Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
Received on October 25, 1999; revised on December 27, 2000; accepted on January 25, 2001.
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Abstract |
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The 35-kDa glycoprotein ancrod (from Malayan pit viper venom) containing 36% complex N-glycans with the antennae NeuAc2,3Galß1,3GlcNAcß- acted as the best macromolecular acceptor substrate (Km: 45 µM), as examined with FTB. On desialylation the acceptor efficiency dropped to
50% (Km for asialo ancrod: 167 µM). Sialylglycoproteins, such as carcinoembryonic antigen, fetuin, and bovine
1-acid glycoprotein, were better acceptors than asialo fetuin. On the contrary, fetuin triantennary glycopeptide containing predominantly NeuAc
2,3Galß1,4GlcNAcß- was only 55% active as compared to the asialo glycopeptide (Km: 1.43 and 0.63 mM, respectively). Thus, the human lung tumor
1,3/4-L-FT has the potential to generate clustered sialyl Lewis a and Lewis b determinants in N-glycans and sialyl Lewis x determinant in mucin core 2 structures.
Key words: fucosyltransferases/kinetic properties/mucinous lung adenocarcinoma/specificities/sialyl Lewis a, x, and b determinants
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Introduction |
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The cellular expression of the Lewis blood group associated antigens Lea, Leb, and sialyl Lea is confined largely to endodermally derived tissues, such as lining epithelia and glandular epithelia (Szulman and Marcus, 1973; Oriol et al., 1986
). The coexistence of
1,3- and
1,4-fucosyltransferase activities in tissues expressing Lewis antigens make it difficult to assess the extent to which the Lewis gene encoded enzyme contributes to the synthesis of Lewis xrelated structures. The
1,3/4 fucosyltransferase of human A431 epidermoid carcinoma cell line exhibited only 11% activity toward Galß1,4GlcNAc as compared to its activity on Galß1,3GlcNAc (Johnson et al., 1993
). A recent investigation on human lung carcinoma cells NCI-H69 and PC9 indicated that the total fucosyltransferase activity in these cells was primarily comprised of Fuc-T IV and Fuc-T VI (Sherwood and Holmes, 1999
). The present article reports the partial purification, acceptor specificities, and kinetic properties of two molecular forms of
1,3/4-L-fucosyltransferase from human lung tumor (poorly differentiated mucinous adenocarcinoma of lung) displaying high substrate affinity for clustered units of 3-sialyl Galß1,3GlcNAcß- in asparagine linked carbohydrate as well as for mucin core 2 structure containing 3-sialyl Galß1,4GlcNAcß- unit, in addition to the novel
1,2-L-fucosylating activity.
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Results |
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Characterization of the product arising from Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me by the action of FTB
The [14C] fucosyl compound isolated from the acceptor, Galß1,3GlcNAcß1,3Galß-O-Me, by the action of FTB had the same mobility as the authentic synthetic compound, Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me in two different solvent systems (Figure 2, panels I and II). The [14C] fucosylated product arising from Galß1,3(Fuc
1,4)GlcNAcß1,3Galß-O-Me moved as a single spot with lower mobility than the [14C] fucosylated product arising from Galß1,3GlcNAcß1,3Galß-O-Me in two different solvent systems. This was shown by locating the radioactivity by scraping the gel off the plates for counting, as well as by direct autoradiography (Figure 2, panels I, II, and III). When treated with
1,2-fucosidase (Prozyme), almost complete release of [14C] Fuc from the [14C] fucosylated product of Galß1,3(Fuc
1,4) GlcNAcß1,3Galß-O-Me was observed (Figure 2, panel IV). Both FTA and FTB exhibited the same pattern of activities toward the acceptor, Galß1,3(Fuc
1,4)GlcNAcß1,3Galß-O-Me (Figure 3A) and the Km values were FTA, 2.5 mM and FTB, 1.0 mM (Figure 3B).
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Association of novel 1,2-L-fucosylating activity with human lung adenocarcinoma
1,4-L-fucosyltransferase
FTB was used as the enzyme source. When 1,4-L-fucosyltransferase activity was measured with increasing concentration of the acceptor, 3-O-sulfoGalß1,3GlcNAcß-O-Al, in the presence of Galß1,3(4,6 di-O-Me)GlcNAcß-O-Al (3.0 mM), which is an acceptor for the novel
1,2-fucosylating activity, an inhibition of the former activity as well as mutual inhibition of the latter activity were observed (Figure 7A). Conversely, when the novel
1,2-L-fucosylating activity was measured with increasing concentration of the acceptor, Galß1,3(4,6 di-O-Me)GlcNAcß-O-Al, in the presence of 3-O-sulfoGalß1,3GlcNAcß-O-Al (3.0 mM), mutual inhibition of both activities occurred (Figure 7B).
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Discussion |
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The striking features of the two molecular forms of this enzyme from human lung tumor are as follows: The ratio of their activities with simple type I and type 2 LacNAc acceptor substrates closely resembles those of 1,3/4-fucosyltransferases purified from human milk (Johnson et al., 1992
) and the culture media of human A431 epidermoid carcinoma cell line (Johnson et al., 1993
), the activity being more than 10-fold greater toward type 1 acceptor. But there are subtle differences between these enzymes and the lung tumor FTA and FTB. Human milk and A431 cell line
1,3/4-FTs showed considerably reduced activity (46%) when sialic acid is linked to
2,3 to the terminal ß-galactosyl residue of type 1 structure, whereas FTA and FTA exhibited almost the same extent of activity with
2,3-sialylated Type 1 acceptors. 3'-Sialyl LacNAc type 2 was one third as active as 3'-sialyl LacNAc type 1 in case of
1,3/4FTs of human milk and A431 cell line, whereas this activity was only about one tenth with FTA and FTB, which is quite similar to the behavior of FTA and FTB toward asialo acceptors. In contrast to > twofold activity exhibited by A431 cell line
1,3/4-FT toward 2'-fucosyllactose, both FTA and FTB were only
60% active toward this acceptor as compared to 3'-sialyl LacNAc type 1 acceptor. Furthermore, we made a remarkable observation that 3'-sialyl LacNAc type 2 in mucin core 2 structure, namely, NeuAc
2,3Galß1,4GlcNAcß1,6(Galß1,3)GalNAc
-O-Me, is four to five times active as NeuAc
2,3Galß1,4GlcNAcß-O-Bn, and the acceptor efficiency is 43% and 63% that of NeuAc
2,3Galß1,3GlcNAcß-O-Bn toward FTA and FTB, respectively. In fact, even other substitutions (such as methyl or sulfate group) on C-3 OH of ß1,4-linked Gal of mucin core 2 increased the efficiency of these acceptors about five to eightfold (see Table II).
In an earlier study (Chandrasekaran et al., 1995b) we have shown an association of a novel
1,2-L-fucosylating activity, which leads to the expression of blood group Lewis b determinant from Lewis a,with the Lewis type
1,3/4-L-fucosyltransferase. In the present study we have shown that FTA and FTB can fucosylate the acceptors Galß1,3(Fuc
1,4)GlcNAcß-O-Al, Galß1,3(4-O-Me)GlcNAcß-O-Al, Galß1,3(4,6-di-O-Me)Glc-NAcß-O-Al, and Galß1,3(Fuc
1,4)GlcNAcß1,3Galß-O-Me. The efficiencies of accepting Fuc by Galß1,3(Fuc-
1,4)GlcNAcßGal-O-Me and Galß1,3(4,6-di-O-Me)Glc-NAcß-O-Al are remarkably equal or greater than that of Galß1,3GlcNAc. Johnson et al. (1993)
found Galß1,3(Fuc
1,4)GlcNAcß1,3Galß1,4Glc as equally good as Galß1,3GlcNAc as an acceptor for A431 carcinoma cell line
1,3/4-FT. Unfortunately, they did not attempt to locate the transferred radioactive Fuc in the product, but might have assumed its location as the Glc moiety in parallel to the product from 2'-fucosyl lactose.
A determination of Km values for a number of synthetic acceptors indicated that the most efficient one was the LacNAc type 1 acceptor containing a sulfate substituent on C-3 of Gal followed by the acceptors containing methyl or sialyl groups as C-3 substituents (Km: 0.10 mM, 0.40 mM, and 2.50 mM for FTA and 0.047 mM, 0.16 mM, and 0.67 mM for FTB, respectively). The Km determined for another efficient acceptor 3'-sialyl LacNAc type 2 in mucin core 2 structure, namely, NeuAc2,3Galß1,4GlcNAcß1,6(Galß1,3)GlcNAc
-O-Me, was 3.33 mM for FTA and 0.77 mM for FTB. In general FTA was lethargic in its activity as compared to FTB, probably due to its larger size. Johnson et al. (1993)
used carbohydrate acceptors containing no aglycans in their study on cell line A431
1,3/4-FT, and the Km values they reported were generally much higher than the values we obtained in the present study for FTA and FTB. This could be explained by their data that the one acceptor with an aglycan Galß1,3GlcNAc-O-(CH3)8COOMe they examined was superior to Galß-1,3GlcNAc (Km 0.3 mM and 3.0 mM, respectively).
Among the high molecular weight acceptors, ancrod serves as the best acceptor for 1,3/4-FT of human lung tumor. Ancrod, a thrombin-like serine protease isolated from the venom of the Malayan pit viper Agkistrodon rhodostoma (Nolan et al., 1976
), is a glycoprotein (36% carbohydrate by weight) of 35 kDa containing complex type N-glycans (di-, tri-, and tetraantennary carrying NeuAc
2,3Galß1,3GlcNAcß- in a molar ratio [mol/100 mol] 23:40:12) (Pfeiffer et al., 1992
). Thus, clusters of oligosaccharides containing NeuAc-
2,3Galß1,3GlcNAcß- seem to be the best target of this enzyme for action. After the removal of sialic acid, the activity of the enzyme on the asialo acceptor, that is, asialo ancrod, dropped to
50%. On the other hand, fetuin triantennary glycopeptide, which has been shown to contain NeuAc-
2,3Galß1,4GlcNAcß- in two antennae and a combination of NeuAc
2,3Galß1,4GlcNAcß- and NeuAc
2,3Galß1,3GlcNAcß- on the third (Townsend et al., 1986
), was less efficient than the corresponding asialo acceptor. This would suggest that NeuAc
2,3Galß1,4GlcNAcß- cluster in a triantennary chain has a negative influence on this enzyme activity. The enzyme appears to like sialylated glycoproteins; this becomes evident from the finding that fetuin, bovine
1-acid glycoprotein, and carcinoembryonic antigen served as better acceptors than asialo fetuin.
When the acceptor substrate specificity of the Lewis enzymes was characterized by site-directed mutagenesis (Depuy et al., 1999) it was found that Trp111 of hypervariable stem domain is responsible for the specificity of fucose transfer to H-type 1 acceptors, and the acidic residue Asp112 is essential for this enzyme activity. It was also found that more than Trp and Asp are probably necessary to change the specificity of bovine fut bencoded
1,3-L-fucosyltransferase from type 2 activity to type 1 (Depuy et al., 1999
). Consistent with this observation, a very small increase in the number of cells producing Lea and sialyl Lea antigens was observed in cells transfected by a Fuc-T VI chimera containing Fuc-T III subdomains 4 and 5 (positions 103153) (Legault et al., 1995
). It was also shown that two amino acid changes in Fuc-T V (Asp 86
His and Thr87
Ile) increased the type 1 activity of the recombinant enzyme (Nguyen et al., 1998
). The molecular phylogeny of fucosyltransferase genes suggests that the common ancester of FUT 3 and FUT 5 genes has acquired the capacity to use type 1 acceptors without loss of the activity to use type 2 acceptors (Oulmouden et al., 1997
). It is interesting to note that the
1,3/4-fucosyltransferase of human lung tumor seems to have a very low capacity to fucosylate type 2 acceptors, indicating that other independent mutations might have contributed to this change.
Lo-Guidice et al. (1995) identified a sulfotransferase in human respiratory mucosa responsible for the 3-O-sulfation of terminal Gal in LacNAc, containing mucin carbohydrate chains. Subsequently the same group (Lo-Guidice et al., 1997
) reported the occurrence of the carbohydrate chain 3-O-SulfoGalß1,3(Fuc
1,4)GlcNAcß1,3Galß1,3GalNAc
- in respiratory mucins of a nonsecretor (O, Lea+b) patient suffering from chronic bronchitis. Consistent with their findings, the present study has found that 3-O-SulfoGalß1,3GlcNAcß- is a high affinity acceptor substrate for human lung tumor (mucinous adenocarcinoma)
1,3/4-L-fucosyltransferase.
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Materials and methods |
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Glycoproteins and glycopeptides
Fetuin, asialo fetuin, bovine 1 acid glycoprotein, bovine IgG, and apotransferrin were purchased from Sigma. Fetuin triantennary glycopeptide, fetuin triantennary asialoglycopeptide, bovine IgG diantennary glycopeptide, and carcinoembryonic antigen were available from our earlier studies (Chandrasekaran et al., 1983
, 1995b). Asialo apotransferrin was made by heating apotransferrin at 80°C in 0.1 N HCl for 1 h, neutralizing with 0.1 N NaOH, dialyzing against water in the cold room for 24 h, and then lyophilizing. Highly purified ancrod (a thrombin-like serine protease) (Nolan et al., 1976
; Pfeiffer et al., 1992
) was a generous gift from Dr. Chris Nolan of Abbott Labs (Chicago, IL). Asialo ancrod was prepared from ancrod as described above.
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., 1995a
, 1997). The chemical synthesis of NeuAc
2,3Galß1,4GlcNAcß1,6(Galß1,3) GalNAc
-O-Me will be reported elsewhere.
Assay of 1,4-fucosyltransferase
The incubation mixtures run in duplicate contained 50 mM HEPESNaOH, 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 this 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 before. 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 sample values did not vary more than 5%.
Gel chromatography
Two milliliters of the Affinity GelGDP purified 1,3/4-fucosyltransferase preparation from human lung tumor 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 6.3, containing 0.1% Triton X-100 and 0.02% NaN3. Fractions of 2 ml at a flow rate of 6 ml per h were collected, and 10 µl of alternate fraction were assayed for
1,4-fucosyltransferase activity using 2-O-MeGalß1,3GlcNAcß-O-Bn as the acceptor.
PAGE
Ready-made polyacrylamide gels (10% resolving gel and 4% stacking gel containing no SDS) were used for both native as well as SDSPAGE. Bio-Rad Mini-Protean II Electrophoresis Cell was used. Native PAGE was performed in the cold room with Trisglycine pH 8.3 containing 0.1% Triton X-100. The enzyme sample 100 µl was mixed with 100 µl of 2x buffer containing glycerol, Triton X-100, and bromophenol blue applied in equal volume (33 µl) to each of the middle six wells and run at 30 mA/gel using Bio-Rad Power Pac 1000 at 200 constant volts. After electrophoresis each gel was removed and cut into 3-mm slices (22 slices total). Each gel slice was shaken in the cold room in 200 µl of 0.1 M HEPESNaOH, pH 7.0, containing 2% Triton X-100 and 0.1% NaN3 for 24 h using speci mix (Thermolyne). The gel eluates were assayed for 1,4-fucosyltransferase activity by using the acceptor 2-O-MeGalß1,3GlcNAcß-O-Bn. SDSPAGE was performed at room temperature in Tris glycine, pH 8.3, containing 0.1% SDS. The sample was mixed with equal volume of 2x sample loading buffer containing SDS, mercaptomethanol, glycerol, and bromophenol blue and denatured in boiling water bath for 5 min before the electrophoretic run. The gels were stained using a Bio-Rad silver staining kit.
Identification of the products arising from Galß1,3GlcNAcß1,3Galß-O-Me and Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me by the action of FTB
A 10-fold reaction mixture (200 µl) containing Galß1,3GlcNAcß1,3Galß-O-Me and another containing Galß1,3(Fuc1,4)GlcNAcß1,3Galß-O-Me were incubated for 4 h at 37°C. After incubation the reaction mixtures were diluted with 1 ml water 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 thin-layer chromatography on silica gel GHLF (Analtech, 250 microns, scored 20 cm x 20 cm) plates developed in two different solvent systems; 1-butanol/acetic acid/water (3:2:1) and 1-propanol/NH4OH [25%]/water (12:2:5), along with Galß1,3GlcNAcß1,3Galß-O-Me and Galß1,3(Fuc
1,4)Glc-NAcß1,3Galß-O-Me markers. The radioactive content of 0.5 cm width segments scraped into scintillation vials and soaked in 2 ml water was determined by liqiud scintillation spectroscopy. Autoradiography was carried out at 70°C using Biomax MR film (Kodak) after spraying plates with EnHance (DuPont).
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Acknowledgment |
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Abbreviations |
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Footnotes |
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References |
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