor markers are also expressed in the normal counterparts, but it is well known that the carbohydrate structure from cancer cells is considerably different from that of nonmalignant cells (Fukuda, 1996
). Alpha-fetoprotein and human chorionic gonadotrophin are typical examples of this event, where the difference of binding for a specific lectin is used for discriminating benign lesions from cancers (Amano et al., 1988
; Yamashita et al., 1993
). As for PSA, however, few studies have been performed because of the difficulty in obtaining sufficient amounts of it for carbohydrate structural analyses. Specifically, carbohydrate alteration in serum PSA from cancer patients has not been demonstrated. This may be partly due to the molecular diversity of serum PSA, which contains both free PSA and PSA-
1-antichymotrypsin complex (Christensson et al., 1993
).
In the present study, we performed a systematic carbohydrate structural analysis on the human seminal fluid PSA, and demonstrated the existence of sialic acid 2-3-galactose linkage in addition to the sialic acid
2-6 linkages, which had been considered the sole sialic acid linkage in most of the previous studies. Considering that cancer-associated carbohydrate alterations have been detected by lectin affinity chromatography of PSA (Prakash and Robbins, 2000
; Sumi et al., 1999
), we tested several different lectins to develop an improved assay system for discriminating cancer-associated PSA from that of benign lesions. We found that free serum PSA from prostate cancer patients exhibits increased binding to Maackia amurensis agglutinin (MAA) than that from benign prostate hypertrophy patients, suggesting that the MAA binding of serum PSA could be used as a novel diagnostic measure for prostate cancer.
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Results |
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Con A column chromatography followed by the PSA assay showed that the relative amount of branched N-glycans containing more than two antennas (Con Aunbound fraction) of free PSA was significantly higher in prostate cancer patients than that in benign prostate hypertrophy patients (p < 0.05) (Figure 3). In contrast, the high-mannose fraction of free PSA was significantly higher in benign prostate hypertrophy patients than that in prostate cancer patients (p < 0.05). There were no significant differences in biantennary and hybrid fractions in free PSA between prostate cancer and benign prostate hypertrophy patients.
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Among the lectins examined, the MAA-bound fraction of free PSA in prostate cancer patients was significantly higher than that in benign prostate hypertrophy patients (p < 0.001) (Figure 4). However the difference in the MAA-bound fraction of total PSA from prostate cancer patients and benign prostate hypertrophy patients was not significant. On the other hand, the SNA-bound fraction of free PSA in benign prostate hypertrophy patients was significantly more than that in prostate cancer patients (p < 0.05) (Figure 4). These results suggest that in prostate cancer 2,3-linked sialic acid replaced some
2,6-linked sialic acid in PSA N-glycans. There were no significant differences in the bound fraction of AAL and LcH lectins between prostate cancer and benign prostate hypertrophy patients (Figure 4). These results combined indicate that the amount of
2,3-linked sialic acid is increased in free PSA derived from prostate cancer patients compared to benign prostate hypertrophy. Conversely,
2,6-linked sialic acid in free PSA decreased in prostate cancer patients compared to benign prostate hypertrophy.
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Discussion |
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PSA is a glycoprotein with one asparagine (N)-linked oligosaccharide, and it is well known that malignant transformation is often associated with an aberrant glycosylation on the cell surface (Fukuda, 1996). Previously, it has been demonstrated that the cancer-associated alteration of the N-glycans on PSA was characterized by an increase in highly branched oligosaccharides (Prakash and Robbins, 2000
; Sumi et al., 1999
). This is consistent with the findings that the increased branching of N-glycans, which is formed by N-acetylglucosaminlytransferase V (GnT-V), was associated with cancer cells (Dennis et al., 1987
; Hubbard, 1987
; Pierce and Arango, 1986
; Saitoh et al., 1993; Yamashita et al., 1984
). Although the significance of sialic acid residue for the cancer-associated alteration of carbohydrates on PSA has also been suggested (Huber et al., 1995
), the precise terminal sialyl structure of PSA carbohydrates still remain to be elucidated. In the present study, we showed that the major core structure of N-glycans on the seminal fluid PSA, the normal counterpart of cancer-associated PSA, is biantennary N-glycans. This is consistent in principle with the previous reports showing biantennary N-glycans in PSA (Bélanger et al., 1995
; Okada et al., 2001
; Prakash and Robbins, 2000
; Sumi et al., 1999
). In addition, we revealed the terminal sialic acid linkage as a mixture of sialic acid
2-6 galactose and sialic acid
2-3 galactose with a 3:1 ratio.
Previously, the carbohydrate structure of human seminal PSA was investigated to detect only two components that are biantennary N-glycans with or without a fucose (Bélanger et al., 1995). Although the exact reason is unknown, the discrepancy between their results and ours may be due to, at least in part, the difference in the analyzing methods used. Bélanger et al. (1995)
performed the structural analysis of carbohydrates using nuclear magnetic resonance. Because this technique requires a large amount of samples, the minor components might have escaped from their analysis. In the present study, pyridylaminated N-glycans were analyzed by HPLC followed by two kinds of sialidase digestion for determination of the terminal sialic acid linkage. This process facilitated the precise structural analysis because different N-glycans can be distinguished using relatively small amounts of materials.
In our study, the statistically significant differences in the core carbohydrate structures of free serum PSA (branched N-glycans and high-mannose N-glycans) were observed between prostate cancer patients and benign prostate hypertrophy patients (Figure 3). However, there still were considerable overlaps. By contrast, we discovered that MAA-bound fraction of free PSA is significantly increased in prostate cancer patients compared with benign prostate hypertrophy patients, showing a distinct difference with no overlap between the two groups (Figure 4). Because MAA lectin specifically reacts with sialic acid 2-3 galactose terminal structure (Wang and Cummings, 1988
), this characteristic carbohydrate moiety of free serum PSA reflects a distinctive increase of sialic acid
2-3 galactose residue in the sera from prostate cancer patients.
It has been reported that PSA purified from LNCap prostate cancer cells contain poorly sialylated N-glycans (Peracaula et al., 2003). In contrast, PSA from prostate cancer patients contains increased
2,3-linked sialic acid, as shown in the present study. Although it is not clear why this discrepancy was obtained, it is possible that the LNCap cell line used in their study may be deviated from the parent cell line after many passages.
By contrast, unlike free PSA, total PSA failed to show any significant difference in MAA binding between prostate cancer and benign prostate hypertrophy patients. This discrepancy might be attributable to the presence of serum protease inhibitors. 1-Antichymotrypsin is a glycoprotein that contains bi- and triantennary N-glycans (Laine et al., 1991
). The lectin-bound fraction assayed by the total PSA detection kit may be affected by the carbohydrates attached to
1-antichymotrypsin, obscuring the difference in N-glycans attached to PSA between prostate cancer and benign prostate hypertrophy patients.
In conclusion, our present study showed that sialic acid 2-3 galactose residue coexists with sialic acid
2-6 galactose residue in the seminal fluid PSA and that there is a distinct increased binding to MAA lectin in free serum PSA isolated from prostate cancer patients compared to benign prostate hypertrophy patients. The distinction can be simply achieved by MAA affinity chromatography followed by estimating an amount of free and complex PSA by enzyme-linked immunosorbent assay. Although further studies are needed to define the clinical application, the MAA-bound fraction of free PSA may provide a promising diagnostic tool for a better discrimination of prostate cancer from benign prostate hypertrophy.
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Materials and methods |
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Prostate cancer cell line
The human prostate cancer cell line LNCaP was obtained from the American Type Culture Collection (Rockville, MD) and maintained with RPMI 1640 medium with 10% fetal calf serum. The culture medium after culturing in the serum-free medium (Opti-MEM, Gibco-BRL, Grand Island, NY) for 48 h was used as a source of the cancer-associated PSA for lectin binding assays.
Human benign prostate hypertrophy tissue
The human benign prostate hypertrophy tissue was obtained from a patient who underwent retropubic prostatectomy at the Department of Urology, Sendai National Hospital (Sendai, Japan). Histopathologic examination proved no malignancy in the surgical specimen.
Human serum samples
Serum samples were obtained from 15 randomly selected patients with newly diagnosed prostate cancer at various clinical stages (4 cases at T2N0M0, 5 at T3N0M0, and 6 at T3N1M1) and 15 patients with benign prostate hypertrophy whose total serum PSA levels were between 4.0 and 10.0 ng/ml. Clinical tumor staging was based on the TNM Atlas (4th edition, International Union against Cancer, 1997). These patients were treated at the Department of Urology, Sendai National Hospital. The diagnosis for all of the patients was confirmed by the histopathologic findings of the transurethral resection or prostate needle biopsy. The median age of patients was 72 years with a range of 5582 years for prostate cancer patients and 69 years with a range of 5281 years for benign prostate hypertrophy patients. The mean total PSA concentration of the prostate cancer patients and the benign prostate hypertrophy patients was 89 ng/ml (range 5.0128.0 ng/ml) and 8.8 ng/ml (range 5.010.0 ng/ml), respectively. Informed consent was obtained from all patients who provided the human materials.
Preparation of PA-N-glycans from human PSA
A complex type biantennary N-glycan-PA was prepared from the recombinant human erythropoietin as described previously (Kanazawa et al., 1999). The human seminal fluid PSA (300 µg) was denatured by boiling for 3 min in 240 µl 50 mM TrisHCl buffer, pH 7.0, containing 0.5% of sodium dodecyl sulfate and 1% 2-mercaptoethanol. After cooling down, 120 µl 7.5% NP-40 was added to the solution, and PSA was digested with 5 U N-glycosidase F (Boehringer Mannheim, Mannheim, Germany) at 37°C for 16 h. The completion of the reaction was confirmed by its migration on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The released N-glycans were separated from the protein portion and detergents by using reverse phase HPLC column (Vydac C4, 0.46 x 250 mm) equilibrated with 12% acetonitrile containing 0.1% trifluoroacetic acid at a flow rate of 1.0 ml/min. The unbound fraction was pooled and then lyophilized. The residue was dissolved in an aliquot of water and applied to a Bio-Gel P-4 column (1.0 x 45 cm, BioRad, Hercules, CA) equilibrated with 10 mM ammonium acetate buffer, pH 6, and the orcinol-positive fractions were collected and lyophilized. N-glycans thus obtained were pyridylaminated by the method described previously (Kuraya and Hase, 1992
). Excess reagents were removed by gel filtration using a HW-40F column (1.5 x 45 cm, BioRad) equilibrated with 10 mM ammonium acetate, pH 6.2.
HPLC
Anion exchange chromatography was performed using a Mono Q column (5 mm x 50 mm, Pharmacia, Basking Ridge, NJ) equilibrated with 10 mM ammonium in water, pH 9, at a flow rate of 1.0 ml/min at 25°C. After applying PA-glycans from human PSA to the column, PA-glycans bound to the column were eluted by increasing the concentration of ammonium acetate, pH 8.0, from 0 to 0.2 M in 40 min. PA-glycans were detected by fluorescence (excitation wavelength, 310 nm; emission wavelength, 380 nm).
Reverse phase HPLC was carried out using a Shimpack CLC-ODS column (6 x 150 mm, Shimadzu, Tokyo) equilibrated with 0.05% 1-butanol in 10 mM phosphate buffer, pH 3.8, at a flow rate of 1.0 ml/min at 55°C. After injecting a sample, the concentration of 1-butanol was raised linearly to 0.5% over 60 min. PA-sugar chains were monitored by measuring the fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm).
PA-glycans fractionated by anion exchange HPLC were digested with two kinds of sialidases; NANase I (Glyko, Novato, CA), which releases only 2-3-linked sialic acid and NANase II (Glyko) which releases both
2-3- and
2-6-linked sialic acids from glycans. PA-sugar chains were digested with 10 mU NANase I or 20 mU NANase II in 50 mM sodium phosphate buffer, pH 6.0, at 37°C for 16 h. Neutral PA-glycans were incubated with 20 mU of jack bean ß-galactosidase (Seikagaku, Tokyo) in 0.1 M citrate phosphate buffer, pH 4.0, or with 20 mU jack bean ß-N-acetylhexosaminidase (Seikagaku) in 0.1 M citrate phosphate buffer, pH 5.0, at 37°C for 16 h.
MALDI-TOF MS
MALDI-TOF mass spectra were acquired on a Voyager DE STR (Perceptive Biosystems, Albertville, MN) equipped with a nitrogen laser. Spectra were obtained in the linear mode at an accelerating voltage of 20 kV. The delayed extraction was used with a nominal pulse delay of 200 ns. As the matrix, 2,5-dihydroxybenzoic acid in water (15 mg/ml) was used for all samples. Equal volumes (5 µl) of the sample and matrix solution were mixed, from which 1 µl was applied on a sample plate. Each spectrum obtained was the average of 150200 scans. The oligosaccharides produced sodium adduct ions, and [M+Na]+ or [M+2Na]2+ were detected in a positive ion mode.
Lectin column chromatography and PSA assay
Con A, LcH, AAL, SNA, and MAA conjugated to agarose were purchased from EY Laboratories (San Mateo, CA). Lectin affinity column chromatography was performed as described previously with some modifications (Barak et al., 1989; Chan and Gao, 1991
; Kobata and Yamashita, 1993
). Briefly, columns of agarose-bound lectins were equilibrated with buffers containing 0.02% Tween 20, 0.02% sodium azide, and 0.5% bovine serum albumin. Serum samples were applied to the column and kept at room temperature for 30 min, followed by washing with five column volumes of the buffer. This fraction was designated as the lectin-unbound fraction. The lectin-bound fraction was eluted with five column volumes of hapten sugars (see following description of methods). For the precolumn, unbound, and bound fractions, total PSA and free PSA were measured by DPC Immulite HS-PSA and DPC Immulite free-PSA (Diagnostic Products, Los Angeles, CA), respectively. Total PSA includes PSA associated with serum protease inhibitors, whereas free PSA does not (Christensson et al., 1993
). The relative amount of the lectin-bound fraction against the precolumn amount was calculated.
For Con A column chromatography, samples were separated into four fractions: the unbound fraction (first fraction), the fraction eluted with 10 mM -methyl glucose (second fraction), the fraction eluted with 10 mM
-methyl mannose (third fraction), and the fraction eluted with 0.3 M
-methyl mannose (fourth fraction). Because PSA has no O-glycosylation site but one N-glycosylation site (Bélanger et al., 1995
), branched N-glycans containing more than two antennae are eluted in the first fraction, biantennary N-glycans in the second fraction, hybrid type N-glycans in the third fraction, and high-mannose type N-glycans in the fourth fraction, respectively. The percentage of each fraction relative to the precolumn amount was calculated. Statistical analysis was carried out by Mann-Whitney's U-test.
Preparation of PSA-positive fractions from benign prostate hypertrophy tissue and LNCaP culture medium
The prostate tissue from human benign prostate hypertrophy was minced and homogenized with 20 mM phosphate buffer, pH 7.4. Homogenate was centrifuged at 22,000 x g for 20 min, and the supernatant was collected. The tissue extract or LNCaP culture medium was fractionated by sequential precipitation with 25% and 70% saturated ammonium sulfate, and the resulting precipitate was dialyzed against 20 mM acetate buffer, pH 5.6. Each of the crude fractions was applied to a CM-Toyopearl cation exchange column (Tosoh, Tokyo) equilibrated with 20 mM acetate buffer, pH 5.6. After the pass-through fraction was washed with the starting buffer (300 ml), the adsorbed proteins were eluted with a linear gradient from 0 to 0.5 M of NaCl. Fractions containing proteins detected by absorbance at 280 nm were combined and subjected to the analysis for detecting the presence of PSA by immunoblotting method. After each sample (total of 35 mg protein) was separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (12.5% separation gel), the gel was blotted to polyvinylidene difluoride membrane (Immobilon-PSQ, Millipore, Bedford, MA). The membrane was incubated with rabbit anti-human PSA antibody (Dako, Copenhagen), followed by horseradish peroxidaselabeled anti-rabbit IgG antibody (Cedarlane Lab, Hornby, Ontario, Canada) and then developed with Konica immunostain HRP-1000. The PSA-positive proteins of LNCaP culture medium (LN-C7, 28.5 kDa) and benign prostate hypertrophy tissue (BP-C5, 29 kDa), prepared as described, were used for the surface plasmon resonance analysis.
Surface plasmon resonance analysis
Surface plasmon resonance was measured with an SPR670 system (Nippon Laser Electronics, Nagoya, Japan) using the sensor chip formed with self-assembly membrane by dithiodibutyric acid. After activating with ethyl-dimethylaminopropyl-carbodiimide hydrochloride and N-hydroxysuccinimide, the sensor chip was equilibrated with a flow buffer (10 mM phosphate buffered saline, pH 7.2) at a flow rate of 15 ml/min at 25°C. Then the anti-human PSA antibody (1:50 dilution) was loaded and covalently fixed onto the sensor chip. The remaining active residues on the chip were blocked with successive treatment of 1 M ethanolamine (pH 7.2) and 100 µg/ml of bovine serum albumin. After loading the PSA-positive fractions, 0.1 M H3PO4 was injected to wash the nonspecific binding materials out. To the antibody-selected PSA ligands, 500 µg/ml of two lectins, SNA and MAA (EY Laboratories), were loaded and reacted for 4 min (association phase), followed by washing with the flow buffer (dissociation phase).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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