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 June 18, 2002; revised on September 6, 2002; accepted on September 23, 2002
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Abstract |
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Key words: Capan-1 / human ribonuclease / Lewis antigens / N-acetylgalactosamine / pancreatic cancer
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Introduction |
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Human pancreatic ribonuclease (RNase 1) is a secretory glycoprotein found in most human tissues, but mainly in the pancreas, where it is expressed by acinar cells (Futami et al., 1997; Fernández-Salas et al., 2000
; Peracaula et al., 2000
) prior to secretion into the pancreatic ducts. RNase 1 is also expressed by pancreatic adenocarcinoma tissues and secreted by pancreatic adenocarcinoma cell lines, presumably of ductal origin (Fernández-Salas et al., 2000
; Peracaula et al., 2000
).
Pancreatic adenocarcinoma is one of the carcinomas with the worst prognosis and is very difficult to diagnose. It is the fourth to fifth leading cause of cancer-related mortality in Western countries (Mangray and King, 1998; Greenlee et al., 2001
). A variety of serum tumor markers, especially CA19-9, a Lewis blood grouprelated mucin, have been proposed for diagnosis and follow-up of this type of neoplasia, but their application remains experimental (Lillemoe et al., 2000
). The identification of a specific early tumor marker would result in substantial improvements in the survival and treatment of the patients affected by pancreatic adenocarcinoma.
RNase 1 has been detected in serum by enzymatic and immunological methods, and its elevation in pancreatic cancer suggested that it might be a useful indicator of this carcinoma. However, although RNase 1 levels in pancreatic adenocarcinoma sera are usually higher than in healthy sera, the differences are not specific enough to use the absolute amount of RNase 1 as a tumor marker (Reddi and Holland, 1976; Weickman et al., 1984
; Kurihara et al., 1984
; Kemmer et al., 1991
; Kobayashi and Kawakubo, 1994
; Tabarés, 2000
).
Monosaccharide composition analysis of RNase 1 glycans from healthy pancreas indicated higher levels of fucose and absence of sialic acid in comparison to RNase 1 secreted from a human pancreatic tumor cell line, Capan-1 (Ribó et al., 1994; Fernández-Salas et al., 2000
). In addition, enzyme-linked immunosorbent assays (ELISAs) of RNase 1 from healthy pancreas and Capan-1 using anticarbohydrate antibodies (Fernández-Salas et al., 2000
) showed a differential expression of Lewis antigens in the two samples. These data suggest that the altered glycosylation of RNase 1 could permit differentiation between the RNase 1 produced by pancreatic acinar cells and the RNase 1 produced by pancreatic adenocarcinoma cells. Moreover, aberrantly glycosylated RNase 1 in serum could provide a tumor marker of pancreatic adenocarcinoma. To establish a firm analytical basis for these findings and to provide an insight into the perturbations in the levels of glycosylating enzymes in the tumor cells, a more detailed analysis of the glycosylation of normal and tumor-associated RNase 1 was performed.
Accordingly, using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we compared the oligosaccharides released from RNase 1 purified from pancreas and from two human pancreatic adenocarcinoma cell lines, Capan-1 and MDAPanc-3. The compositions of the oligosaccharide pools were completely different, suggesting that different glycosyltransferases are involved in the pancreatic tumor cell glycan processing pathways. Some of the glycosylation changes in RNase 1 correspond to blood group-related carbohydrate antigens, in particular the Lewis antigens. Lewisy antigen was only detected in RNase 1 from healthy pancreas. In contrast, RNase 1 from tumor cells expressed charged structures, such as sialyl-Lewisx or sialyl-Lewisa antigens, differences that could easily be identified by western blot analysis with antibodies against the Lewis structures. We present the details of the altered glycosylation pattern of RNase 1 that allow distinction between normal and a tumor situation. These findings suggest that the detection of glycosylation changes both in RNase 1 and in other serum glycoproteins secreted by carcinoma cells could be of great value in the discovery of new tumor markers.
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Results |
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Characterization of oligosaccharides: overall profiles of RNase 1 glycans from pancreas by normal phase HPLC analysis
Oligosaccharides from the different electrophoretic bands of RNase 1 from normal pancreas were released by in situ digestions with PNGase F, fluorescently labeled with 2-aminobenzamide (2AB) and subjected to normal phase HPLC and both matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography (LC)-electrospray (ESI) MS. From the many RNase 1 fractions analyzed, glycan profiles of only five typical examples are shown (P1, P3, P5, P7, and P9) that correspond to RNase 1 fractions with different levels of glycosylation (Figure 1e). Glycans were analyzed on the basis of their elution positions, measured in glucose units (GU) (Guile et al., 1996) and from their masses as obtained from MALDI-TOF and LC-ESI MS. Fourteen structures with GU values from 6.8 to 10.0 (Peaks 112, Figure 1e and Tables I and II) were present in all HPLC fractions (P1P9), although in varying amounts. Additional glycans with GU units from 10.3 to 14.4 were present in the more highly glycosylated RNase 1 fractions, P1P6 (155%), where two or three glycosylation sites were occupied (Figure 1e). Fractions P7P9, in which only Asn-34 was glycosylated, contained mainly bianntenary glycans with GU values between 6.8 and 10.0. Increasing amounts of the larger glycans (with GU>10) were detected where Asn-76 and/or Asn-88 sites were occupied, indicating that both of these sites could accommodate larger sugars than Asn-34. As the number of occupied sites increases, so does the overall complexity of the individual glycans.
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Digestions of the defucosylated glycans from the RNase 1 fractions, with either Streptococcus pneumoniae galactosidase (SPG) (specificity for galactose linked ß1-4) (Figure 3e) or bovine testes ß-galactosidase (BTG) (specificity for galactose linked ß1-3/4>6) (Figure 3f) in the enzyme array, produced the same results, that is, A2, A2B, A3, and A4. This indicates that all galactose residues are linked ß1-4 to GlcNAc.
Finally, treatment with S. pneumoniae ß-N-acetylhexosaminidase (SPH, specificity for GlcNAc linked ß1-2>4 to mannose) resulted in the formation of (Man)3(GlcNAc)2 from A2 structures (Figure 3g). When A3 structures were present, (GlcNAcß1-4)(Man)3(GlcNAc)2 was also obtained as a product of incomplete digestion. The difficulty in removing the GlcNAc 1-4 attached to mannose (Guile et al., 1998) indicated that these glycans were mainly branched on the 3-antenna. A4 structures were digested to a glycan with a GU value of 6.13, corresponding to A3. Only the GlcNAc, which was linked ß1-2 to the mannose of the 3-antenna, was removed, because the enzyme has difficulty removing ß1-2 linked GlcNAc from a mannose to which a second GlcNAc is ß1-6 linked (Yoshima et al., 1980
).
Detailed analysis of fucosylation in RNase 1 glycans from pancreas
The fucosylated structures were further characterized by sequential exoglycosidase digestion of the glycan pool using AMF (specificity for fucoses linked 1-3/4) and BKF (broad specificity for
-fucoses). AMF digested one or two fucose residues from the biantennary glycans (peaks 5, 8a, 9, 10ab, 11, 12) (decrease in elution position of around 0.8 GU per fucose) (Figure 4b). The triantennary glycans contained from one to three fucose residues. The fucoses were assumed to be attached to GlcNAc in the
1-3 rather than in the
1-4 position as galactose had already been linked to GlcNAc in the ß1-4 position (see previous discussion). Treatment with BKF removed from one to four fucose residues linked either
1-2 to galactose or
1-6 to the internal GlcNAc (core fucose) (Figure 3d). To establish the linkage of these fucose residues, the glycosylation pattern of a digestion with AMF+BTG was compared with that from a digestion with AMF+BTG+BKF (Figure 4c, d). If fucose was linked to galactose at the nonreducing termini, BTG would not be able to remove the galactose. On the other hand, if fucose was attached to outer arm GlcNAc, then digestion with AMF+BTG would remove the terminal galactose residue. The structures of the major polyfucosylated peaks could then be determined. Peaks 8, 10, and 12 (Figures 1
4, Tables I and II) contained glycans with two fucoses linked
1-2 to outer arm galactose and one core fucose; peaks 6, 9, and 11 glycans had one fucose
1-2 linked to outer arm galactose, and one core fucose; and peaks 1, 5, 8 and 10 contained only one core fucose. All glycans with outer arm fucose either
1-3 or
1-2 linked also contained fucose linked
1-6 to the core GlcNAc, as is commonly the case (D. R. Wing, personal communication).
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Oligosaccharide sequencing of RNase 1 glycans from the pancreatic adenocarcinoma cell line Capan-1: RNase 1 purification
RNase 1 fractions were purified from Capan-1 cell culture media following the method described by Fernández-Salas et al. (2000) with some modifications. After collection and concentration of the Capan-1 conditioned medium, RNase 1 glycosylated fractions were first purified by heparin affinity column chromatography, as in RNase 1 purification from pancreas. RNase 1 fractions were then pooled and further purified by a MONO-S cationic exchange column and a Vydac C4 reversed-phase column. Pure ribonuclease fractions eluted from reversed-phase chromatography were identified by ribonuclease activity in a zymogram (Bravo et al., 1994
), and their homogeneity was tested by SDSPAGE (Figure 5a). Glycosylated RNase1 fractions (CP2-CP5) were used for glycan analysis.
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As described for pancreatic RNase 1 fractions, the different electrophoretic behavior of the RNase 1 fractions from Capan-1 reflected different levels of glycosylation. Glycosylated bands with a high apparent molecular mass had two or three glycosylation sites occupied, whereas low-apparent-molecular-mass glycosylated fractions had only one site occupied (Fernández-Salas et al., 2000). Thus, glycosylated fractions of apparent molecular mass higher than 24 kDa (CP2a, CP3ab, CP4ab, CP5ab) contain more than one occupied glycosylated site, whereas CP4c and CP5c correspond to RNase 1 fractions with only one site occupied (Figure 5a). Band CP5d contains unglycosylated RNase 1.
Normal phase HPLC of Capan-1 oligosaccharides showed that all these RNase 1 fractions contained glycans with GU values ranging from 6.8 to 10.0 with similar profiles (Figure 5b). In addition, the more highly glycosylated RNase 1 bands CP2a, CP3a, CP4a, and CP5a contained a proportion of larger glycans (GU from 10 to 14). The lower bands, CP3b, CP4b, and CP5b, contained smaller amounts of these larger glycans, but none were detected in the lowest bands CP4c and CP5c.
Exoglycosidase sequencing of Capan-1 RNase 1 glycans: CP3a glycans
Capan-1 RNase 1 glycan pools released from these bands were digested by exoglycosidase arrays. All the different glycan pools gave similar results, which are discussed in the following (Figure 6).
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Analysis of fucosylation in RNase 1 glycans from Capan-1
Further digestion of the glycans from band CP3a with AMF (Figure 6a, panel c) indicated the presence of one or more fucose residues, linked either 1-3 or
1-4. Additional digestion with BKF removed additional fucoses (data not shown). To determine the linkage of these fucoses (i.e., whether they are outer arm fucoses linked
1-2 to galactose or
1-6 to the core GlcNAc) a digestion with ABS+AMF+BTG was compared with that from ABS+AMF+BTG+BKF (Figure 6a, panels d and e). Glycans with GU ranging from 6.8 to 10 produced a similar glycan pattern in both digestions, but with a shift of 0.4 GU units for all peaks except one (unshaded peak, Figure 6a, panel d) when the digestion contained BKF. These data indicated that this peak alone had no core fucose. The observation that all galactose residues were removed with ABS+AMF+BTG shows that no fucose was linked to these external galactoses in these structures, and, furthermore, the fucose residues removed with BKF corresponded to a core fucose (Figure 6a, panel e). However, glycans with GU higher than 10 contained several fucose residues that were linked either
1-2 or
1-6.
Analysis of core structures of RNase 1 glycans from Capan-1
Digestions of the glycan pool with SPG (specificity for galactose linked ß1-4) (data not shown) and BTG (specificity for galactose linked ß1-3/4) (Figure 6a, panel e and Figure 6b, panel a) gave slightly different results indicating the presence of galactose linked both 1-3 and 1-4). These data indicate that fucoses released by AMF were linked in both the 1-3 or 1-4 positions.
Some of the glycan structures obtained after BTG or SPG digestions, in particular the major peak at GU 5.74 (Figure 6a, panel e and Figure 6b, panel a), appeared to contain the complex biantennary bisected glycan A2B. Comparing the profile of the glycans after a further digestion with either jack bean ß-N-acetylhexosaminidase (JBH) or SPH showed that most of this peak was digested with SPH but not with JBH (Figure 6b, panels b and c). Digestions on standards have shown that JBH does not digest glycans with bisecting GlcNAc, whereas SPH does, consistent with the assignment of A2B to the major component of the peak at GU 5.74.
SPH is not very efficient at removing GlcNAc linked ß1-4 to mannose but will remove a bisecting GlcNAc (as discussed). However, SPH does not digest GalNAc residues. Thus, a further digestion with JBH, which does remove GalNAc, was carried out (Figure 6b, panel d). Digestion with both SPH and JBH resulted in Man3, Man4, and Man5 peaks (Figure 6b, panel d), which indicated the presence of both GalNAcs and hybrid glycans. These data were confirmed by MS (see following discussion). Hybrid glycans represented about 35% of the total structures, with approximately equal proportions of Man4 and Man5 hybrids. Digestion of the intact glycan pool with jack bean -mannosidase shifted several peaks, confirming the presence of hybrid mannose structures (data not shown).
MS analysis of Capan-1 RNase 1 glycans
MALDI and ESI mass spectra of the glycans from band CP5c (Figure 7, Table III) contained several major ions with a high percentage of HexNAc. The most prominent of these had compositions of Hex3HexNAc6dHex2 (mass 2157.8) and, particularly Hex3HexNAc6dHex3 (mass 2303.9) (Figure 7a). The exoglycosidase digestions had shown the presence of fucoses in these glycans, so dHex is assigned as a fucose throughout the rest of the discussion. The ESI tandem mass spectrometry (MS/MS) spectra of these compounds (see the following) contained prominent B-type fragment ions (Domon and Costello, 1988, fragmentation nomenclature) at m/z 407.2 (Figure 8) corresponding to a composition of HexNAc2. Glycans with this composition have been found on several glycoproteins (Green et al., 1985
; Green and Baenziger, 1988
; Chan et al., 1991
; Van-den-Eijnden et al., 1995
) and contain antennae consisting of GalNAc-GlcNAc rather than Gal-GlcNAc. The inability of SPH to digest these structures, even though they were sensitive to JBH, was consistent with the presence of the terminal GalNAc residue. The amount of material available was insufficient to confirm this by gas chromatography MS.
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The structure producing the [M+2H]2+ ion m/z 1141.6 (Hex3HexNAc6Fuc3) (Figure 8) appears from the enzyme digests to be a trifucosylated complex biantennary glycan containing two GalNAc-GlcNAc antennae. Although the glycan was probably a mixture of isomers, depending on the positions of fucose substitution, the MS/MS spectrum confirmed the biantennary structure. The prominent fragment at m/z 407.2 (Figure 8) corresponded to a composition of HexNAc2 and was absent from the spectra of the hybrid structures, indicating that it did not originate from the core. Where Gal-GlcNAc chains are present, a corresponding ion at m/z 366.2 is produced from the antennae (Figure 9). A corresponding singly charged ion at m/z 1875.9 was formed by loss of two unsubstituted HexNAc residues. It is most likely that this fragmentation occurs as a single cleavage rather than as two independent losses of single HexNAc residues thus supporting the occurrence of an unsubstituted GalNAc-GlcNAc chain. This conclusion is supported by the MS/MS spectrum of the 2AB derivative of a trifucosylated biantennary (Gal-GlcNAc containing antennae) from human parotid gland that is known to contain mainly outer arm fucose residues on the same (3) antenna (Gillece-Castro et al., 1991; Guile et al., 1998
). This compound also fragmented by cleavage at the GlcNAc residue of the antenna with loss of Gal-GlcNAc. Ions were also present in the spectrum (Figure 8) at m/z 553.3 and 699.3 corresponding to the compositions of (HexNAc)2 with one and two fucose residues, respectively, thus locating two of the fucose residues to one of the antennae. The weak fragment at m/z 699.3 corresponding to a composition of HexNAc2Fuc2 showed that at least some of the molecules contained two outer arm fucose residues attached to the same antenna. The location of the third fucose residue on the reducing-terminal GlcNAc residue (core fucose) was indicated by the presence of the singly charged Y-type fragment ion at m/z 488.3 ([GlcNAc(Fuc)-2AB+H]+).
Fragmentation of the compound with a [M+2H]2+ ion at m/z 1068.4 (Hex3HexNAc6Fuc2) indicated a similar structure but with one less outer arm fucose residue (singly charged ions at m/z 407.2 [HexNAc2], 553.3 [HexNAc2Fuc], and 488.3 [HexNAc(Fuc)-2AB]). It was not possible from the spectrum to determine the location of the fucose residue attached to the antennae.
MS/MS spectra of the compounds containing four HexNAc residues suggested that several isomers existed for most masses. The high content of hexose, up to seven residues, detected by MALDI MS and ESI MS in compounds of this type suggested that these compounds were hybrid structures, and this was confirmed by the fragmentation patterns. A representative spectrum is shown in Figure 9 for the compound with a composition of Hex5HexNAc4Fuc ([M+2H]2+ ion at m/z 954.9). The abundant Y-type ion at m/z 488.3 (GlcNAc[Fuc]-2AB) confirmed the presence of a core-fucose residue. This spectrum was unlike that of a reference sample of a core-fucosylated biantennary glycan, a common N-linked glycan with this composition. The most abundant fragment ion was the singly charged ion at m/z 204.1 (HexNAc). This ion, along with the fragment at m/z 1704.8 (singly charged), which corresponds to the loss of one HexNAc from the precursor ion, and the very low abundance of the ion at m/z 407.2 (GlcNAc2) as compared to Figure 9, indicates the presence of an unsubstituted terminal GlcNAc residue but absence of the GalNAc-GlcNAc moiety. The ion at m/z 366.2, corresponding to Hex-HexNAc, was consistent with the presence of a terminal Hex-HexNAc group. Loss of this moiety gave the ion at m/z 1542.8. An abundant singly charged Y-type fragment at m/z 1218.6 corresponded to the loss of three hexose residues from the ion at m/z 1704.8 (loss of HexNAc) or two hexoses from m/z 1542.8, consistent with a hybrid structure (cleavage at the core mannose). It was unclear, however, if the spectrum represented a mixture of these, or other isomers.
Additional information on the structure of these glycans came from the results of the digests with ABS, AMF, BKF, BTG, SPH, and JBH (Figure 6) which showed that both Man4- and Man5-containing hybrid structures were present. In addition, MALDI and ESI MS data indicated that compounds containing four HexNAc residues existed with three to seven hexose residues. This distribution would correspond to hybrid glycans with from zero to two mannose residues attached to the mannose of the 6-arm (Man3Man5 structures) together with up to two galactose residues attached to the two noncore GlcNAc residues that comprised the 3-antenna. Thus, the fully substituted (seven-hexose-residues) glycan would contain five mannose and two galactose residues, and the least substituted compound would only contain the core mannose residues. This distribution of hexose residues confirmed the presence of two HexNAc residues on the 3-antenna and the absence of bisecting structures in at least some of these glycans.
Although the compounds with seven hexose residues must have contained two Gal-GlcNAc residues on the 3-antenna, their structures are unambiguous. However, for compounds with unsubstituted GlcNAc residues, there is the possibility that this GlcNAc residue was attached in the bisecting position between 1-3 and
1-6 arms. A fragment ion thought to be unique for the presence of bisecting GlcNAc (GlcNAc-Man-GlcNAc-GlcNAc [Fuc]-2AB) was present at m/z 1056.5 in the spectrum shown in Figure 9, suggesting the presence of a bisect, but none of the other spectra that were examined contained an ion at this mass, strongly indicating that most contained two GlcNAc residues on the 3-antenna and no bisecting GlcNAc.
Summary of Capan-1 RNase 1 glycans
HPLC, exoglycosidase digestions, and mass spectra analyses have shown that RNase 1 from Capan 1 possesses a range of complex, bisected, and hybrid N-glycans, some of which contain GalNAc residues. The main core structures, deduced by MS/MS fragmentation, were hybrid glycans with a branched 3-antenna and biantennary structures containing mainly Gal-GlcNAc chains (see Figure 10). Both types of compounds were substituted with up to three fucose residues and some were additionally sialylated (Table III). Bisected biantennary structure A2G2B was deduced by exoglycosidase analysis, and their substitution with fucoses and sialic acid was determined by LC-ESI MS (Figure 10 and Table III).
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The results of exoglycosidase digestions on the glycans from fraction M2 are shown in Figure 11b. Digestion with sialidase (ABS) suggested the presence of sialic acids, whereas fucosidase digestions indicated the presence of fucose linked 1-3 or 1-4 to the antennae and core fucose. Treatment with galactosidases (SPG and BTG) gave similar digestion products, indicating that most galactose residues were linked ß1-4 to GlcNAc. The glycans obtained after galactosidase digestion are likely to be A2 and A2B from their GU values, and there were also other structures of higher GU that may be hybrid glycans. The low levels of glycans precluded a rigorous analysis in these MDAPanc3 RNase 1 fractions. However, the general conclusion that can be drawn from the available data is that the major glycans are most likely to be core and outer arm fucosylated complex biantennary oligosaccharides, some of which could contain sialic acid.
Western blotting and ELISA analysis of RNase 1fractions with antibodies against Lewis antigens
Some of the major differences in the glycan structures from pancreas, Capan-1, and MDAPanc-3 RNase 1 were confirmed by immunoblotting using antibodies against Lewis antigens (Figure 12).
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To evaluate if these glycosylation processing differences could be detected by western blotting, high (two to three occupied sites) and medium (one to two occupied sites) glycosylated RNase 1 fractions from healthy pancreas, Capan-1, and MDAPanc-3 were probed with antibodies to Lewisy, sialyl-Lewisx, and sialyl-Lewisa antigens. Glycosylated RNase 1 fractions (150250 ng) were loaded onto an SDSPAGE gel and transferred to a PVDF membrane. The conditions of the transfer were modified from the standard ones (Trisglycine, pH 8.5, 20% methanol) to produce a maximum yield for the transfer. Bicarbonate-carbonate, pH 9.9, 20% methanol was used as a transfer buffer because basic proteins such as RNase and lysozyme are more efficiently transferred under conditions of high pH (data not shown).
Highly glycosylated fractions of RNase 1 from Capan-1 were positive for sialyl-Lewisx while, in contrast, most glycosylated RNase 1 fractions from pancreas were positive for Lewisy (Figure 13a, b). MDAPanc-3 RNase 1 fractions were negative for Lewisy and sialyl-Lewisx but were immunoreactive with anti-sialyl-Lewisa antibodies (Figure 13c). The membranes were stripped and reprobed with anti-RNase 1 polyclonal antibodies to corroborate that the Lewis positive bands were from the RNase 1 fractions. These results were also confirmed by ELISA analysis: 1050 ng of medium and highly glycosylated fractions from pancreas were positive for Lewisy and negative for sialyl-Lewisx or sialyl-Lewisa fractions consistent with our previous data (Fernández-Salas et al., 2000) (data not shown). Capan-1 and MDAPanc-3 fractions were positive for sialyl-Lewisx and sialyl-Lewisa, respectively (Figures 13d, e).
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Discussion |
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Capan and MDAPanc-3 cells express SLex and SLe a antigens on their cell surfaces
Altered expression of carbohydrate antigens has been described in both pancreatic adenocarcinoma tissues (Kim et al., 1988; Philipsen et al., 1991
; Sinn et al., 1992
; Zhang et al., 1997
; Nakamori et al., 1999
) and in pancreatic tumor cell lines (Mas et al., 1998
; Hosono et al., 1998
). Increased expression of Lewisx, sialyl-Lewisx, and sialyl-Lewisa has been detected in pancreatic adenocarcinoma tissues (Kim et al., 1988
; Sinn et al., 1992
; Nakamori et al., 1999
). In normal tissues, duct cells and centroacinar cells abundantly expressed type I structures, such as Lewisa and Lewisb (Philipsen et al., 1991
), and Lewisy has been detected in both ductal and acinar cells (Philipsen et al., 1991
; Kim et al., 1988
). Lewisy was also expressed in pancreatic cancers (Kim et al., 1988
; Zhang et al., 1997
). Several tumor pancreatic cell lines presented an enhanced expression of sialyl-Lewisa and sialyl-Lewisx structures on the cell surface (Mas et al., 1998
; Hosono et al., 1998
). In this article, we extended these findings to MDAPanc-3 and Capan-1 cells, both metastatic pancreatic tumor cell lines, demonstrating that they express SLex and SLea antigens on their surfaces, too. Increased sialylation of tumor cell surface molecules is a well-known phenomena; sialic acid, typically found at the termini of sugar chains of glycoconjugates, is an important monosaccharide that can serve as ligand in important recognition phenomena such as lectin binding (Varki, 1997
).
Differential expression of Lewis antigens in tumor and normal secreted RNase 1: SLex and SLea in tumor RNase 1 and Ley in normal RNase 1
The changes in glycosyltransferase activities in tumor cells, which were apparent from the analysis of their cell surface glycosylation, were also reflected in their secreted glycoproteins, which use the same biosynthetic pathway. In this article, the analysis of the oligosaccharides attached to a pancreatic glycoprotein, RNase 1, secreted by both normal and tumor pancreatic cells (Capan-1 and MDAPanc-3), revealed two completely different glycosylation patterns. One of the most significant differences was the presence of acidic glycans in tumor RNase 1. In Capan-1 RNase 1, these charged oligosaccharides fractions, which represent about a quarter of the total glycans, were shown to contain sialic acid and gave rise to SLex epitope. These data are consistent with the structures found by ELISA on the cell surface of these Capan-1 cells.
In contrast to RNase 1 glycans from tumor cells, N-glycans of RNase 1 from normal pancreatic cells presented a higher content of fucose and no sialic acid. These data are in agreement with the glycosylation described for glycopeptides derived from human pancreatic secretion (which includes RNase 1) that also contained high levels of fucose and no sialic acid (Yoshihara et al., 1995). The fucosylated glycans in RNase 1 were mainly biantennary, but triantennary, tetraantennary, and some polylactosamine glycans, all extensively fucosylated, were also detected. These larger glycans, completely absent in the RNase 1 from tumor cells, were identified in the highly glycosylated fractions of pancreas RNase 1, suggesting that they are probably located at Asn-76 and/or Asn-88.
Some of these N-glycans from normal RNase 1 contained the Ley epitope because they terminated in fucose 1-2 linked to terminal galactose instead of sialic acid (Figure 12). ELISA and western blotting analysis confirmed their presence. In contrast, SLex and SLea were detected in RNase 1 from Capan-1 and MDAPanc-3 cells, respectively (Figure 13). The detection of the different epitopes in normal and tumor RNase 1 by commercially available monoclonal antibodies (mAbs) suggests that this method may be used to detect these differences in other tissues or biological fluids.
Our data suggest that there is a high level of 1-2-fucosyltransferase activity in normal pancreatic cells in contrast to Capan-1 tumor cells where there is increased
2-3-sialyltransferase activity. These results agree with studies described for other human metastatic pancreatic cancer cells where it was reported that the expression of sialyl-Lewisx and sialyl-Lewisa is regulated by a diminution of the activity of
1-2 fucosyltransferase and an increase of
1-3/
1-4-fucosyltransferases and
2-3-sialyltransferase (Mas et al., 1998
; Aubert et al., 2000
).
Normal and Capan-1 pancreatic RNase 1 and N-acetylglucosaminyltransferases (GnTs)
In normal RNase 1, bisected GlcNAc was present only in 10% of the complex biantennary structures. In RNase 1 from Capan-1, bisecting structures were also detected, although not quantified. Bisecting GlcNAc is attached by GnT III, and its activity is increased in pancreatic adenocarcinoma (Nan et al., 1998), together with that of GnT IV and V. These authors also showed that RNase 1 from pancreatic cancer patient serum bound to wheat germ agglutinin lectin more than serum RNase 1 from healthy donors, suggesting that the tumor RNase 1 glycans contained an increased level of bisecting GlcNAc.
Other N-glycans from Capan-1 contained hybrid structures with two antennae on the 3-arm that would arise from the activity of GnT IV that is also increased in human pancreatic adenocarcinoma (Nan et al., 1998). Increased glycan branching is a common feature of malignant cells and leads to further termini that can be sialylated.
Capan-1 RNase 1 has N-linked glycans containing ß-linked GalNAc
Some of the tumor N-glycan structures contain ß-linked GalNAc. The carbohydrate attachment of this oligosaccharide is controlled by ß1-4-N-acetylgalactosaminyl transferase activity, and it is not commonly found in N-glycans. However, it has been identified in the glycoproteins from the pituitary gland (Green et al., 1985; Green and Baenziger, 1988
; Chan et al., 1991
; Van-den-Eijnden et al., 1995
) and from other vertebrate sources, such as bovine milk, rat prolactin, or kidney epithelial cells (Varki et al., 1999
). ß-linked GalNAc has also been reported in melanoma tissues (Chan et al., 1991
; Kuo et al., 1998
), suggesting that the expression of this carbohydrate is related to a malignant transformation.
We have shown that in pancreatic cancer, both cell surface glycoproteins and secreted RNase 1 are aberrantly glycosylated. Glycan processing is cell type-specific; therefore other glycoproteins secreted from pancreatic tumors would also be expected to show disease-associated glycosylation changes. The results presented in this article offer the possibility of distinguishing between a normal pancreas and one containing a tumor by detecting the altered glycosylation of circulating glycoproteins. Elevated serum levels of RNase 1 are not specific enough to be used as a marker for pancreatic carcinoma (Reddi and Holland, 1976; Weickman et al., 1984
; Kurihara et al., 1984
; Kemmer et al., 1991
; Kobayashi et al., 1994
; Tabarés, 2000
). The tumor-associated glycosylation changes in glycoproteins, such as RNase 1, which is secreted and shed into the blood stream, may be detected by mAbs or lectins against specific carbohydrate epitopes, such as SLex and SLea, opening a new way of using this protein as a tumor marker of pancreatic cancer.
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Material and methods |
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RNase 1 fractions were further purified by two chromatographic steps: HiTrap Heparin column (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) and Vydac C4 (Vydac, Hesperia, CA) reversed-phase chromatography. The HiTrap Heparin column was preequilibrated with 50 mM TrisHCl, 20 mM NaCl, pH 7 before sample injection. The column was eluted with 20 column volumes of a linear NaCl gradient (0.020.6 M) followed by 10 column volumes of a second linear NaCl gradient (0.61.5 M). Ribonuclease fractions were eluted between 0.375 and 0.75 M NaCl, dialyzed against MilliQ water, and freeze-dried. Reversed-phase chromatography with a Vydac C4 column was carried out as described previously (Ribó et al., 1994).
RNase 1 purification from conditioned cell media
Capan-1 (American Type Culture Collection no. HTB-79) and MDAPanc-3 cell lines (Frazier et al., 1990) were cultured in 10% synthetic serum basal medium supplement (Seromed/Biochrom KG, Berlin, Germany). These cells were adapted to grow in this medium following the protocol described by Fernández et al. (1994)
.
RNase 1 was purified from the conditioned media of Capan-1 and MDAPanc-3 cultures using a modification of the protocol of Fernández-Salas et al. (2000). At the first step of purification, instead of an anionic-exchange column (DEAE-Sepharose CL-6B, Amersham Pharmacia Biotech), the concentrated media were loaded in a HiTrap Heparin column in an HPLC system (AKTA, Amersham Pharmacia) as described for RNase 1 purification from pancreas. The next two chromatographic steps, cationic exchange on a MONO-S HR 5/5 column (Amersham Pharmacia Biotech) and reversed-phase on a Vydac C4 column, were performed following the protocol described previously (Fernández-Salas et al., 2000
). After each chromatographic step, ribonuclease activity and sample purity were checked by zymography (Bravo et al., 1994
) and by SDSPAGE with silver staining (Blum et al., 1987
), respectively.
Release and purification of N-linked oligosaccharides
N-glycans were released from purified ribonuclease 1 fractions by in situ digestion of the protein in SDSPAGE gel bands with N-glycosidase F (PNGase F, Roche, Mannheim, Germany) as described earlier (Küster et al., 1997). Briefly, purified ribonuclease 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, (Pool, Dorset, UK).
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 Signal labelling kit (OGS, Abingdon, Oxon, UK).
HPLC
Normal-phase HPLC was performed using a TSK-Gel Amide-80 4.6 mmx250 mm column (Tosoh Biosep LLC, Montgomeryville, PA) on a 2690 Alliance separations module (Waters, Milford, MA) 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).
Weak anion-exchange HPLC (Guile et al., 1994) was performed using a Vydac 301VHP575 7.5x50 mm column (Anachem, Luton, Bedfordshire, UK) 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; Newcastle disease virus neuraminidase (EC 3.2.1.18), 200 mU/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; JBH (EC 3.2.1.30), 10 mU/ml; and jackbean -mannosidase (EC 3.2.1.24), 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, UK) and oligosaccharides were analyzed by normal phase HPLC.
For incubations with jack bean -mannosidase, the enzyme was added, incubated overnight, and then a second aliquot was added and incubated for a further 8 h. The enzyme was then denatured by boiling for 5 min, the sample was spun, and the supernatant was analyzed by normal phase HPLC.
MALDI-TOF MS
Positive ion MALDI-TOF mass spectra were recorded with a Micromass TofSpec 2E reflectron-TOF mass spectrometer (Micromass, Manchester, UK) fitted with delayed extraction and a nitrogen laser (337 nm). The acceleration voltage was 20 kV; the pulse voltage was 3200 V; and 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 LC-ESI MS
LC-ESI 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 normal phase HPLC column was packed with stationary phase material from a GlycoSep N column (Oxford GlycoSciences, Abingdon, Oxon, UK). The operating conditions for the mass spectrometer were as follows: 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 tandem mass spectrometry (MS/MS) scan time 1 s, survey scan 9501600 with detection mass range m/z 503500; and mass selection resolution about 3 Da. The MS to MS/MS automatic switching was initiated when the nominated peak intensity rose above 4. Switching back to MS mode occurred after 30 s or when the peak intensity fell below 1. The mass of this peak was then ignored by the automatic switching routine for 30 s. An automatic collision energy (CE) profile was used with CEs of 1432 V. Results from the different collision energies were combined for data evaluation.
N-glycan analysis by ELISA and western blotting
mAbs F3 (anti-Lewisy) and KM-93 (anti-sialyl-Lewisx) were from Calbiochem (Darmstadt, Germany). The mAb against sialyl-Lewisa was a generous gift from C. de Bolós (Bolós et al., 1995). Peroxidase-conjugated goat anti-mouse immunoglobulins, IgG+IgM, was from Jackson Immunoresearch Laboratories (West Grove, PA). Polyclonal antibodies against human recombinant RNase 1 were obtained by standard procedures (Peracaula et al., 2000
).
ELISA. 1050 ng of purified samples, classified according to their glycosylation (highly glycosylated, 23 occupied sites, and medium-low glycosylated, 12 occupied sites), were bound overnight at 4°C to 96-well polystyrene plates in carbonate/bicarbonate buffer, pH 9.6. After washing twice with saline (0.9% NaCl solution)Tween 0.05%, plates were blocked with 1% nonfat milk, 0.05% (v/v) Tween in phosphate buffered saline (PBS) for 1 h at room temperature. The wells were then washed once with salineTween 0.05% and were incubated for 2 h at room temperature with the above primary MAbs at 1/50 dilution in PBS, 0.05% Tween, except for anti-sialyl-Lewisa (1/2 dilution). Plates were washed four times with salineTween 0.05% and the second antibody, peroxidase-conjugated goat anti-mouse antiserum 1:2000 in PBS, 0.05% Tween, was added and allowed to stand for 1 h at room temperature. Plates were washed again four times with salineTween 0.05%, and detection was performed using a chemiluminescence kit (SuperSignal West Dura, Pierce, Rockford, IL) in an automated chemiluminescence reader (Roche). Data are expressed as the mean±SD of assays performed in triplicate. Negative controls were wells without antigen, or without the first antibody, and wells with lysozyme as antigen.
Western blotting. Analyses of Lewis antigens were performed following standard western blot protocols. Briefly, RNase 1 fractions were pooled in high and medium glycosylated fractions, electrophoresed in a 15% SDSPAGE gel and transferred to a polyvinylidine difluoride membrane (Roche). The transfer buffer was 3 mM carbonate/10 mM bicarbonate, pH 9.9, 20% methanol. The transfer was performed at 100 V for 3 h. Filters were blocked in 3% (w/v) nonfat milk, 0.1% Tween in Tris-buffered saline and incubated for 2 h with anti-sialyl Lewisx (1/50), anti-Lewisy (1/50), or anti-sialyl Lewisa (1/2) in blocking buffer. Secondary antibody, peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs) was added at 1/8000 and incubated for 1 h. Detection was performed using a chemiluminescence kit.
Cell ELISA
Capan-1 (ATCC no. HTB-79) and MDAPanc-3 cell lines (Frazier et al., 1990) were cultured to 90% confluence in Dulbecos's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were seeded on a 96-wells plate at a concentration of 104 cells/well and allowed to grow to confluence. After the cells had been washed three times with PBS (pH 7.2), they were fixed with 2% formaldehyde in PBS for 20 min at room temperature. The assay was carried out following the protocol described by Zhou et al. (1991)
. Briefly, after washing with PBS and blocking the plate wells with 1% bovine serum albumin in PBS, specific mouse mAbs against carbohydrates (anti-sialyl-Lewisx or anti-sialyl-Lewisa at 1/50 and 1/2 dilutions, respectively, in 1% bovine serum albumin in PBS) were added and incubated for 2 h at room temperature. The wells were then washed and incubated with the secondary antibody, peroxidase-conjugated goat anti-mouse IgG 1/2000 diluted in 1% bovine serum albumin in PBS for 1 h at room temperature. The assay was developed with 100 µl/well of 3,3',5,5'-tetramethylbenzidine (BM BluePOD substrate soluble, Roche). The reaction was stopped with 100 µl/well of 1 M H2SO4, and the absorption was measured at 450 nm (against a reference wavelength of 630 nm) in an automated microplate reader (BIO-TEK, Winooski, VT). As negative controls, highly differentiated breast carcinoma cells MCF-7 (Soule et al., 1973
) were used together with plates containing wells without primary or secondary antibodies.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: rafael.llorens{at}udg.es and pauline.rudd{at}bioch.ox.ac.uk
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Abbreviations |
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References |
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