Glycobiology, 2002, Vol. 12, No. 3 153-162
© 2002 Oxford University Press
Biosynthesis of the carbohydrate antigenic determinants, Globo H, blood group H, and Lewis b: a role for prostate cancer cell 
1,2-L-fucosyltransferase
Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Received on May 17, 2001; revised on December 27, 2001; accepted on December 31, 2001.
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Prostate carcinoma LNCaP cells were unique among several human cancer cell lines which include two other prostate cancer cell lines, PC-3 and DU-145, in expressing
1,2-L-fucosyltransferase (FT) as an exclusive FT activity. Affinity gel-GDP and Sephacryl S100 HR columns were used for a partial purification of this enzyme from 3.9 x 109 LNCaP cells (~200-fold; 40% yield). The Km value (2.7 mM) for the LacNAc type 2 acceptor was quite similar to the one reported for the cloned blood group H gene-specified 1,2-FT [Chandrasekaran et al. (1996)
Biochemistry 35, 8914–8924]. N-Ethylmaleimide was a potent inhibitor (Ki 12.5 µM). The enzyme showed four-fold acceptor preference for the LacNAc type 2 unit in comparison to the T-hapten in mucin core 2 structure. Its main features were similar to those of the cloned enzyme: (1) C-6 sulfation of terminal Gal in the LacNAc unit increased the acceptor efficiency, whereas C-6 sialylation abolished acceptor ability; (2) C-6 sulfation of GlcNAc in LacNAc type 2 decreased by 80% the acceptor ability, whereas LacNAc type 1 was unaffected; (3) Lewis x did not serve as an acceptor; (4) the C-4 hydroxyl rather than the C-6 hydroxyl group of the GlcNAc moiety in LacNAc type1 was essential for activity; and (5) the acrylamide copolymer of Galß1,3GlcNAcß-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of 1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galß1,3GalNAcß1,3Gal-O-Me and Galß1,3(Fuc1,4)Glc-NAcß1,3Galß-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucß1,3Gal-NAcß1,3Gal-O-Me was a very efficient acceptor, indicating that the C-6 hydroxyl group of the terminal Gal moiety in Globo H is not essential for the enzyme activity. Thus, the present study was able to demonstrate three different catalytic roles of LNCaP 1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H.
Key words: 1,2-L-fucosyltransferase/Globo H/H-type 1/Lewis b/prostate cancer cells
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Introduction |
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A characteristic feature of tumor progression in distal colon and rectum is the expression of the blood group determinants Lewis b, H-type 2, and Lewis y, which contain the common motif Fuc
1,2Galß-R (Yuan et al., 1985; Hakomori, 1989; Itzkowitz, 1992). An increase of 1,2-fucosyltransferase (FT) activity was also found in colon cancer (Orntoft et al., 1991; Yazawa et al., 1993; Sun et al., 1995). The H gene, which is overexpressed in colon cancer tissues, is the gene responsible for the synthesis of H blood group antigen of erythrocytes (Sun et al., 1995). Goupille et al. (1997) transfected a weakly tumorigenic clone derived from a rat colon carcinoma cell line with the cDNA encoding for the human H FT and found that the expression of 1,2-fucosylated structures on a variant of CD44 adhesion molecule may be responsible for the aggressiveness of colon carcinoma cells.
Globo H is another structure containing Fuc1,2Galß- and has been found at the cancer cell surface as a glycolipid and as glycoprotein (Bremer et al., 1984; Menard et al., 1983; Livingston, 1995; Adobati et al., 1997). Globo H has been characterized by immunohistochemistry using the murine monoclonal antibody MBr1 (Bremer et al., 1984; Zhang et al., 1997). There is enhanced expression of MBr1-positive antigens on both primary and metastatic prostate cancer specimens (Zhang et al., 1998). Slovin et al. (1999) showed that Globo H is one of several candidate antigens present in prostate cancer cells that can serve as targets for immune recognition and treatment strategies.
An examination of several cancer cell lines by this laboratory revealed that the breast cancer cell line MCF-7 and the prostate cancer cell line LNCaP expressed 1,2-FT activity. Both 1,2- and 1,3-FT activities were found in MCF-7. In contrast to two other prostate cancer cell lines, DU145 (Mickey et al., 1977) and PC-3 (Kaighn et al., 1979) expressing 1,3-FT activity, LNCaP expressed exclusively 1,2-FT activity. Furthermore, LNCaP has been shown to maintain the characteristics of prostate carcinoma synthesizing secretory human prostatic acid phosphatase and organ-specific prostate antigen responding to sex hormones and containing the y chromosome (Horoszewicz et al., 1983). In view of the fact that Globo H Fuc1,2Galß1,3GalNAcß1,3Gal1,4Galß1,4Glc is overexpressed in prostate cancer, it is of interest to study 1,2-FT, which is expressed by prostate cancer cells. The present article deals with partial purification and characterization of 1,2-L-FT present in LNCaP cells.
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Identification of
1,2-L-FT as an exclusive FT activity in LNCaP cellsThe FT activities of LNCaP cells were compared with those of two other prostate cancer cell lines DU145 and PC3, the breast cancer cell line MCF-7 and the ovarian teratocarcinoma cell line PA-1 by using a number of synthetic acceptors (see Table I). The use of Galß-O-Bn, which is a specific acceptor for measuring
1,2-L-FT, indicated that only LNCaP and MCF-7 expressed 1,2-FT. DU145, PC-3 and PA-1 were active toward Galß1,4GlcNAcß-O-Al and were almost inactive toward Galß1,3GlcNAc, indicating that they express exclusively 1,3-L-FT activity. MCF-7 showed more than fivefold activity toward Galß1,4GlcNAcß-O-Al as compared to its activity with Galß-O-Bn, indicating that 1,3- and 1,2-L-FT activities were the major and minor activities, respectively. On the other hand, LNCaP utilized the acceptors Galß-O-Bn, Galß1,3GlcNAc, and Galß1,4GlcNAc and their glycosides to the same extent, indicating that 1,2-L-FT is the only fucosylating activity in LNCaP cells. In support of this contention, LNCaP was almost inactive toward the acceptors specific for measuring 1,3- and 1,4-L-fucosylating activities such as NeuAc2,3Galß1,4GlcNAcß-O-Bn, 2-O-MeGalß1,3GlcNAcß-O-Bn, and so on (see Table I). But LNCaP exhibited high activity (148.3%) toward 6-O-SulfoGalß1,3GlcNAcß-O-Al, which had been shown earlier by our laboratory (Chandrasekaran et al., 1996) as a high-affinity acceptor for the cloned 1,2-L-FT.
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Partial purification and acceptor-substrate specificity analysis of
1,2-L-FT uniquely expressed by LNCaP cellsThe enzyme has been purified ~200 fold with 40% recovery (Table II) by using Triton X-100 extraction, affinity Gel-GDP chromatography, and then fractionation on Sephacryl S-100 HR column. When the final purified enzyme preparation was examined with various synthetic compounds as acceptors (Table III), it was found that LacNAc types 1 and 2 were better than blood group T-hapten (Galß1,3GlcNAcß-O-Al: 120.0%; Galß1,4GlcNAcß-O-Al: 97.9% and Galß1,3GalNAc
-O-Al: 64.8%). Replacement of terminal ß-Gal with ßGalNAc resulted in loss of activity (GalNAcß1,4GlcNAcß-O-Bn: 2.8%). Compounds with terminal -Gal did not serve as acceptors. The GlcNAc moiety of LacNAc structure has influence on the enzyme activity (Galß1,3GlcNAcß-O-Al: 120.0%; Galß1,3Galß-O-Al: 42.8%). Furthermore, the C-4 OH of the GlcNAc moiety is essential for the enzyme activity (Galß1,3[4-O-Me]GlcNAcß-O-Bn: 13.8%; Galß1,3[6-O-Me]GlcNAcß-O-Bn: 88.3%; and Galß1,3[4–6-di-O-Me]GlcNAcß-O-Bn: 9.7%).
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As compared to T-hapten, the Globo H backbone structure was twofold more active (Galß1,3GalNAc
-O-Al: 64.8%; Galß1,3GalNAcß1,3Gal-O-Me: 124.8%). Furthermore, we found that the C-6 OH group of the terminal Gal moiety in Globo H structure is not needed for the enzyme activity (D-Fucß1,3GalNAcß1,3Gal-O-Me: 142.1%). LacNAc structures in extended chains were quite active (Galß1,3GlcNAcß1,3Gal-ß1,4Glc: 107.6%; Galß1,4GlcNAcß1,6Man-1,6Man1,6Man: 114.5%). Compounds containing terminal Lewis x structure were inactive (Galß1,4[Fuc1,3]GlcNAcß1,6Man1,6Man: 2.1%; Galß1,4[Fuc1,3]GlcNAc-ß1,3Galß-O-Me: 0%).
The most interesting observation was that ß-glycosides of Lewis a were inactive acceptors, whereas Lewis a linked ß1,3 to Gal was equally active as Galß-O-Bn (Galß1,3[Fuc1,4[GlcNAcß-O-Al: 4.1%; Galß1,3[Fuc1,4]GlcNAcß-O-Naph: 0.7%; Galß1,3[Fuc1,4[GlcNAcß-O-Me: 0%; Galß1,3[Fuc1,4]Glc-NAcß1,3Galß-O-Me: 109.0%). In this context, it is important to note that GlcNAc substitution on C-3 OH of the terminal Gal moiety abolished the acceptor activity (GlcNAcß1,3Galß-O-Me: 0.1%; GlcNAcß1,3Galß1,4Glc: 1.4%); these data strongly suggest that Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me is being 1,2 fucosylated on the terminal Gal moiety.
As anticipated, LacNAc structure in mucin core 2 was found as a better acceptor than the Gal moiety linked ß1,3 to GalNAc (Galß1,4GlcNAcß1,6[3-O-MeGalß1,3]GalNAc-O-Bn: 117.9%; 3-O-MeGalß1,4GlcNAcß1,6[Galß1,3]GalNAc-O-Bn: 78.6%). It was also noticed that a terminal ß1,6-linked GlcNAc moiety in mucin core 2 inhibits the enzyme activity (3-O-MeGalß1,4GlcNAcß1,6[Galß1,3]GalNAc-O-Bn: 78.6%; Galß1,3[GlcNAcß1,6]GalNAc-O-Al: 35.2%).
Among the acrylamide copolymers of allyl glycosides containing terminal ß-Gal moiety, Galß1,3GlcNAcß-O-Al/AA-CP exhibited the highest affinity for the enzyme (39.3% activity at 0.05 mM concentration as compared to Galß-O-Bn at 3.0 mM concentration).
Next, we examined the influence of sulfate or sialyl group on the enzyme activity using various sulfated or sialylated synthetic compounds (Table IV). C-6 sulfation of the terminal Gal moiety increased the acceptor ability (6-O-SulfoGalß1,3GlcNAcß-O-Al: 151.1%; 6-O-SulfoGalß1,4Glc-NAcß-O-Me: 165.4%). C-6 sulfation of GlcNAc in LacNAc type II decreased the acceptor activity (Galß1,4[6-O-Sulfo]Glc-NAc: 36.8%; Galß1,4[6-O-Sulfo]GlcNacß1,6Man-O-Me: 18.0%). On the other hand, C-6 sulfation of GlcNAc in LacNAc type I increased the acceptor efficiency (Galß1,3[6-O-Sulfo]GlcNAcß-O-Bn: 131.6%; Galß1,3[6-O-Sulfo]GlcNAc-ß1,3Galß-O-Al: 185.7%). C-6 sialylation of the terminal Gal moiety abolished the acceptor ability (NeuAc2,6Gal-ß1,3GlcNAcß-O-Bn: 1.5%). The compounds 6-O-Sulfo-Galß1,4(Fuc1,3)GlcNAcß-O-Bn, GalNAcß1,4(6-O-Sulfo)Glc-NAc-ß-O-Me, 6-O-SulfoGalß1,3(NeuAc2,6)GalNAc-O-ONP, and 6-O-SulfoGlcNAcß1,3Galß-O-Me were identified as inactive acceptors (see Table IV). The Globo H backbone structure on 3-O-sulfation of terminal Gal became an inactive acceptor (3-O-SulfoGalß1,3GalNAcß1,3Gal-O-Me: 0.8%). Neither C-6 sulfation of the GlcNAc moiety nor C-3 sulfation of one of the terminal Gal moieties of the mucin core 2 acceptors had any appreciable influence on their acceptor activities, as was evident from a comparison of their acceptor efficiencies to that of the corresponding non-sulfated compounds (see Table IV).
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The kinetic properties of LNCaP
1,2-L-FTMg2+ and Mn2+ stimulated to a maximum of 100% the LNCaP
1,2-L-FT activity at 30 mM and 10 mM concentrations (Figure 1) respectively, whereas Ca2+ stimulation was 50% at 20 mM. There was no inhibition of this activity by any of the divalent metal ions at the maximum concentration (50 mM) tested.
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The pH optimum for this enzyme activity was found to be 6.8 (Figure 2) as demonstrated with two different acceptors, namely, Galß1,4GlcNAcß-O-Al and Galß1,3GalNAcß1,3Gal
-O-Me. The two acceptors showed an identical pattern of activities over the pH range tested, indicating that the same enzyme utilizes both acceptors. An identical pattern of inhibition was observed when the enzyme activity was measured with three different acceptors: Galß-O-Bn, Galß1,4GlcNAcß-O-Al, and Galß1,3GalNAcß1,3Gal-O-Me (Figure 3) in the presence of varying concentrations of N-ethylmaleimide and the Ki was calculated as 12.5 µM. This data strongly suggested that the same enzyme 1,2 fucosylates both LacNAc and Globo H backbone structures.
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Demonstration of the enzyme specificity toward LacNAc structures
The [14C] fucosylated products arising from the mucin core 2 acceptor containing two terminal Gal moieties (namely, Galß1,4GlcNAcß1,6(Galß1,3)GalNAc
-O-Bn) were isolated by Sep-Pak C18 cartridge method and then subjected to fractionation on peanut agglutinin–agarose column (see Figure 4). It was found that ~80% of the radioactive product exhibited specific binding to the column, indicating its structure as 14C-Fuc1,2Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bn and the remaining 20% as Galß1,4GlcNAcß1,6(14C-Fuc-1,2Galß1,3)GalNAc-O-Bn.
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Further evidence for the
1,2-L-fucosylation of LacNAc and Globo H backbone structures by the same enzymeThe enzymatic transfer of [14C]Fuc to the acceptors Galß1,4GlcNAcß-O-Al and Galß1,3GalNAcß1,3Gal
-O-Me at varying concentration was measured separately both in absence and in presence of 3.0 mM sulfated acceptors, namely, 6-O-SulfoGalß1,4GlcNAcß-O-Me, 6-O-Sulfo Galß1,3Glc-NAcß-O-Al, or 6-O-SulfoGalß1,3GalNAc-O-Al (see Figure 5). It was found that only the sulfated LacNAc acceptors inhibited profoundly the transfer of [14C]Fuc to both Galß1,4GlcNAcß-O-Al and Galß1,3GalNAcß1,3Gal-O-Me in an identical fashion, whereas 6-O-SulfoGalß1,3GalNAc-O-Al showed very low inhibition in both cases. The Km values (mM) using the acceptors Galß1,4GlcNAcß-O-Al and Galß1,3GalNAcß1,3Gal-O-Me, respectively, were as follows: 2.7 and 1.6; in presence of (1) 6-O-SulfoGalß1,4GlcNAcß-O-Me (8.0 and 6.7), (2) 6-O-SulfoGalß1,3GlcNAcß-O-Al (6.7 and 5.0); and (3) 6-O-Sulfo-Galß1,3GalNAc-O-Al (3.3 and 2.1).
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Identification of the fucosyl linkage as
1,2 and also the novel 1,2-L-fucosylating activity of LNCaP 1,2-L-FTThe [14C] fucosylated compounds arising from the acceptors Galß1,3GlcNAcß1,3Galß-O-Me, Galß1,3(Fuc
1,4)GlcNAc-ß1,3Galß-O-Me, Galß1,3GalNAcß1,3Gal-O-Me and D-Fuc-ß1,3GalNAcß1,3Gal-O-Me were isolated by Dowex-1-Cl method followed by Biogel P2 chromatography. The acceptors moved as single spots on thin-layer chromatography (TLC) (Figure 6, panel I). As anticipated, Galß1,3(Fuc1,4)Glc-NAcß1,3Galß-O-Me had lower mobility than Galß1,3Glc-NAcß1,3Galß-O-Me; D-Fucß1,3GalNAcß1,3Gal-O-Me had higher mobility than Galß1,3GlcNAcß1,3Gal-O-Me. Each radioactive product moved as a single spot exhibiting lower mobility than the parent acceptor (Figure 6, panel II). The radioactive product from Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me moved slower than the product from Galß1,3GlcNAcß1,3GalNAcß1,3Galß-O-Me. The radioactive product from D-Fucß1,3GalNAcß1,3Gal-O-Me moved faster than the product from Galß1,3GalNAcß1,3Gal-O-Me. Furthermore, we have shown by autoradiography that the radioactive products moved as single spots, exhibiting the same mobility difference (Figure 6, panel IV). Treatment of these radioactive products with 1,2-L-fucosidase (Prozyme, CA) resulted in a complete release of [14C]Fuc (Figure 6, panel III).
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We found that the acceptor specificities of LNCaP
1,2-L-FT were quite identical to those of the cloned human blood group H gene-specified 1,2-L-FT reported earlier by our laboratory (Chandrasekaran et al., 1996). The main features were as follows: (1) The Km values for LacNAc type 2 for both enzymes were almost the same (cloned enzyme:1.67 mM; LNCaP enzyme: 2.70 mM; (2) the specificities of both enzymes are directed mainly toward LacNAc structure (fourfold as compared to T-hapten Galß1,3GalNAc); (3) C6 sulfation of terminal Gal increased the acceptor efficiency, whereas C6 sialylation abolished the acceptor ability; (4) C6 sulfation of GlcNAc in LacNAc type 2 decreased ~80% the acceptor activity, whereas LacNAc type 1 was not affected at all; (5) Lewis x did not serve as an acceptor; (6) the C4 hydroxyl group but not the C6 hydroxyl group of the GlcNAc moiety in LacNAc type 1 is essential for the activity; and (7) Galß1,3GlcNAcß-O-Al/AA-CP served as the best acceptor among the various acrylamide copolymers examined.
The present study used additional novel synthetic acceptors and was able to decipher biologically highly significant unique catalytic abilities of this enzyme. The synthetic compounds Galß1,3GalNAcß1,3Gal-O-Me, D-Fucß1,3GalNAcß1,3Gal-O-Me, and Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me served as high-affinity acceptors for this enzyme. Thus this enzyme has the potential to synthesize Globo H (namely, Fuc1,2Galß1,3GalNAcß1,3Gal-) and Lewis b in extended chain (namely Fuc1,2Galß1,3(Fuc1,4)GlcNAcß1,3Galß-). Furthermore, this enzyme utilizes the compound D-Fuc-ß1,3GalNAcß1,3Gal-O-Me very efficiently as an acceptor, indicating that the C6 hydroxyl group of the terminal Gal moiety is apparently not essential for enzyme activity.
The construction of Fuc1,2Galß- linkage known as H determinants is determined by the H and secretor blood group loci known as FUT 1 and FUT 2 respectively (Larsen et al., 1990; Kelly et al., 1995). Sarnesto et al. (1992)) gave evidence for a significant difference in the kinetic properties of the H- and secretor-type 1,2-L-FTs purified from human sera. Valli et al. (1998) studied 1,2-L-FT in human colon adenocarcinoma cell lines and suggested the involvement of secretor-type 1,2-L-FT in the biosynthesis of type 1 chain tumor-associated antigens in human colon carcinoma cells. Nishihara et al. (1999) reported that an augmented expression of Le b antigens in distal colon cancers was caused mainly by up-regulation of the secretor enzyme, and in proximal cancers, it is caused by the upregulation of the H enzyme alone, indicating that both enzymes are involved in cancer. The present study has demonstrated that LNCaP 1,2-L-FT can synthesize Lewis b from Lewis a and Globo H from Galß1,3GalNAcß1,3Gal-. Furthermore, our previous studies (Chandrasekaran et al., 1995, 2001) have demonstrated that Lewis type FT (FT III) can synthesize Lewis b from Lewis a. Thus, the biosynthesis of Lewis b determinant and Globo H can be outlined in a scheme (Figure 7).
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We found in the present study that the LNCaP
1,2-FT could utilize both Galß1,3GlcNAc and Galß1,4GlcNAc resulting in type 1 and type 2 H structures. However, it could catalyze the synthesis of Lewis b from Lewis a, but not of Lewis y from Lewis x. The explanation for this discrepancy comes from the conformational analysis, as discussed.
The conformational analysis of Imberty et al. (1995)) indicates that the torsion angle at the Galß1,4GlcNAc linkage in the Lewis x trisaccharide has a maximum of –103.6, whereas this value for the Galß1,3GlcNAc linkage in the Lewis a trisaccharide is 144.1. The maximum torsion angle at the Fuc1,3GlcNAc linkage is –150.7 and at the Fuc1,4GlcNAc linkage is –97.7. The research group further reported that, in contrast to Lewis x, a second conformational family exists in the case of the Lewis a trisaccharide, even if it is not energetically favored. They explained that when this conformational family occurs, it is correlated to a conformational change in the Fuc1,4GlcNAc linkage, resulting in a change of 
value from –97.7 to –170.7. Pérez et al. (1996) studied the conformation of two crystallographic independent molecules of the Lewis x trisaccharide and found that these two molecules differ essentially in the torsion angles at their Galß1,4GlcNAc linkages by 10°. This observation could suggest the possible existence of two or possibly more conformational species of the Lewis a trisaccharide differing in their glycosidic torsion angles at the Galß1,3 GlcNAc linkage. Thus, all the conformational analysis data would indicate that the Lewis a trisaccharide exhibits a conformation that is favorable for enzymatic action leading to the formation of the Fuc1,2 Gal linkage. Additionally, in support of this contention, we have very recently found that gastric carcinoma 1,2-FT can utilize Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me but not Galß1,4(Fuc1,3)GlcNAcß1,3Galß-O-Me as an acceptor substrate (Chandrasekaran et al. unpublished data).
Now, we have shown that the synthesis of Lewis b is facilitated by the dual catalytic roles of 1,2-FT and 1,3/4-FT and that of Globo H by 1,2-L-FT, and these enzymes are encountered in normal tissues. It is tempting to conclude that the overexpression of these carbohydrate antigens in cancer is not caused by an aberrant or novel 1,2-L-FT but may be due to an overexpression of these normal enzymes in cancer. However, there is a possibility for the existence of an aberrant or novel 1,2-FT converting Lewis x to Lewis y, and this particular enzyme may also convert Lewis a to Lewis b.
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Materials and methods |
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Cell culture
The breast carcinoma cell line MCF-7, the prostate cancer cell line DU 145, and the ovarian teratocarcinoma cell line PA-1 were grown in minimal essential medium. The prostate carcinoma cell lines LNCaP and PC-3 were grown in RPMI 1640 and Leibovitz’s medium, respectively (all media were supplemented with 10% fetal bovine serum and the antibiotics penicillin, streptomycin, and emphtoericin B) in 250-ml T-flasks under conditions as recommended by the American Type Culture Collection. Furthermore, LNCaP cells were grown in 2-L roller bottles to obtain the large amount of cells needed for the purification of
1,2-L-FT. The cells were homogenized with 0.1 M Tris–Maleate, pH 7.2, containing 2% Triton X-100 and 0.1% NaN3 using a Dounce all-glass hand-operated homogenizer. The homgenate was centrifuged at 16,000 x g for 1 h at 4°C. Protein was measured on the supernatants by the BCA micro method (Pierce Chemical) with bovine serum albumin as the standard. The supernatants were adjusted to 5 mg protein/ml by adding the necessary amount of extraction buffer and then stored frozen at –20°C until use.
Partial purification of 1,2-L-FT from human prostate carcinoma LNCaP cells
LNCaP cells (3.9 x 109) were homogenized with 30 ml of 0.1 M Tris–Maleate, pH 7.2, 2% Triton X-100, and 0.1% NaN3 using a Dounce all-glass hand-operated homogenizer; subjected to mixing in the cold room using speci-mix for 2 h; and then centrifuged at 16,000 x g at 4°C. The pellet was rehomogenized with 20 ml of the extraction buffer, mixed for 1 h, and centrifuged as before. The combined supernatant was passed through affinity Gel-GDP (25 ml bed volume) that had been equilibrated with the extraction buffer. After the entry of the sample, the affinity column was washed with 75 ml of the extraction buffer and then eluted with 100 ml of the extraction buffer containing 0.5 mM GDP. The eluate was concentrated by Amicon ultrafiltration to 5 ml using PM10 membrane and dialyzed against 500 ml of the extraction buffer for 48 h at 4°C with three changes. The purified enzyme preparation was applied to a Sephacryl S-100 HR column (2.5 x 118.0 cm) at 4°C equilibrated and eluted with 0.1 M Tris–Maleate, pH 7.2, containing 0.1% Triton X-100 and 0.02% NaN3. Fractions of 2 ml at a flow rate of 6 ml/h were collected, and 10-µl aliquots of alternate fractions were used for measuring 1,2-L-FT activity using Galß-O-Bn as the acceptor. The enzyme activity emerged as a single peak after the void volume. These fractions were pooled, concentrated, and dialyzed as described and then stored frozen at –20°C.
Synthetic compounds
We have already reported the synthesis of several compounds used in the present study (Jain et al., 1993, 1994, 1998; Chandrasekaran et al., 1995). The chemical synthesis of Galß1,3GalNAcß1,3Gal-O-Me and D-Fucß1,3GalNAc-ß1,3Gal-O-Me will be reported elsewhere.
Assay of FT
The incubation mixtures run in duplicate contained 50 mM HEPES–NaOH, pH 7.5, 5 mM MnCl2, 7 mM ATP, 3 mM NaN3, the acceptor (3.0 mM unless otherwise stated), 0.05 µCi of GDP-[U-14C]Fuc (specific activity 290 mCi/mmol) and enzyme in a total volume of 20 µl; the control incubation mixtures had everything except the acceptor. At the end of incubation for 2 h at 37°C, the mixture was diluted with 1.0 ml of water and passed through a Dowex-1-Cl column (1 ml in a Pasteur pipet) (Chandrasekaran et al., 1992). The column was washed twice with 1 ml water; the breakthrough and wash that contained the [14C] fucosylated neutral acceptor were collected together in a scintillation vial and the radioactive content was determined using 3a70 scintillation fluid (Research Products International, Mount Prospect, IL) and a Beckman LS9000 instrument. The Dowex column was then eluted with 3.0 ml of 0.2 M NaCl to obtain the [14C] fucosylated product from sialylated/sulfated acceptors and then counted for radioactivity as described. Corrections were made by subtracting the radioactivity in the water and NaCl eluates of the control incubation mixtures from the values of the corresponding eluates of the tests. Duplicate samples did not vary more than 5%.
Effect of pH and divalent cations on LNCaP 1,2-L-FT
HEPES–NaOH buffer in the pH range 6.0–8.4 (final concentration in reaction mixture, 0.1 M) was used under the standard incubation conditions. For seeing the effect of divalent metal ions, the incubation mixture contained varying concentrations (0–50 mM) of Mg acetate, Mn acetate, or Ca acetate under standard incubation conditions.
Inhibition by N-ethylmaleimide
The enzyme was preincubated for 30 min at 37°C with varying concentration of NEM and then assayed under standard incubation conditions.
Testing for competitive inhibition
We took advantage of the fact that the radioactive product arising from the competitive acceptor, namely, the monosulfated compound, binds to the Dowex-1 column, whereas the product from the non-sulfated neutral acceptor can be washed out from the column with water. The concentration of the neutral acceptor was varied from 0 to 3.0 mM and that of the sulfated acceptor was kept constant (3.0 mM).
Peanut agglutinin–agarose affinity chromatography
A column of 5 ml bed volume of peanut agglutinin agarose (Vector Lab, Burlingame, CA) was employed using 10 mM HEPES, pH 7.5, containing 0.1 mM Ca2+, 0.01 mM Mn2+, and 0.1% NaN3 as the eluting buffer as recommended by the manufacturer. The fractionation was done at room temperature. The [14C] fucosylated product mixture from Galß1,4GlcNAc-ß1,6(Galß1,3)GalNAc-O-Bn was applied to the column in 1.0 ml of the buffer. After the entry of the sample into the column bed, it was allowed to remain in contact with the gel for 20 min before starting elution with the same buffer. Fractions of 1 ml were collected. The bound material was eluted with the same buffer containing 0.2 M galactose.
Identification of the products arising from Galß1,3GlcNAcß1,3Galß-O-Me, Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me, Galß1,3GalNAcß1,3Gal-O-Me, and D-Fucß1,3GalNAcß1,3Gal-O-Me by the action of LNCaP FT
Tenfold reaction mixtures (200 µl) containing each acceptor separately were incubated for 4 h at 37°C. After incubation the reaction mixtures were diluted with 1 ml water and then passed through Dowex-1-Cl as described. The breakthrough plus water eluates were lyophilized to dryness, dissolved in 1 ml water and then subjected to chromatography on a Biogel P-2 column (1.0 x 116 cm) utilizing 0.1 M pyridine acetate, pH 5.4, as the eluting buffer. Fractions appearing under the first peak containing the [14C] fucosyl compound were pooled in each case, lyophilized to dryness and dissolved in 200 µl water. These radioactive samples were subjected to TLC on silica gel GHLF (Analtech, 250 microns, scored 20 cm x 20 cm) plates developed in the solvent system 1-propanol/NH4OH [25%]/water (12:2:5). The radioactive content of half-cm width segments scraped into scintillation vials and soaked in 2 ml water was determined by liquid scintillation spectroscopy. Autoradiography was carried out at –70°C using Biomax-MR film (Kodak) after spraying the TLC plate with EnHance (DuPont).
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Acknowledgment |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentAbbreviationsReferences |
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This work was supported by grant CA35329 awarded by the National Cancer Institute.
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Abbreviations |
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TopAbstractIntroductionResults and discussionMaterials and methodsAcknowledgmentAbbreviationsReferences |
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FT, fucosyltransferase; T-hapten: Galß1,3GalNAc
-; TLC, thin-layer chromatography.
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1 To whom correspondence should be addressed
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