2 Unitat de Bioquímica i Biologia Molecular, Departament de Biologia, Universitat de Girona, Campus de Montilivi s/n. 17071, Girona, Spain
3 Glycobiology Institute, Department of Biochemistry, Oxford University, Oxford OX1 3QU, United Kingdom
Received on October 23, 2002; revised on November 29, 2002; accepted on December 12, 2002
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Abstract |
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Key words: N-glycosylation / LNCaP cells / prostate cancer / prostate-specific antigen / tumor marker
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Introduction |
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Several approaches have been developed to improve the specificity of PSA tests (see Milford Ward et al., 2001, and references cited herein) to avoid diagnosing false positives as prostate cancer. The most common approach is to measure the ratio of free PSA (fPSA)/total PSA (tPSA) (free PSA plus PSA complexed to
-1-antichymotrypsin), which is lower in prostate cancer; this procedure is used in clinic diagnoses, although it still gives false positives. Other alternatives are PSA density (ratio of PSA concentration to prostate volume), the detection of pro-PSA and kallikrein 2 (Stephan et al., 2000
; Peter et al., 2001
), or PSA velocity (change in PSA concentration over time), but unfortunately none of these approaches has clearly shown a substantial improvement in the ability to distinguish between BPH and PCa.
A deeper knowledge of the PSA molecule is required to examine whether some of its biochemical characteristics, like glycosylation, could allow the distinction of PSA from normal and tumor origins and, therefore, be useful in developing new prostate cancer markers. PSA is a glycoprotein with a single N-oligosaccharide chain attached to Asn-45. The structure of PSA glycans from the seminal fluid of healthy donors have been partially characterized by 1H-nuclear magnetic resonance (NMR) (Bélanger et al., 1995) and oligosaccharide sequencing (Okada et al., 2001
). PSA glycans have been described as sialylated complex biantennary glycans, mostly core fucosylated and with a minor presence of GalNAcs in the antennae, the proportion of which seemed to be increased in the highest pI PSA fractions (Okada et al., 2001
). However, the type and extent of sialylation of these glycans were not determined.
Because oncogenesis leads to a remarkable alteration of cellular glycosylation, tumor-secreted glycoproteins may reflect the altered glycosylation pattern of cancer cells and are likely to be good candidates for tumor markers (Lis and Sharon, 1993; Kim and Varki, 1997
; Dennis et al., 1999
; Orntoft and Vestergaard, 1999
). In that context, we have recently described that human pancreatic RNase 1, a glycoprotein secreted mostly by pancreatic cells, has completely different oligosaccharide chains when produced by pancreatic tumor cells (Fernández-Salas et al., 2000
; Peracaula et al., 2003
), suggesting a new possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma. These results prompted us to investigate the glycosylation pattern of other tumor-secreted proteins, such as PSA.
To establish whether the glycosylation of PSA, which is secreted either by normal and tumor prostate cells, could be altered in prostate tumors, we analyzed and compared the glycosylation pattern of PSA purified from seminal fluid of healthy donors with PSA produced by prostate tumor cells from a human prostate tumor cell line, LNCaP. Human prostate carcinoma cell lines are very useful models for characterizing markers for PCa and for elucidating the mechanisms of these markers. Few prostate tumor cell lines have been established; among them, the LNCaP cell line (established from a prostate cancer metastasis in the lymph node) (Horoszewicz et al., 1980) is the most commonly used model for the study of PCa because it retains some of the most prominent differentiated features of the human prostate cell, including the synthesis of the prostate-specific proteins such as PSA (Kumar et al., 2000
). PSA glycans from LNCaP cells had been partially characterized by oligosaccharide polyacrylamide gel electrophoresis (PAGE) (Prakash and Robbins, 2000
); tumor PSA appears to contain glycans with some triantennary structures apart from the biantennary structures that were also present in normal PSA. Chromatofocusing analysis (Wu et al., 1998
) showed that LNCaP PSA had higher pI than normal PSA, indicative of a minor content of sialic acid in tumor PSA.
To confirm and clearly establish the glycosylation differences between PSA from normal prostate and from LNCaP cells, a detailed analysis of the oligosaccharides of PSA of normal and tumor origin was carried out, including high-performance liquid chromatography (HPLC), exoglycosidase digestions analysis, and mass spectrometry (MS). Our results show significant differences between PSA from the normal and tumor situations. In particular, major differences, which could also be detected by specific antibodies against carbohydrate epitopes, were the complete absence of sialic acid and the presence of higher fucose and GalNAc content in LNCaP PSA.
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Results |
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Overall profiles of PSA glycans (high and low pI PSA) from normal seminal fluid by NP-HPLC analysis
Glycosylated PSA samples (low and high pI) were electrophoresed in sodium dodecyl sulfate (SDS)PAGE gels under reducing conditions and used for glycan analysis (Figure 1a). PSA oligosaccharides were released from gel bands by in situ digestion with PNGase F and fluorescently labeled with 2-aminobenzamide (2AB). Normal-phase (NP) HPLC of PSA-labeled oligosaccharides showed that they contained glycans with glucose unit (GU) values ranging from 7.0 to 8.6 for low pI PSA and from 6.2 to 7.9 for high pI PSA (Figure 1b).
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Sequential oligosaccharide digestions of PSA glycans (low pI PSA) from normal seminal fluid
First, NP-HPLC analysis of an aliquot of the glycan pool from low pI PSA before and after digestion with exoglycosidase arrays was carried out (Figure 2).
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Analysis of sialylation of low pI PSA glycans from normal seminal fluid
To determine the extent and type of sialylation of each of the individual PSA glycans, weak anion exchange chromatography (WAX) was carried out (Figure 3). The column was calibrated with N-glycans (neutral, monosialylated, disialylated, and trisialylated) from bovine fetuin (Figure 3a). 2AB-labeled N-glycan pools from PSA untreated (Figure 3b) or treated with NANI (Figure 3c) were separated according to charge. An increase in the proportion of monosialylated glycans was detected after digestion by NANI, indicating that some of the PSA disialylated glycans contained one sialic acid linked 2-3. Three pools were collected from each chromatographic WAX run: 05 min, contained neutral oligosaccharides; 79 min, contained monosialylated; and 1012 min, contained disialylated structures. Subsequent analysis of each pool by NP-HPLC chromatography (data not shown) revealed which glycan structures were neutral or contained one or two sialic acids (Figure 2a and Table I).
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Digestion with S. pneumoniae galactosidase (SPG, specificity for galactose linked ß1-4) (data not shown) in the enzyme array produced the same results as when using bovine testes ß-galactosidase (BTG, specificity for galactose linked ß1-3/4>6) (Figure 2d), indicating that all the galactose residues are linked ß1-4 to GlcNAc.
Treatment with S. pneumoniae ß-N-acetylhexosaminidase (SPH, specificity for GlcNAc linked ß1-2>4 to mannose) resulted in the formation of FcGlcNAc2Man3 from the FcA2 structure, GlcNAc2Man3 from the A2 structure, and FcA1GalNAc1 and A1GalNAc1 from the glycans that contained GalNAc (Figure 2e). Most of the structures (83%) contained one fucose 1-6 linked to the core GlcNAc.
The core fucose was digested when a further treatment with bovine kidney fucosidase (BKF, broad specificity for -linked fucoses) was carried out (Figure 2f), resulting in the formation of two structures: the pentasaccharide core, GlcNAc2Man3, and A1GalNAc1 in a proportion of 75% to 25%, respectively.
Finally, a further treatment with jack bean ß-N-acetylhexosaminidase (JBH), which digests terminal GalNAc and GlcNAc (Figure 2g), digested the A1GalNAc1 to GlcNAc2Man3.
MS-ESI-LC analysis of low pI PSA glycans from normal seminal fluid
Electrospray ionization (ESI) mass spectra of the glycans from low pI PSA contained three major ions of masses 1099.9, 1245.4, and 1265.9 with compositions Hex5HexNAc4Fuc1Neu5Ac1, Hex5HexNAc4Fuc1Neu5Ac2, and Hex4HexNAc5Fuc1Neu5Ac2, respectively (Table I). These masses agree with the assignments derived from the digestion data.
In conclusion, the major glycan structures of low pI PSA were complex biantennary structures containing mono- or disialylated glycans with sialic acid linked 2-6 and/or
2-3. About 25% of glycans contained GalNAc. Fucose was present linked
1-6 to the core GlcNAc in 83% of the structures.
Sequential oligosaccharide digestions of PSA glycans (high pI PSA) from normal seminal fluid
The structural determination of the glycans from high pI PSA was carried out by NP-HPLC analysis of an aliquot of the glycan pool before and after digestion with exoglycosidase arrays (Figure 4).
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Further treatment with BTG (Figure 4c) indicated the presence of the following neutral glycans: FcA2, FcA2GalNAc1, A2, and FcA1. These structures were corroborated with subsequent digestions with BKF and SPH (Figure 4d, e).
High pI PSA glycans appear to be mostly monosialylated, which explains the higher pI of this PSA fraction. Some of the structures to which sialic acids are attached correspond to oligosacharides with incomplete antennae, that is, FcA2Gal1, FcA1Gal1, and FcA2GalNAc1. The proportion of structures containing GalNAc is around 20%, similar to that found for the low pI PSA glycans. In the same way, fucose was only detected linked 1-6 to GlcNAc.
Culture of LNCaP cells in different conditions
LNCaP cells were grown in presence and absence of 10% fetal bovine serum (FBS). In the latter case, cells were stimulated with dihydrotestosterone (DHT) to increase PSA expression.
The conditioned media of LNCaP cells grown in 10% FBS produced 1.5 µg/ml PSA, determined by sandwich enzyme-linked immunosorbent assay (ELISA), a similar concentration to the one described by Prakash and Robbins (2000) when they stimulated these cells with DHT. In contrast, PSA concentration from LNCaP cells grown without FBS was only 0.6 µg/ml, a lower concentration explained by the androgen-dependent behavior of LNCaP cells for both growing and secreting PSA (Corey et al., 1998
; Langeler et al., 1993
; Vaïsänen et al., 1999
). When these LNCaP cells growing in the absence of FBS were supplemented with 125 nM DHT, PSA concentration rose to 3.5 µg/ml.
Purification of PSA from the tumor cell line LNCaP
A new method was set up to purify to homogeneity PSA from LNCaP-conditioned media. The purification procedure was carried out slightly differently when cells were growing in the absence or presence of FBS. In the first case, only two chromatographic steps were required to purify PSA; in the latter case two extra chromatographic steps were added to obtain pure PSA separated from the other contaminating proteins contained in FBS.
When purifying PSA from LNCaP media without FBS, the first step was an affinity-chromatography using a Cibacron-Blue 3GA column. This column has been reported to bind to several proteins such as enzymes with known affinities to nucleotide cofactors. Previous assays have indicated that this resin binds strongly to PSA, which was eluted by high ionic strength. A typical elution profile is shown in Figure 5a. Positive fractions that contained PSA were detected by ELISA sandwich assay that has a sensitivity of 15 ng/ml. These fractions were then pooled and rechromatographed in a reverse-phase column C-4 (Figure 5b). The PSA fraction eluted at 40% of acetonitrile. PSA purity after these two chromatographies was checked by SDS electrophoresis silver-stained gel and western blot (Figure 6), where a major band of about 32 kDa corresponded to the purified protein. A minor band of a slightly lower molecular weight was also detected. One hundred fifty milliliters of LNCaP medium without FBS and stimulated with DHT yielded 50 µg of pure PSA, whereas 150 ml LNCaP medium without FBS and hormone yielded 1015 µg protein.
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N-terminal sequencing analysis of LNCaP PSA
N-terminal sequence analysis of purified PSA from LNCaP media was performed to further characterize the protein. Most PSA molecules contained the mature protein, with a regular NH2 terminus. Pro-PSA forms (zymogen forms) were also identified in about one third of the molecules: a more abundant -5 (LILSR) form and a minor -7 (APLILSR) form.
Characterization of oligosaccharides of PSA from LNCaP tumor cells
Overall profiles of PSA glycans from LNCaP tumor cells by NP-HPLC analysis
Oligosaccharides from the electrophoretic bands of PSA purified from LNCaP cells grown in different conditionsthat is, with FBS, without FBS, with presence and absence of DHT (Figure 7a,b)were released by in situ digestions with PNGase F, fluorescently labeled with 2AB and subjected to NP-HPLC and matrix-assisted laser desorption ionization and time-of-flight MS (MALDI-TOF MS).
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Sequential oligosaccharide digestions of PSA glycans from LNCaP tumor cells
NP-HPLC analysis of an aliquot of the glycan pools from PSA fractions before and after digestion with exoglycosidase arrays was carried out (Figures 8 and 9).
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Further digestion with SPH (Figure 9c) resulted in the formation of FcGlcNAc2Man3 from FcA2, FcA1GalNAc1 from FcA2GalNAc1, and FcA1Gal1F1(12) from FcA2- Gal1F1(12). Peaks 3 (FcA2GalNAc2) and 5b (FcA2Gal1- GalNAc1F1[12]) were undigested because GalNAc needs JBH to be digested. The proportion of glycan structures containing GalNAc was 65%.
Next, digestion with BKF (Figure 9d) indicated that all glycans contained core fucose because all shifted at least 0.4 GU. Some glycans had extra fucoses linked 1-2 to galactose (FcA1Gal1F1[12] and FcA2GalNAc1Gal1F1 [12]) and were digested to GlcNAc2Man3 and A1GalNAc1, respectively.
Finally, a further JBH treatment (Figure 9e) digested glycans with GalNAcs to the pentasaccharide core structure, GlcNAc2Man3.
MALDI analysis of PSA glycans from LNCaP tumor cells
MALDI analysis of glycans from tumor PSA is shown in Figure 10 and Table II. The major ions corroborated the structures described by exoglycosidase arrays analysis.
Summary of PSA glycans from LNCaP tumor cells
PSA glycans from LNCaP were all neutral and fucosylated. Major glycans are listed in Table II. Fucosylation was found to be core fucose in nearly all glycan structures, and about 15% contained outer-arm fucose linked 1-2 to galactose residues, which gives rise to the H2 epitope (Fuc
1-2Galß1-4GlcNAc, Figure 8) (Oriol, 1995
). The proportion of glycan structures that contain GalNAc was 65%.
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Discussion |
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PSA from LNCaP cells contains zymogen forms
About two-thirds of the PSA from LNCaP cells consisted of normal NH2 terminus PSA, and about one-third contained two zymogen forms. Vaïsänen et al. (1999) described higher amounts of the pro-PSA forms in LNCaP media that could be explained by the absence of human kallikrein 2 in their PSA preparation. Human kallikrein 2 is likely to activate any pro-PSA form into mature PSA (Vaïsänen et al., 1999
; Lövgren et al., 1999
). Different proforms of PSA in serum of prostate cancer patients have been identified as a possible diagnostic for distinguishing PCa from BPH, because pro-PSA forms were more abundant in PCa tissues (Mikolajczyk et al., 2000
). However, the detection of this pro-PSA form in sera and its diagnostic value still remain uncertain (Peter et al., 2001
).
Major glycosylation differences in PSA from normal and tumor origins
The glycosylation analysis of PSA from normal seminal fluid and from the prostate cell line LNCaP revealed major differences in their glycan structures. Significant changes mostly affected the outer ends of the oligosaccharide chains. PSA glycans from normal and tumor sources were both complex biantennary structures that differed in their content of GalNAc, sialic acid, and fucose, giving rise to distinct carbohydrate epitopes.
Changes in GalNAc content
An increase in levels of GalNAc (from 25% to 65%) was found in PSA from LNCaP cells. GalNAc is not commonly found in N-glycans of vertebrates. However, it has been identified in some glycoproteins from the pituitary gland and from other vertebrate sources, such as bovine milk, rat prolactin, or kidney epithelial cells (Manzella et al., 1996; Varki et al., 1999
). ß-Linked GalNAc has also been reported in melanoma tissues (Chan et al., 1991
; Kuo et al., 1998
); we have recently described it in some of the tumor N-glycan structures from pancreatic adenocarcinoma cells Capan-1 (Peracaula et al., 2003
), suggesting that the expression of this carbohydrate is related to a malignant transformation.
Differential expression in sialic acid: sialylated glycans are absent in tumor PSA
One of the most interesting differences between PSA from normal and tumor origin was the content of sialic acid. In contrast to LNCaP glycans that did not contain sialic acid in their structures, sialic acid was detected in nearly all glycans from the most abundant PSA fraction (low pI PSA) from seminal fluid. Bélanger et al. (1995) had already described by 1H-NMR that the major glycoform was a disialylated biantennary complex structure, with a core fucose, FcA2Gal2Neu5Ac2(26). More recently, Okada et al. (2001) characterized the N-glycans of the two PSA isoforms (low and high pI PSA) from seminal fluid but without analyzing their type and extent of sialylation. Here we carried out a detailed analysis of the sialic acids. Interestingly, in addition to sialic acidlinked
2-6, we also detected sialic acidlinked
2-3 in some of the disialylated structures of low pI PSA, indicating the activity of more than one sialyltransferase on PSA (Varki et al., 1999
). The different pI of both PSA isoforms is indicative of their content of sialic acid. Low pI PSA had about 50% of each, mono- and disialylated structures, whereas high pI PSA only contained monosialylated structures.
LNCaP PSA was reported to have a higher pI than normal PSA from seminal plasma (Huber et al., 1995; Wu et al., 1998
; Herrala et al., 1998
). These differences are consistent with the lack of sialic acid we described for LNCaP PSA glycans. Similar differences in PSA pIs have been reported between serum PSA of benign hyperplasia and prostate tumor patients, with higher pIs in the latter case (Huber et al., 1995
), suggesting that the glycosylation changes described in PSA from prostate tumor cells may be reflected in serum PSA.
Differential expression in fucose: H2 antigen is present only in tumor PSA
Fucose content was also altered between PSA from normal seminal fluid and prostate tumor cells. Most normal PSA glycans (83%) contained core fucose linked 1-6 to GlcNAc. This proportion was increased in PSA LNCaP glycans where nearly all structures were core fucosylated. Moreover, 1015% of the glycan structures from LNCaP PSA had another fucose linked
1-2 to an outer-arm galactose, giving rise to the H2 epitope (Fuc
1-2Galß1-4GlcNAc) (Oriol, 1995
). The presence of the H2 epitope agrees with the reported expression of
1-2-fucosyltranferase in LNCaP cells (Marker et al., 2001
; Chandrasekaran et al., 2002
). In these studies, Marker et al. (2001)
suggest that the activity of
1-2-fucosyltranferase may contribute to pathological prostatic growth. Thus, as it had been described for the lack of sialic acid in prostate tumor cells, the H2 epitope could be expressed in the tumor situation and be used for tumor PSA identification.
Changes in glycosylation, in particular fucosylation and sialylation, have been reported in prostate tumor tissues. By immunohystochemistry studies, carbohydrate antigens of Lewis class II, especially Lewisy, known to be minimal or absent in benign secretory epithelial cells, are highly expressed in tumor tissues (Martensson et al., 1995; Zhang et al., 1997
; Culig et al., 1998
). In addition, the up-regulation of sialyl Lewisx correlates with poor prognosis of the tumor (metastasic prostate cancer) (Jorgensen et al., 1995
). Lectin studies on tumor tissues have also revealed a different glycosylation pattern on their N- and O-linked glycoproteins (Arenas et al., 1999
). A strong expression of fucose residues detected by the lectins Ulex europaeus agglutinin and Aleuria aurantia agglutinin staining was described for N-linked glycoproteins of PCa tissues. Some of these changes could be reflected in prostate tumorsecreted glycoproteins such as PSA, which does present a higher fucose content, and could be used for identifying tumor PSA.
In conclusion, the need to identify PSA when it originates from tumor cells to distinguish between benign and malign prostatic pathologies has prompted us to analyze the glycosylation pattern of normal and tumor PSA. The differences reported, especially those referring to the content of sialic acid and fucose, suggest a possible way to discriminate between both situations, although further investigations are needed to reveal whether these glycosylation differences could be reflected in serum PSA and their usefulness for diagnosis purposes.
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Materials and methods |
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PSA from human seminal fluid (low pI PSA and high pI PSA) were purchased from Lee Scientific (St. Louis, MO).
Electrophoresis and western blotting
Electrophoresis in 12% SDSpolyacrylamide mini-gels was performed at room temperature according to the method of Laemmli (1970). The gels were silver stained following the method of Blum et al. (1987)
. Molecular mass standards were obtained from Invitrogen (Carlsbad, CA). All samples were reduced with 5% 2-mercaptoethanol before analysis.
Analysis of PSA was performed following standard western blot protocols. PSA samples were electrophoresed in a 12% SDSPAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) at a constant voltage of 30 V, overnight at 4°C, in 192 mM glycine/Tris 25 mM/methanol 20%. Filters were blocked in 3% (w/v) nonfat milk and 0.1% Tween in Tris-buffered saline and incubated for 1 h with a rabbit polyclonal antibody against PSA (Dako, Glostrup, Denmark) diluted 1:2000 in blocking buffer. Secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL) was added at 1/10,000 and incubated for 1 h. Detection was performed using a Super Signal West Dura chemiluminescence kit (Pierce).
Cell culture conditions
LNCaP cells (ATCC CRL-1740) were a generous gift from Dr. A. Berenguer of the Department of Urology (Hospital Universitario de Getafe, Madrid, Spain).
LNCaP cells (106) at passage 7075 were cultured and maintained in RPMI 1640 with glutamine (Gibco, Paisley, Scotland, United Kingdom) supplemented with 10% FBS (Linus [Cultek], Madrid, Spain) and 50 µg/ml gentamicin (Gibco). All cell cultures were incubated at 37°C in a humidified atmosphere (95%) and 5% CO2 for 2 or 3 days. The supernatant was removed, and the cells were washed with PBS. LNCaP cells were grown in one of the following media for 2 or 3 days: (1) RPMI 1640 with glutamine supplemented with 10% FBS; (2) RPMI 1640 with glutamine without FBS and supplemented with 125 nM DHT (Sigma, St. Louis, MO); or (3) RPMI 1640 with glutamine without FBS and without DHT. The different media were collected, centrifuged, and stored at -20°C.
Chromatographic purification of PSA from LNCaP cells medium grown with FBS
Cell culture media was filtered through a 0.22-µm membrane (Gelman-Pall, Ann Arbor, MI, ) and concentrated 10 times with a tangential filtration device by using a 5000-Da cut-off polysulfone membrane (Millipore). The concentrated medium was dialyzed against buffer A: 25 mM Tris-HCl/20 mM NaCl, pH 7.5, by using 3500 Da cut-off tubing (Spectra-Por, Rancho Domínguez, CA, ), and was loaded into a Cibacron-Blue 3GA (Sigma) affinity column at a flow rate of 0.2 ml/min that had been preequilibrated using the same buffer. The unbound protein was eluted with 4 column volumes (CVs) of buffer A at a flow rate of 0.5 ml/min. PSA fractions were eluted with a 25 mM2 M NaCl linear gradient (4 CVs), followed by 3 CVs of buffer B: 25 mM Tris-HCl/2 M NaCl, pH 7.5, and 3 CVs of buffer C: 25 mM Tris-HCl/1 M NaSCN, pH 7.5. Five- to ten-milliliter fractions were collected after the gradient start. The fractions collected were tested by sandwich ELISA, and PSA-containing fractions were pooled and freeze-dried.
The next chromatographic step was a gel filtration chromatography in Biogel P60 (BioRad, Hercules, CA). The buffer used for sample dilution, column equilibration, and protein elution is buffer D: 50 mM Tris-HCl/200 mM NaCl, pH 7.5. The flow rate was maintained at 0.3 ml/min. Two-milliliter fractions were collected and assayed for PSA presence by sandwich ELISA. Positive fractions for PSA were pooled, dialyzed against water, and freeze-dried.
The next chromatographic step was Cibacron-Blue column 1 ml (Amersham Pharmacia, Little Chalfont, United Kingdom) in an HPLC system (AKTA, Amersham Pharmacia), using the same buffers (A, B, and C) and protocol as previously described.
Finally, a reversed-phase chromatography column (214 TP-RP C-4; Vydac, Hesperia, CA, ) was performed in an HPLC system. The sample was dissolved in 0.5 ml Milli-Q-grade water. The starting and column equilibration buffer was 90% buffer E: 0.1% trifluoroacetic acid in Milli-Q-grade water/10% buffer F: 0.1% trifluoroacetic acid in acetonitrile. The flow rate was 0.5 ml/min. The following linear step gradient was used: 1025 min, 1025% eluent F; 2575 min, 2550% eluent F; 7585 min, 50100% eluent F. Fractions were tested for PSA by sandwich ELISA, and the positive ones were pooled and freeze-dried.
Chromatographic purification of PSA from LNCaP cells medium grown without FBS
A two-step chromatographic purification protocol was carried out after filtering the conditioned media through a 0.22-µm membrane. First, PSA was purified by Cibacron-blue column and then by a reversed-phase chromatography column, following the same protocols as described.
Amino-terminal sequence analyses were performed by automated Edman degradation on a Beckman LF3000 Protein Sequencer at the Proteomics facility of the Institut de Biomedicina i Biotecnologia (Universitat Autònoma de Barcelona, Spain).
Release and purification of N-linked oligosaccharides
N-glycans were released from purified PSA by in situ digestion of the protein in SDSPAGE gel bands with PNGase F, (Roche) as described earlier (Küster et al., 1997). Briefly, purified PSA fractions were separated by electrophoresis under reducing conditions and visualized by Coomassie staining. Bands containing the glycoprotein were excised from the gel, reduced, alkylated, and treated with PNGase F to release the N-linked glycans. Wide range molecular markers were from Sigma.
Fluorescent labeling of the reducing terminus of oligosaccharides
Oligosaccharides were fluorescently labelled with 2AB by reductive amination (Bigge et al., 1995) using the Oxford GlycoSciences (Abingdon, Oxford, United Kingdom) Signal labeling kit.
HPLC
NP-HPLC was performed using a TSK-Gel Amide-80 4.6 mmx250 mm column (Tosoh Biosep, Montgomeryville, PA) on a 2690 Alliance separations module (Waters, Milford, CT) equipped with a Waters temperature control module and a Waters 474 fluorescence detector. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 30°C. Gradient conditions were a linear gradient of 2058% A, over 152 min at a flow rate of 0.4 ml/min. Samples were injected in 80% acetonitrile. Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to create a dextran ladder, as described previously (Guile et al., 1996).
WAX HPLC (Guile et al., 1994) was performed using a Vydac 301VHP575 7.5x50 mm column (Anachem, Luton, Bedfordshire, United Kingdom) according to the modified methodology described by Zamze et al. (1998)
. Briefly, solvent A was 0.5 M formic acid, adjusted to pH 9 with ammonia solution. Solvent B was 10% (v/v) methanol in water. Gradient conditions were a linear gradient of 05% A over 12 min at a flow rate of 1 ml/min, followed by 521% A over 13 min, then 2150% A over 25 min, 80100% A over 5 min, then 5 min at 100% A. Samples were injected in water.
Simultaneous oligosaccharide sequencing by exoglycosidase digestions
All enzymes were purchased from Glyko (Novato, CA). The 2AB-labeled oligosaccharides were digested in a volume of 10 µl for 18 h at 37°C in 50 mM sodium acetate buffer, pH 5.5, using arrays of the following enzymes: ABS (EC 3.2.1.18), 1 U/ml; NANI (EC 3.2.1.18), 1 U/ml; AMF (EC 3.2.1.111), 3 mU/ml; BKF (EC 3.2.1.51), 1 U/ml; SPG (EC 3.2.1.23), 0.1 U/ml; BTG (EC 3.2.1.23), 1 U/ml; SPH (EC 3.2.1.30), 120 mU/ml; and JBH (EC 3.2.1.30), 50 U/mL. After incubation, enzymes were removed by filtration through a protein binding nitrocellulose membrane (Pro-Spin 45 µm CN filters, Radley and Co., Essex, United Kingdom), and oligosaccharides were analyzed by NP-HPLC.
MALDI-TOF MS
Positive ion MALDI-TOF mass spectra were recorded with a Micromass TofSpec 2E reflectron-TOF mass spectrometer (Micromass, Manchester, United Kingdom) fitted with delayed extraction and a nitrogen laser (337 nm). The acceleration voltage was 20 kV; the pulse voltage was 3200 V; the delay for the delayed extraction ion source was 500 ns. Samples were prepared by adding 0.5 µl of an aqueous solution of the sample to the matrix solution (0.3 µl of a saturated solution of 2,5-dihydroxybenzoic acid in acetonitrile) on the stainless steel target plate and allowing it to dry at room temperature. The sample/matrix mixture was then recrystallized from ethanol (Harvey, 1993).
HPLC-ESI-MS
ESI LC/MS data were obtained with a Waters CapLC HPLC system interfaced with a Micromass hybrid quadrupole time-of-flight mass spectrometer fitted with a Z-spray electrospray ion source and operated in positive ion mode. A 1x150 mm microbore NP-HPLC column was packed with stationary phase material from a GlycoSep N column (Oxford GlycoSciences). The operating conditions for the mass spectrometer were: source temperature 100°C, desolvation temperature 120°C,desolvation gas flow 200 L/h, capillary voltage 3000 V, cone voltage 30 V, TOF survey scan time 1 s, mass range m/z 502300; TOF MS/MS scan time 1 s, survey scan 9501600 with detection mass range m/z 503500, mass selection resolution about 3 Da.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: pauline.rudd{at}bioch.ox.ac.uk or rafael.llorens{at}udg.es
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Abbreviations |
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References |
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