Key words: bile salt-dependent lipase/cancer (pancreas)/feto-acinar pancreatic protein/glycosylation/J28 glycotope
The fetoacinar pancreatic protein or FAPP is an oncofetal glycovariant of bile salt-dependent lipase (Escribano and Imperial, 1989; Mas et al., 1993a) or BSDL, a lipolytic enzyme present in the pancreatic secretion and involved in the duodenal hydrolysis of cholesteryl esters (Lombardo and Guy, 1980). FAPP is a specific component of the pancreatic acinar cells which is associated with the ontogenesis and development of the human pancreas. This protein was further defined as a member of the oncodevelopment-associated pancreatic antigens. The earliest expression of FAPP is seen in undifferentiated mesenchymal cells and in nascent acini, at the beginning of the morphological differentiation of the pancreas which occurred after 9-10 weeks of gestation (Albers et al., 1987). Maximal synthesis appeared at the time of intense acinar cell proliferation, between 15 and 25 gestation weeks. The expression of FAPP declines progressively thereafter and reaches, in adults, levels far lower than those observed in fetuses (Albers et al., 1987). However, FAPP has not been detected in other fetal tissues (Albers et al., 1987) and in any adult tissues except the pancreas of patients suffering with a cancer (Escribano et al., 1986). FAPP was first identified using a polyclonal antiserum in the Syrian golden hamster (Benedi et al., 1984) and was further characterized in human pancreas with a murine monoclonal antibody referred to as mAbJ28 (Escribano et al., 1986). Serum FAPP concentration, detected with mAbJ28, is enhanced in pancreatic pathologies especially adenocarcinoma and the mAbJ28 recognized a structure found in human tumoral tissues (Escribano et al., 1986) and in certain human pancreatic tumor cell lines (SOJ-6, BxPC-3) (Mazo et al., 1991; Miralles et al., 1993). This structure seems weakly expressed or absent in some other cell lines such as MiaPaCa-2 and Panc-1 (Mazo et al., 1991) and in normal pancreas (Escribano et al., 1986). It has been shown that the epitope recognized by mAbJ28 (termed J28 glycotope) is a carbohydrate-dependent antigenic structure (Escribano and Imperial, 1989). FAPP and BSDL offer both O- and N-linked carbohydrate structures (Escribano and Imperial, 1989; Guy et al., 1981), of which the O-linked ones are involved in the J28 glycotope structure (Mas et al., 1997a). O-Linked carbohydrate structures of FAPP and BSDL are located within tandem repeated identical mucin-like sequences, each carrying a site for O-glycosylation (Wang et al., 1995). We have further shown, that fucose residues were important elements of the J28 glycotope structure (Mas et al., 1997a). Even though this glycotope accommodates a fucosylated structure, it differed from Lewis b and Lewis y blood group. We have also shown that sialidase treatment of FAPP did not abolished the mAbJ28 reactivity; therefore, J28 antigenic structure may also differ from that of sialyl-Lewis x and sialyl-Lewis a. All these fucosylated oligosaccharide structures are regarded as tumor-associated antigens.
Human pancreatic cancer is characterized by an alteration in fucose-containing surface blood group antigens such as H antigen, Lewis b, Lewis y, and sialyl-Lewis a and sialyl-Lewis x. These determinants can be synthesized by sequential action of [alpha]3-sialyltransferases or [alpha]2-fucosyltransferases (Fuc-T) and [alpha]3/4-Fuc-T on (poly)N-acetyllactosamine chains (Kukowska-Latallo et al., 1990; Larsen et al., 1990; Kumar et al., 1991; Lowe et al., 1991; Koszdin and Bowen, 1992; Weston et al., 1992a,b; Natsuka et al., 1994; Sasaki et al., 1994; Kelly et al., 1995). It has been shown that the core-2 (poly)N-acetyllactosamine branch formed by core-2 [beta]6-N-acetylglucosaminyltransferase (or Core2GlcNAc-T for short) is critical for the synthesis of both sialyl-Lewis a and sialyl-Lewis x on O-linked glycans (Shimodaira et al., 1997; Whitehouse et al., 1997). The presence of Core2GlcNAc-T can be correlated with the presence of sialyl-Lewis a or sialyl-Lewis x in O-linked carbohydrate structures detected on carcinoma cells. Therefore, fucosyltransferases and Core2GlcNAc-T are key enzymes regulating the synthesis of these carbohydrate determinants. In a recent study, we have shown that fucosyltransferase activities varied between normal and tumoral pancreatic tissues (Mas et al., 1998). A substantial decrease of [alpha]2-Fuc-T activity associated with enhanced [alpha]3- and [alpha]4-Fuc-T activities was observed in tumoral compared to normal pancreatic tissues (Mas et al., 1998). The presence of the J28 determinant on mucin-like structures of BSDL encompassing high amount of O-linked glycans enable structural determination by physical methods (NMR, mass spectroscopy, etc.). The aim of this work was therefore to determine which glycosyltransferase activities, overexpressed in tumoral tissues, could be involved in the formation of the J28 determinant. We have focused on Core2GlcNAc-T, FUT3, and FUT7 enzymes and showed that Core2GlcNAc-T was critical for the building of the J28 glycotope. Further fucosylation of the core2 branch by FUT3 appeared as essential element of this carbohydrate determinant structure. Glycosyltransferase activities in tumoral pancreatic cells
A survey of the literature (Mazo et al., 1991; Miralles et al., 1993) indicated that the J28 glycotope can be detected on BSDL expressed by SOJ-6 and BxPC-3 but not on the protein found in MiaPaCa-2 and Panc-1 cells. The J28 glycotope is absent in the normal human pancreatic tissue whereas it is strongly expressed in tumoral pancreatic tissue (Escribano et al., 1986). Because this glycotope involved fucose residues, we first have determined the Fuc-T activities in lysates of SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1 cells and compared these activities with those detected in normal human pancreatic tissue homogenates (Table I). It appeared that Fuc-T activities on phenyl [beta]-d-galactoside, N-acetyllactosamine and lacto-N-biose which are representative of [alpha]2-, [alpha]3-, and [alpha]4-Fuc-T, respectively, were largely decreased in MiaPaCa-2 and Panc-1 cells. The [alpha]2-Fuc-T activity was substantially decreased in the four cell lines compared to that recorded in human pancreatic tissues. Conversely, the activity of [alpha]4-Fuc-T was significantly increased in SOJ-6 and BxPC-3 compared to MiaPaCa-2, Panc-1, and human normal tissues.
Another glycosyltransferase, the core-2 [beta]6-N-acetylglucosaminyltransferase or Core2GlcNAc-T, has been associated with malignant transformation (Shimodaira et al., 1997). This enzyme, which represents an important regulatory step for the extension of O-linked sugars, allows the formation of Gal[beta]1-3/4GlcNAc[beta]1-3 repetitive sequences (Shimodaira et al., 1997) which may be used as acceptor for fucosyltransferases. As also shown on Table I, the activity of Core2GlcNAc-T recorded using Gal[beta]1-3GlcNAc-O-p-nitrophenyl was very high in SOJ-6 cells, while undetectable in normal pancreatic tissues. Taken altogether, these results suggested that the Core2GlcNAc-T activity associated with [alpha]3- or [alpha]4-Fuc-T activities could be involved in the building of the fucosylated J28 oncofetal glycotope, carried out by tandemly repeated identical sequences of BSDL (Mas et al., 1997a), each of them bearing O-glycosylated structure (Wang et al., 1995). Cloning of the cDNA encoding the C-terminal repeats of BSDL
The first objective was to obtain the cDNA encoding exon 11 of BSDL. This exon codes for the sixteen repeated sequences located on the C-terminal region of the protein (Reue et al., 1991). For this purpose, two primers covering the sequence of exon 11 of BSDL, from nucleotide 1169 to the termination codon were used in RT-PCR experiments. A ~1.1 kb fragment was thus obtained, the sequence of which represents 1080 bp and matches 100% that of the human BSDL (Reue et al., 1991). The amino acid sequence deduced from this fragment corroborated the corresponding sequence of BSDL, from Phe364 to Phe723. Sixteen identical sequences (Figure
Figure 1. HCA plots comparison of the human pancreatic BSDL and the C-terminal peptide of BSDL. The cDNA fragment obtained by RT-PCR performed on human pancreatic mRNA and using Cter BSDL-5[prime] and Cter BSDL-3[prime] probes was purified on 1% agarose gel and subcloned into pCR2.1 vector and sequenced using M13 forward and reverse primers. The deduced amino acid sequence was compared to the known protein sequence of the human BSDL. In the HCA plots, the protein sequences are written as a duplicated [alpha]-helical net and the contour of clusters of hydrophobic residues are automatically drawn. The [alpha]-helical net has been demonstrated to offer the best correspondence between the positions of hydrophobic clusters and regular secondary structures (Woodcock et al. 1992). The standard one-letter code for amino acids is used except for proline (regular secondary structure breaker), glycine (the less constraint amino acids), serine, and threonine (which can be at the protein surface in the case of C-terminal peptide) which are represented by open stars, open diamonds, squares with solid dots, and open squares, respectively. The vertical dotted lines indicated the 16 identical repeated sequences in which O-glycosylation sites on threonine residue are indicated by solid squares. Broken arrows represent sequences deleted for the need of the artwork.
Table I. CHO-K1 cells clones expressing glycosyltransferases
Under our assay conditions, CHO-K1 cells expressed no detectable Core2GlcNAc-T, [alpha]2-Fuc-T (determined on phenyl [beta]-d-galactoside, data not shown), [alpha]3-Fuc-T, and [alpha]4-Fuc-T activities (see below, Figure
Figure 2. Recapitulative route for CHO-K1 transfection. Parental type CHO-K1 cells were transfected using lipofectamine mediator with plasmids comprising cDNA encoding Core2GlcNAc-T, FUT3, FUT7 or the C-terminal peptide of BSDL (Cter). These transfections lead to the selection of CHO-C2, CHO-F3, CHO-F7, and CHO-Cter clones. CHO-C2 clone was further transfected with FUT7 or FUT3 and with the C-terminal peptide cDNA to give CHO-C2/F7/Cter, CHO-C2/F3/Cter, and CHO-C2/Cter clones. CHO-F3 and CHO-F7 clones were also transfected with the C-terminal peptide cDNA to obtain CHO-F3/Cter and CHO-F7/Cter clone. By the end, six clones were selected, CHO-Cter, CHO-F7/Cter, CHO-F3/Cter, CHO-C2/F3/Cter, CHO-C2/F7/Cter, and CHO-C2/Cter. All these clones secrete the recombinant C-terminal peptide of BSDL.
Figure 3. Glycosyltransferase activities of the selected clones. Clones obtained as summarized in Figure 2 were assayed for transfected glycosyltransferases. (A) The Core2GlcNAc-T activity was determined in cell lysates obtained from parental CHO-K1 cells, CHO-C2, CHO-C2/Cter, CHO-C2/F3/Cter, and CHO-C2/F7/Cter clones. The activity was assayed using Gal[beta]1-3GlcNAc-O-p-nitrophenyl as acceptor (values are mean ± SD of at least three independent determinations). (B) The [alpha]3-Fuc-T activity was determined in cell lysates obtained from parental CHO-K1 cells, CHO-C2/Cter, CHO-F7, CHO-F7/Cter, and CHO-C2/F7/Cter clones. The [alpha]3-Fuc-T was assayed on 3[prime]-sialyl-N-acetyllactosamine as acceptor (values are mean ± SD of at least three independent determinations). (C) The [alpha]3-fucosyltransferase ([alpha]3-Fuc-T) activity was determined in cell lysates obtained from parental CHO-K1 cells, CHO-C2/Cter, CHO-F3, CHO-F3/Cter, and CHO-C2/F3/Cter clones. The [alpha]3-Fuc-T was assayed using N-acetyllactosamine as acceptor (values are mean ± SD of at least three independent determinations). (D) The [alpha]4-Fuc-T activity was determined in cell lysates obtained from parental CHO-K1 cells, CHO-C2/Cter, CHO-C2/F3/Cter and CHO-C2/F7/Cter clones. The [alpha]4-Fuc-T was assayed on lacto-N-biose as acceptor (values are mean ± SD of at least three independent determinations). Glycosyltransferase activities of selected clones
The Core2GlcNAc-T activity was determined in clones transfected with the cDNA encoding this transferase. As shown on Figure Secretion of the C-terminal peptide of BSDL
CHO-K1 cells, CHO-Cter, CHO-C2/Cter, CHO-F3/Cter, CHO-F7/Cter, CHO-C2/F3/Cter, and CHO-C2/F7/Cter clones were progressively accustomed to grow in Opti-MEM medium, in the absence of FCS. All these clones, except CHO-K1 cells, expressed the C-terminal peptide of BSDL. The construction comprising the cDNA encoding this peptide encompassed the V-J2-C region of the mouse IgK chains which might drive expressed C-terminal peptide toward secretion in the cell culture medium. Therefore, these transfected cells were allowed to secrete the C-terminal peptide for at least 24 h. At the end of the production period, the cell-free medium was concentrated by lyophilization. Dry material was resuspended in water and analyzed by SDS-PAGE and Western-blot using pAbL64 as primary antibodies. After SDS-PAGE and Coomassie blue staining, two peptides migrating at approximately 78 and 83 kDa (Figure
Figure 4. SDS-PAGE analysis of recombinant C-terminal peptide of BSDL produced by different clones. Clones obtained as described in Figure 2 were accustomed to Opti-MEM medium and allowed to concentrate recombinant C-terminal peptide of BSDL in the cell-medium. The cell-free medium of the selected six clones and that of parental CHO-K1 cells were withdrawn and concentrated by lyophilization. Then proteins (~100 µg/lane) were separated on SDS-PAGE (7.5% acrylamide) and stained with Coomassie blue.
Figure 5. Western blot analysis of recombinant C-terminal peptide of BSDL produced by different clones. Peptides (~100 µg/lane) obtained as described in Figure 4 were separated on 10% acrylamide SDS-PAGE and electrotransferred onto a nitrocellulose membrane. Replicas were then developed with pAbL64. (A) or mAbJ28 (B). Note that CHO-C2, CHO-F3, and CHO-F7 that were not transfected with the cDNA encoding C-terminal peptide were reactive with neither antibody and consequently did not produced, as expected, the C-terminal peptide (not shown). Expression of the oncofetal J28 glycotope
The next issue was to determine whether CHO cells transfected with the various glycosyltransferases and the C-terminal peptide of BSDL were able to express the J28 glycotope. For this purpose, the secreted product of each clone above obtained (see Figure
Figure 6. Reactivity of C-terminal recombinant peptides of BSDL with mAbJ28. The reactivity of recombinant C-terminal peptides of BSDL produced by CHO-C2/Cter (open circles), CHO-C2/F3/Cter (open squares), and CHO-C2/F7/Cter (open triangles) was determined by ELISA using mAbJ28 as primary antibody. In vitro fucosylation of the C-terminal peptide produced by CHO-Cter and CHO-C2/Cter cell clones
To ascertain the implication of FUT3 and Core2GlcNAc-T enzymes in the building of the J28 glycotope, C-terminal peptides produced by CHO-Cter and CHO-C2/Cter clones were in vitro fucosylated by recombinant soluble FUT3 (rsFUT3). This enzyme was isolated from concentrated cell culture medium of CHO-K1 transfected with the cDNA encoding FUT3. The transmembrane domain and part of the stem region were deleted from the FUT3 cDNA to generate a soluble form of the enzyme. SDS-PAGE followed by Western blotting analysis using anti-FLAG-M1 monoclonal antibody, showed a band migrating at 32 kDa (data not shown). The cell culture media were assayed for fucosyltransferase activity using type 2 acceptor (N-acetyllactosamine). The activity was 86.1 ± 1.7 pmol/min/mg protein, meaning that rsFUT3, secreted by transfected cells, was active. Therefore, peptides produced by CHO-Cter and CHO-C2/Cter clones were assayed for fucosylation using rsFUT3. The fucose transfer was then determined by ELISA. As shown on Figure
Figure 7. In vitro fucosylation of recombinant C-terminal peptide by recombinant soluble FUT3. Recombinant C-terminal peptides of BSDL produced by CHO-C2/Cter and CHO-Cter were fucosylated in vitro with the recombinant soluble form of FUT3 (rsFUT3), and analyzed by ELISA using mAbJ28 as primary antibody. C-Terminal peptide produced by the CHO-Cter clone incubated without (open triangles) or with 100 µg rsFUT3 (solid triangles). C-Terminal peptide produced by the CHO-C2/Cter clone incubated without (open circles) or with 100 µg rsFUT3 (solid circles).
When peptides expressed by CHO-C2/Cter and CHO-C2/F3/Cter clones were defucosylated with 0.2 unit of bovine kidney [alpha]2/3/4-fucosidase (under conditions already described; see Mas et al., 1997a), the reactivity of these peptides towards mAbJ28 was lost (not shown). These data confirm that fucoses are important elements of the structure recognized by mAbJ28 (Mas et al., 1997a). These fucose residues can be transferred to the glycan by endogenous fucosyltransferases (although not detectable in CHO cells under our condition assay) and as suggested by the reactivity of the peptide originating from CHO-C2/Cter clone. Fucose can also be transferred to glycans by exogenous or transfected Fuc-T. Obviously, [alpha]3/4-Fuc-T (FUT3) which increased the reactivity of the peptide produced by CHO-C2/Cter clone could be one of those fucosyltransferases involved in the fucose transfer to Gal[beta]1-3/4GlcNAc branch initiated by Core2GlcNAc-T. The activity of the latter transferase also appeared as essential to the formation on the oncofetal J28 glycotope.
Characterization of specific markers for pancreatic cancers, the diagnosis of which still remains a virtual death sentence for the patient, is an actual challenge. It is now well established that tumors perform aberrant glycosylation and several useful epitopes directed against carbohydrate-dependent structures have been defined by monoclonal antibodies. Many of these monoclonal antibodies were originally generated against malignant secretory epithelial cells of the pancreas, including SPan-1 (Kobayashi et al., 1991), Du-Pan-2 (Lan et al., 1987), CA19-9 (Magnani et al., 1983), CA50 (Masson et al., 1990), and CAR-3 (Prat et al., 1989), among others. These antibodies generally displayed distinct pattern of tissue staining, often being more highly expressed in certain tissues and tumors than others. All these epitopes were used more or less successfully to diagnose pancreatic cancer. However, the pancreatic cancer diagnosis using some of these antibodies depends upon the genotype of patients. For example CA19-9 measurement is more useful than is Du-Pan-2 determination for Lewis-positive patients but is without interest for Lewis-negative ones (Narimatsu et al., 1998), albeit Du-Pan-2 carbohydrate determinant seems to be the precursor of CA19-9 (Kawa et al., 1994). SPan-1 determination seems more adequate for pancreatic cancer diagnosis in Lewis-negative patients (Kawa et al., 1994). Therefore, blood group-related antigens ABH, Lewis a, sialyl-Lewis a, Lewis b, Lewis x, sialyl-Lewis x, and Lewis y might be associated with other markers to increase the specificity of the diagnosis (Pour et al., 1988). Accordingly, the structure of the J28 glycotope which is specific of pancreatic adenocarcinoma (Escribano et al., 1986) should be better defined before use for diagnosis.
The synthesis of blood group-related tumor associated antigens is regulated by glycosyltransferases such as fucosyltransferases or Fuc-T (Kukowska-Latallo et al., 1990; Larsen et al., 1990; Kumar et al., 1991; Lowe et al., 1991; Koszdin and Bowen, 1992; Weston et al., 1992a,b; Natsuka et al., 1994; Sasaki et al., 1994; Kelly et al., 1995). Two main Fuc-T families have been characterized to date: the [alpha]2-Fuc-T family which includes FUT1 and FUT2 and the [alpha]3-Fuc-T family with FUT3, FUT4, FUT5, FUT6, and FUT7, which are involved in the synthesis of H and Lewis related antigens, respectively. FUT1 and FUT2 encoded by the H gene (Larsen et al., 1990) and the secretor gene (Kelly et al., 1995), respectively, are [alpha]2-Fuc-T. FUT3, an [alpha]3/4-Fuc-T, contributes to the synthesis of all Lewis structures (Kukowska-Latallo et al., 1990). FUT4 is the myeloid [alpha]3-Fuc-T, the activity of which leads to the synthesis of Lewis x and Lewis y (Kumar et al., 1991; Lowe et al., 1991). FUT5 and FUT6, involved in Lewis x and sialyl-Lewis x formation are [alpha]3-Fuc-T (Koszdin and Bowen, 1992; Weston et al., 1992a,b), as is FUT7 which fucosylates sialylated type 2 structures to give sialyl-Lewis x determinant. The [alpha]2-, [alpha]3-, and [alpha]4-Fuc-T activities were detected in normal and in tumoral pancreatic tissues (Mas et al., 1998). Data indicated that the activity of [alpha]2-Fuc-T was largely decreased in tumoral pancreas (Mas et al., 1998) and in tumoral pancreatic cell lines (Mas et al., 1998; this study). However, [alpha]3-Fuc-T and [alpha]4-Fuc-T, which generate sialyl-Lewis x, Lewis a, and sialyl-Lewis a were constant or significantly increased in SOJ-6 and BxPC-3 cells (Mas et al., 1998). The expression of [alpha]3- and [alpha]4-Fuc-T activities associated with the [alpha]2-Fuc-T decrease in these two cell lines can be correlated with the membrane expression of sialyl-Lewis x and sialyl-Lewis a on the cell surface of BxPC-3 and SOJ-6 cell lines (Kukowska-Latallo et al., 1990), and of sialyl-Lewis a in pancreas carcinoma (Iwai et al., 1993; Kaji et al., 1995). However, neither of these carbohydrate determinants were detected on MiaPaCa-2 and Panc-1 cells (Mas et al., 1998). Difucosylated glycotopes, Lewis b and Lewis y, are expressed in normal pancreas but seem absent in pancreatic carcinoma (Ichihara et al., 1993; Yago et al., 1993; Lantini and Cossu, 1997). These data suggested that the expression of sialyl-Lewis a and sialyl-Lewis x paralleled that of the J28 glycotope (Mazo et al., 1991). This glycotope is expressed on the C-terminal mucin-like tail of BSDL (Mas et al., 1997a) and characterizes the oncofetal glycovariant of this enzyme referred to as the fetoacinar pancreatic protein or FAPP (Mas et al., 1993a; Pasqualini et al., 1998). The expression of this determinant is restricted, in adult, to the tumoral pancreas (Escribano et al., 1986; Albers et al., 1987). Little is known about the structure of the carbohydrate dependent J28 glycotope. To date, we have shown that fucosidase treatment of FAPP, abolished the reactivity of mAbJ28 with the protein (Mas et al., 1997a), whereas treatment with sialidase was ineffective. Our data indicated that FAPP was not reactive with antibodies including those specific for Lewis type 1 and type 2 antigenic determinants. The reactivity of FAPP was also negative with the type 3 O-glycosylated precursor T-antigen and the monosialylated Tn antigen, albeit the J28 glycotope is carried out by the O-linked glycans of FAPP (Mas et al., 1997a). In addition, the J28 carbohydrate determinant was related neither to H-type 1, H-type 2, nor H-type 3 carbohydrate structure.
The complexity of mucin carbohydrate precludes direct structural determination. In the present study, we attempted to gain information concerning structural features of the J28 glycotope and investigated which glycosyltransferases could be involved in the establishment of this structure. For this purpose, CHO-K1 cells expressing Core2GlcNAc-T and/or FUT3 and/or FUT7 were transfected with the cDNA coding for the mucin-like C-terminal region of BSDL. In a recent paper, we showed that FAPP sequence did not differ from that of BSDL (Pasqualini et al., 1998). However, a 330 bp deletion located within the sequence encoding tandem repeated sequences leads to the truncation of 10 of these repeated sequences. As a consequence, six sites for O-glycosylation identical to those of BSDL were left on FAPP. As shown here, the oncofetal J28 glycotope can be generated on these repeated sequences independently of their amount. This point clearly indicated that the formation of the J28 glycotope on FAPP depends upon different expression of glycosyltransferases in fetuses and neoplastic tissue as compared to normal counterpart.
Prior to these transfections, we showed that parental CHO-K1 cells and CHO-Cter clone expressed no detectable activity (under our assay conditions) on phenyl [beta]-d-galactoside, N-acetyllactosamine, lacto-N-biose and Gal[beta]1-3GlcNAc-O-p-nitrophenyl acceptors. These substrates are representative of [alpha]2-Fuc-T, [alpha]3-Fuc-T, [alpha]4-Fuc-T, and Core2GlcNAc-T activities, respectively. However, we have previously shown that CHO-K1 cells were able to initiate O-linked glycosylation of transfected BSDL (Bruneau et al., 1997). These cells also present a very low Core2GlcNAc-T activity (Datti and Dennis, 1993) and display a 150-fold increase in activity upon transfection with the cDNA encoding this enzyme. As a first important result, we showed that Core2GlcNAc-T was expressed in pancreatic tumor cell lines except Panc-1, and cannot be detected in normal pancreatic tissues. This enzyme is required to branch GlcNAc residue on the inner GalNAc residue of Gal[beta]1-3GalNAc[alpha]1-O linked structure (Bierhuizen and Fukuda, 1992). This branching allows the possible elongation of the chain to form polymeric (Gal[beta]1-3/4GlcNAc)n[beta]1-6GalNAc structures. Both sialyl-Lewis a and sialyl-Lewis x structures in O-glycans are synthesized via this particular branch (Shimodaira et al., 1997). Therefore, C2[beta]6GnT cDNA was transfected in CHO-K1 followed by the cDNA encoding the BSDL C-terminal peptide. Surprisingly, the C-terminal peptide expressed by the clone thus obtained, CHO-C2/Cter, carried out the J28 determinant. Under these conditions, the antigen peptide migration on SDS-PAGE is slower than that of peptides unreactive to the mAbJ28 such as that expressed by the CHO-Cter clone. Although, we should be aware of that the C-terminal peptide of BSDL likely presents an extended structure and that its SDS-PAGE migration does not necessarily reflect the level of glycosylation, this result corroborates those previously obtained, showing that mAbJ28 always recognizes high-Mr form of BSDL (Mas et al., 1993a). Because CHO clones that do not expressed Core2GlcNAc-T such as CHO-F3/Cter, CHO-F7/Cter, and CHO-Cter, also do not expressed the J28 glycotope, one may strongly suspect that Core2GlcNAc-T is crucial for the expression of this glycotope.
As above mentioned, fucose residues contribute to the binding of mAbJ28 to FAPP (Mas et al., 1997a). The expression of the J28 glycotope was detected in cell line which also significantly expressed sialyl-Lewis a and sialyl-Lewis × structures (Mazo et al., 1991; Mas et al., 1998). Moreover, the significant increase of the [alpha]4-Fuc-T activity in tumoral pancreas and in the two cell lines that express the J28 glycotope, suggested that FUT3 could be overexpressed in pancreatic cancer. Therefore, we have further transfected CHO-C2/Cter clone separately with two Fuc-T involved in the synthesis of sialyl-Lewis a and sialyl-Lewis x, i.e., FUT3 and sialyl-Lewis x, i.e., FUT7. The two clones selected-CHO-C2/F3/Cter and CHO-C2/F7/Cter-expressed the 83 kDa C-terminal peptide of BSDL, on which we were able to detect the J28 glycotope. However, the FUT3 transfected clone showed an increased relative reactivity of the C-terminal peptide towards the mAbJ28 compared to that of CHO-C2/Cter clone. The reactivity of the C-terminal peptide produced by the latter clone was also enhanced in the same proportion by in vitro fucosylation using rsFUT3. Although the FUT7 activity can be measured in CHO-C2/F7/Cter clone, it seems that this Fuc-T was not able to generate a C-terminal peptide more reactive to mAbJ28 than that produced by the CHO-C2/Cter clone. The extremely limited acceptor specificity of FUT7 which readily transfers fucose to 3[prime]-sialyl type 2 structures (Natsuka et al., 1994; Britten et al., 1998) may explain the lack of effect of the transfected enzyme. Because sialyl residues are not involved of the J28 glycotope structure (Mas et al., 1997a), it is possible that FUT7 may not be involved in the formation of this structure.
Taken as a whole, these data indicated that Core2GlcNAc-T is involved in the formation of mAbJ28 target on FAPP. Although not detectable, endogenous FUT3-like activities of CHO cells resulting from the activation of silent genes by transfection processes as documented by Potvin et al., 1990, could further fucosylate these structures. This provides an explanation to the reactivity of the C-terminal peptide produced by the CHO-C2/Cter clone, which is abolished by fucosidase treatment. As a consequence, it appears that both Core2GlcNAc-T and fucosyltransferases are crucial for the establishment of the J28 glycotope structure. Because Gal residues of Gal[beta]1-3/4GlcNAc structures forming the core 2 branch can only be [alpha]2-fucosylated, and that [alpha]2-Fuc-T activity is decreased whereas [alpha]4-Fuc-T activity is increased in tumoral pancreatic tissues and cells, it is possible that GlcNAc residues, which are [beta]1-6 branched by Core2GlcNAc-T on GalNAc residue of the Gal[beta]1-3GalNAc-O-Thr/Ser T antigenic structure, could be [alpha]4-fucosylated by FUT3 to form the J28 glycotope. However, we cannot rule out the possible contribution of FUT5 to the building of the J28 glycotope, since this enzyme, detected in human pancreatic tumoral cells (Mas et al., 1998), also displays an [alpha]4-Fuc-T activity (Costache et al., 1997). Chemicals and reagents
RPMI 1640 medium, Dulbecco's modified Eagle medium (DMEM), Ham's F12 nutriment mixture, Opti-MEM medium, CHO-SFM medium, glutamine, penicillin, streptomycin, G418 or neomycin, trypsin-EDTA, fungizone and fetal calf serum (FCS) were purchased from Life Technologies. Paraformaldehyde and N-acetylglucosamine were from Fluka (Buchs, Switzerland). Agarose was from Amresco (Solon, OH). Benzamidine, BSA, AMP, GDP-fucose, N-acetyllactosamine, lacto-N-biose, phenyl [beta]-d-galactoside, kidney fucosidase, and saponin were obtained from Sigma. Restriction enzymes were from Promega. 3[prime]-sialyl-N-acetyllactosamine was for Oxford Glycosystems (Abington, UK). Triton X-100 was from Pierce. Zeocin was from Invitrogen. Hygromycin and Complete-EDTA-free (a mix of protease inhibitors) were purchased from Boehringer-Mannheim (Germany). GDP-[14C] fucose and UDP-[3H] N-acetylglucosamine were from NEN and PCS from Amersham. Biological materials
Normal human pancreatic tissues came from four donors (females and males aged 60-65) and were a generous gift from Prof. J.R.Delpéro (Institut Paoli-Calmettes, Marseilles, France). Tissues samples were immediately frozen in liquid nitrogen until use. Antibodies
Polyclonal antibodies raised using purified human BSDL were obtained in our laboratory and isolated by affinity chromatography on protein-A Sepharose (Abouakil et al., 1988). These antibodies referred to as pAbL64 were able to react with nonglycosylated BSDL obtained by in vitro translation using pancreatic mRNA and rabbit reticulocytes (Pasqualini et al., 1998). The monoclonal antibody (mAbJ28) specific for the J28 glycotope carried by the oncofetal glycoisoform of BSDL (i.e., FAPP) was a generous gift from Dr. M.J.Escribano (INSERM U 260, Marseilles). Monoclonal mouse antibody (CSLEX-1) specific of the sialyl-Lewis x structure was from Becton-Dickinson. Mouse monoclonal antibodies to FLAG-M1 and to FLAG-M2 were from Kodak, FITC-conjugated anti-mouse goat IgG, FITC-conjugated anti-mouse goat IgM, FITC-conjugated anti-rabbit goat IgG, alkaline phosphatase-conjugated anti-mouse goat IgG, and alkaline phosphatase-conjugated anti-rabbit goat IgG were from Sigma. Cell culture
The Chinese hamster ovary cell line CHO-K1 was supplied by the American Type Culture Collection (ATCC designation CCL 61). CHO-K1 cell line was maintained in Ham's F-12 nutrient mixture. Human pancreatic carcinoma cell lines BxPC-3 (Tan et al., 1986), MiaPaCa-2 (Yunis et al., 1977), and Panc-1 (Lieber et al., 1975) were obtained from ATCC. SOJ-6 (Fujii et al., 1990) cell line was kindly provided by Dr. M.J.Escribano. BxPC-3 and SOJ-6 cells were grown in RPMI 1640 medium whereas MiaPaCa-2 and Panc-1 cells were cultured in DMEM. Mediums were supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (0.1%). Cells were kept at 37°C in an humidified atmosphere of 95% O2 and 5% CO2. When required, CHO cells were accustomed to Opti-MEM or CHO-SFM. Preparation of cell and tissue extracts
Cells were washed in PBS (10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl), harvested with a rubber policeman or with trypsin/EDTA, washed once again in PBS and pelleted by centrifugation. Pellets were washed twice with PBS and lysed for 30 min. at 4°C in 200 µl of lysis buffer (150 mM NaCl, 0.4% Triton X-100 and CompleteTM). Alternatively, cells were sonicated for 10 sec. (4 W, Branson Sonifier) in 10 mM Tris/HCl buffer pH 7.0 with CompleteTM. Human pancreatic tissues were homogenized with a Polytron (Kinematica AG, Switzerland) in 10 mM Tris/HCl buffer (pH 7.0) containing 250 mM sucrose and 5 mM benzamidine. These homogenates were clarified by centrifugation at 14,000 × g for 20 min at 4°C and were either immediately assayed or were stored at -20°C until use. The concentration of protein was determined using the bicinchoninic acid assay (Pierce) using BSA as standard. Plasmids
pCDM8-FUT7 and pCDM7-FUT3 were a generous gift from Dr. J.B.Lowe (University of Michigan, Ann. Arbor, MI). C2[beta]6GnT cDNA was obtained from Dr. M.Fukuda (The Burnham Institute, La Jolla, CA.) and cloned into pcDNA3 vector ended in 3[prime] with the sequence coding for the FLAG epitope (pcDNA3-C2[beta]6GnT-Flag). pLSVHg conferring the resistance to hygromycin was a gift of Dr. B.Malissen (CIML-Marseilles, France). pMAMneo and pSecTag were from Clontech and Invitrogen, respectively. These plasmids carry the gene for the resistance to G418 (or neomycin) and zeocin, respectively. Construction of plasmids
The cDNA encoding the C-terminal tandem repeated sequence of BSDL (Cter-cDNA) was obtained by reverse transcription of human pancreas mRNA. The cDNA(-) pool was amplified by performing a polymerase chain reaction using a pair of primers designed to cover this sequence (Reue et al., 1991) from nucleotide 1169 (Phe364) to nucleotide 2247 (termination codon) (Cter BSDL-5[prime]; 5[prime]-CGTCTAaagcttTTTGAT GTCTACACCGAGTCC and Cter BSDL-3[prime]; 5[prime]-TTTCGTgaattcACGCTAAAACCTAATG AcTgCAGGCATCtG) and using the GC-rich PCR kit from Clontech. These two primers include HindIII and EcoRI overhangs (lowercase letters) for the ligation into the pSecTag vector. Bases were randomly added in 5[prime] of these primers to allow restrictive cleavage of PCR fragments. To eliminate the myc epitope and the sequence coding the 6 histidine residues in 3[prime]-end of the multicloning site of the pSecTag vector, a stop codon was introduced in the primer hybridizing with the 3[prime]-end of the Cter-cDNA. The DNA was amplified using a 35 reaction cycles program as follows: denaturation (94°C, 1 min), annealing (52°C, 1 min) and extension (68°C, 4 min). The reaction was terminated by an incubation at 68°C for 10 min. The cDNA of the soluble form of FUT3 was obtained by PCR using two primers covering the sequence coding for the catalytic domain of the enzyme from nucleotide 142 (Pro48) to nucleotide 1086 (stop codon) (Kukowska-Latallo et al., 1990). These primers (FT3-5[prime]; 5[prime]-ttttaagcttcccagtgggtcctcc and FT3-3[prime]; 5[prime]-aaagaattctctcaggtgaaccaagc cgc) included HindIII and EcoRI restriction sites (small case letters) and were used in PCR programs with 2 units of Taq DNA polymerase (Appligene Oncor, Illkirch, France) in a final volume of 50 µl containing 5 µl 10× PCR buffer (10 mM Tris-HCl pH 9, 50 mM KCl, 1.5 mM MgCl2), 0.35 mM dNTP, 50 pmol of FT3-5[prime], and FT3-3[prime] primers. 1 µl of pCDM7 plasmids containing human FUT3 cDNA (Kukowska-Latallo et al., 1990) was amplified under the following conditions: 94°C for 2 min., 1 cycle; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C for 1 min, 25 cycles. Extension was terminated by incubation at 68°C for 7 min.
PCR fragments were analyzed on 1% agarose gel. After purification using the Geneclean Kit (Bio101), transcripts were subcloned into pCR2.1 TOPO vector (Invitrogen) and sequenced using M13 forward and reverse primers. Once sequenced transcripts (Cter-cDNA and rsFUT3-cDNA) were excised by HindIII and EcoRI digestion and ligated into the pSecTag and pFLAG-CMV1 expression/secretion vector, to give the pSecCter and pFLAG-rsFUT3 plasmids, respectively. All PCR experiments were performed with a Gene AmpPCR System 2400 (PE Applied Biosystem) or with a Robocycler Gradient 96 (Stratagene). Transfection
Stable transfections of CHO-K1 cells were performed with plasmids comprising adequate sequence and using the Lipofectamine mediated transfection procedure according to the manufacturer (Life Technologies). CHO-K1 cells were grown as described above. Semi-confluent cells were typically transfected with 3-4 µg of plasmid DNA, (pSecCter, pFLAG-rsFUT3 and plasmids described above) and, when required, with 1 µg of the plasmid DNA (pMAMneo, pLSVHg) to confer resistance to G418 and hygromycin, respectively. When pcDNA3 or pSecTag plasmids were used, cotransfection with plasmids conferring antibiotic resistance was not necessary, these plasmids encompass gene resistance to neomycin and zeocin, respectively. Antibiotic-resistant cells were selected using several medium exchanges with Ham's F12 containing 10% FCS and the adequate antibiotic (200-500 µg/ml) during a time period of, at least, 4 weeks. Clones transfected with pSecCter were selected with zeocin then clones expressing the C-terminal peptide of BSDL (Cter) were isolated using cloning cylinders and visualized by fluorescence microscopy using pAbL64. Once selected these clones were accustomed to Opti-MEM medium and grown to confluence. Clones expressing the recombinant soluble FUT3 (rsFUT3) were accustomed to CHO-SFM (a specific CHO cells culture medium free of FCS). Confluent cells were grown for at least two days in the absence of FCS and cell culture medium was tested for fucosyltransferase activity after a 10-fold concentration using Centriprep 10-cartridges (Amicon). Fluorescence microscopy
Cells grown to 80% confluence on microscope coverslips were washed three times with PBS buffer and fixed with 3% (vol/vol) paraformaldehyde for 20 min. The excess of paraformaldehyde was eliminated by washing the slides in 1 M glycine (pH 8.5). Between each step, cells were exhaustively rinsed with PBS. Then cells were treated for immunofluorescence as already described (Pasqualini et al., 1998) using adequate primary antibodies and conjugates. Polyacrylamide gel electrophoresis, Western blotting, and dot-blot dilution
Gel electrophoreses (SDS-PAGE) were performed on slab gels of polyacrylamide (7.5 or 10% acrylamide) and 0.1% sodium dodecyl sulfate (Laemmli, 1970) using a Mini-Protean II apparatus from Bio-Rad. Proteins were electrophoretically transferred onto a nitrocellulose membrane (Burnette, 1981) in 0.2 M Tris/HCl, pH 9.2, buffer (10% methanol), at 18 mA overnight in a cold room. Transfer was verified by staining membranes with Ponceau red and replicas were developed using the polyclonal antiserum to pancreatic BSDL (pAbL64) or the monoclonal antibody specific for the J28 glycotope (mAbJ28). The recombinant C-terminal peptide of BSDL (Cter peptide) reactivity with antibodies was quantified by densitometric scannings of Western-blotting or of dot-blotting dilution. For this purpose, peptides were dotted onto nitrocellulose membrane in decreasing rank amount from 200 to 0.4 µg/well and probed with pAbL64 or mAbJ28. Typical lines were obtained by linear regression, the slopes of this lines were representative of the peptide reactivity (in arbitrary units). Alternatively, gels were stained with Coomassie Blue R 250, and destaining was performed in the Coomassie blue solvent (i.e., ethanol/acetic acid/water (2/3/35 by volume)). Glycosyltransferase assays
Standard assays of fucosyltransferases (Fuc-T) were performed in 50 µl final volume of 10 mM Tris/HCl pH 7.0 buffer (10 mM AMP, 10 mM MnCl2), containing 65 µM GDP-[14C]fucose (0.62 GBq/mmol) and 50 µg of cell proteins. Acceptor substrates (N-acetyllactosamine, 20 mM; lacto-N-biose, 20 mM; 3[prime]-sialyl-N-acetyllactosamine, 5 mM or phenyl [beta]-d-galactoside, 25 mM) were then added and the mixture incubated for 3 h. at 37°C. The reaction was terminated by the addition of 50 µl of ethanol and 500 µl of water and an aliquot was counted (LKB scintillation counter) using PCS scintillation fluid. When N-acetyllactosamine, lacto-N-biose, and 3[prime]-sialyl-N-acetyllactosamine were used as acceptors, 500 µl of the mixture were applied to a 1 ml column of AG 1-X8 resin (200-400 mesh, formate form, Bio-Rad) and radiolabeled products were eluted with 1.5 ml of water or 0.15 M NaCl with sialylated acceptor and counted. In the case of normal pancreatic tissues and tumoral pancreatic cell lines, products obtained by enzymatic reactions, as described above, were analyzed by thin layer chromatography. For this purpose, samples were freeze-dried and reconstituted with 10 µl of water. They were then dotted onto 20 × 20 cm precoated cellulose plates (Merck, Darmstadt, Germany). Product separation was performed using ethylacetate/pyridine/acetic acid/water (12:17:3:8 by vol.) as developing solvent for 7 h at room temperature. After development, plates were scanned on a radiochromatogram scanner (Automate TLC-linear analyzer LB2832, Berthold, France) using a 2.5 cm wide lane. Spots were identified using radiolabeled standards (Lewis a, Lewis x, fucosyl-type I, and fucosyl-type II structures) synthesized in our laboratory.
When phenyl [beta]-d-galactoside was used as fucose acceptor, the radiolabeled reaction product was isolated by chromatography on preconditioned reverse-phase Sep-Pak C18 cartridges (Waters-Millipore Corporation). After extensive washing with water (3 × 2 ml), the fucosylated product was eluted with acetonitrile/water (1:1 by vol) and counted.
Core-2 [beta]6-N-acetylglucosaminyltransferase (Core2GlcNAc-T) activity determination was performed in 50 µl of 50 mM MES, pH 7.0 buffer, 1 mM UDP-[3H]-GlcNAc (1.32 GBq/nmol), 0.1 M GlcNAc and 1 mM Gal[beta]1-3GalNAc-O-p-nitrophenyl (Toronto Research Chemical) as acceptor (Yousefi et al., 1991). The reaction was initiated by the addition of 50 µg of cell or tissue proteins and continued for 1 h at 37 °C. The reaction medium was diluted to 5 ml with water, applied to a C18 Sep-Pak column, washed twice with 20 ml water. The reaction product was eluted with 2 ml methyl alcohol and counted.
Controls were performed in the absence of exogenous acceptor to determine endogenous activities which were subtracted from values determined in the presence of added acceptor. Enzyme specific activity was defined as picomoles of sugar transferred from GDP- or UDP-sugar to the specific acceptor per minute and milligram of protein. In vitro fucosylation using recombinant soluble FUT3
In vitro fucosylation was performed in 50 µl final volume of 10 mM Tris/HCl pH 7.0 buffer (0.25% Triton X-100, 10 mM AMP, 10 mM MnCl2), containing 65 µM GDP-fucose and 100 µg of rsFUT3. The reaction was initiated by the addition of 1 mg of glycopeptide as acceptor and terminated for 4 h at 37°C. The reaction was stopped by dilution in cold water. Enzyme-linked immunosorbent assay (ELISA)
Nunc Maxisorb F96 certified immunoplates were coated with 100 µl/well of a solution of pAbL64 diluted to a concentration of 10 µg/ml in 0.1 M carbonate buffer, pH 9.5. The plates covered with adhesive and protected with aluminum sheet, were activated for at least one night at 4°C. Wells were washed three times with 300 µl 10 mM sodium phosphate buffer, pH 7.4, containing 0.05% Tween 20 (PBS/Tween). Wells were saturated with 1% BSA (200 µl/well) in PBS/Tween (30 min, 37°C) and washed three times with PBS/Tween. Then recombinant C-terminal peptides (diluted from 50 µg/ml to 0.05 µg/ml) were added in 100 µl of PBS/Tween. Plates were incubated 2 h at 37°C and then washed; 100 µl of a PBS/Tween solution of mAbJ28 (~5 µg/ml) were added, incubated another 2 h at 37°C, and washed. Alkaline phosphatase conjugated antibodies against mouse IgG diluted in PBS/Tween were added in each well and incubated 30 min at 37°C. Finally after washing, 100 µl of p-nitrophenyl phosphate at 1 mg/ml in 0.2 M Tris/HCl buffer, pH 8.2, containing CaCl2 and MgCl2 (each 1 mM), were added and incubated 1 h at 37°C. Plates were then read with a MR 5000 microplate spectrophotometer (Dynatech, Billingshurst, UK) using a 405 nm filter. Hydrophobic cluster analysis (HCA)
HCA is a graphical protein sequence comparison method based on the detection and comparison of hydrophobic clusters which are presumed to correspond to the regular secondary structure elements constituting the hydrophobic core of globular protein (Gaboriaud et al., 1987; Lemesle-Varloot et al., 1990). The HCA plot were obtained by submitting the protein sequences on program HCA plot from the web site http://www.lmcp.jussieu.fr/~soyer/www-hca/hca-form.html (Callebaut et al., 1997).
This work was supported by grants (Nb 6122 and Nb 9506) from the Association pour la Recherche sur le Cancer (ARC, Villejuif, France) and the financial help from the Ligue Nationale contre le Cancer (Région PACA, Toulon, France) and the Conseil Général des Bouches-du-Rhône (Marseilles, France). E.Pasqualini is the recipient of a fellowship from the Fondation pour la Recherche Médicale (Paris, France). Prof. J.R.Delpéro (Institut Paoli-Calmettes, Marseilles) is greatly acknowledged for his kind gift of human pancreatic tissues. We are also indebted to Drs. M.J.Escribano and M.Fukuda for the release of SOJ-6 cell line and of the cDNA encoding the full length Core2GlcNAc-T, respectively. We also address our special thanks to Dr. J.B.Lowe for the kind gift of pCDM8-FUT7 and pCDM7-FUT3 plasmids.
BSDL, bile salt-dependent lipase (E.C. 3. 1. 1. -); FAPP, feto-acinar pancreatic protein; Fuc-T, fucosyltransferase; Core2GlcNAc-T, core-2 [beta]6-N-acetylglucosaminyltransferase (E.C. 2. 4. 1. 102); CHO, Chinese hamster ovary (cell line); FCS, fetal calf serum.
Introduction
Results
Activities
Substrates
Normal pancreatic tissues(pmol/min/mg protein)
Tumoral pancreatic cell lines (pmol/min/mg protein)
SOJ-6
BxPC-3
MiaPaCa-2
Panc-1
[alpha]2-Fuc-T
Phenyl [beta]-d-galactosidea
26.4 ± 0.7
6.4 ± 0.2
7.7 ± 1.6
0.4 ± 0.3
2.0 ± 0.5
[alpha]3-Fuc-T
N-Acetyllactosaminea
10.9 ± 0.7
6.6 ± 0.4
10.0 ± 1.3
1.9 ± 0.2
3.0 ± 0.1
[alpha]4-Fuc-T
Lacto-N-biosea
6.7 ± 1.3
23.6 ± 1.1
13.7 ± 0.7
0.6 ± 0.1
<0.1
Core2GlcNAc-T
Gal[beta]1-3GlcNAc-O-p-nitrophenyl
n.d.
1071 ± 345
87 ± 17
145 ± 15
n.d.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 25 Aug 1999
Copyright©Oxford University Press, 1999.