Human lung adenocarcinoma {alpha}1,3/4-L-fucosyltransferase displays two molecular forms, high substrate affinity for clustered sialyl LacNAc type 1 units as well as mucin core 2 sialyl LacNAc type 2 unit and novel {alpha}1,2-L-fucosylating activity

E.V. Chandrasekaran, Ram Chawda, John M. Rhodes, Jie Xia, Conrad Piskorz and Khushi L. Matta1

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Human lung tumor {alpha}1,3/4-L-fucosyltransferase (FT) was purified (2000-fold, 29% recovery) from 290 g of tissue by including a chromatography step on Affinity Gel-GDP. Two molecular forms (FTA, larger size carrying 15% {alpha}1,4-FT activity; FTB, the major form with 85% activity) were separated by further fractionation on a Sephacryl S-100 HR column. A difference in the electrophoretic mobilities of these two activities was also found on native polyacrylamide gel electrophoresis (PAGE). Both forms were devoid of typical {alpha}1,2-fucosylating activity but were associated with the novel {alpha}1,2-fucosylating ability of converting the Lewis a determinant to Lewis b. Based on percentage activity toward 2-O-MeGalß1,3GlcNAcß-O-Bn, both forms exhibited the same extent of activity toward various acceptors, which included sulfated, sialylated, or methylated LacNAc type 1 or type 2 as well as mucin core 2 acceptors. However, FTA and FTB exhibited a difference in their ability to act on mucin core 2 3'-sialyl LacNAc (activities 24.2% and 40.8%, respectively, as compared to 2-O-MeGalß1,3GlcNAcß-O-Bn). The unsubstituted LacNAc type 1 acceptors were 15–20 times as active as the corresponding LacNAc type 2 acceptors. The 3-O-substitution on the ß1,4-linked Gal (methyl, sulfate, or sialyl) in mucin core 2 acceptors increased the efficiency of these acceptors five- to eightfold. The most efficient acceptor for FTA and FTB was 3-O-sulfoGalß1,3GlcNAcß-O-Al (Km 100 and 47 µM, respectively). The Km (mM) values for 2-O-methyl Galß1,3GlcNAcß-O-Bn and 3-O-sialyl Galß1,3GlcNAcß-O-Bn were 0.40 and 2.5 (FTA) and 0.16 and 0.67 (FTB), respectively.

The 35-kDa glycoprotein ancrod (from Malayan pit viper venom) containing 36% complex N-glycans with the antennae NeuAc{alpha}2,3Galß1,3GlcNAcß- 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 {alpha}1-acid glycoprotein, were better acceptors than asialo fetuin. On the contrary, fetuin triantennary glycopeptide containing predominantly NeuAc{alpha}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 {alpha}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


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Numerous studies have demonstrated that many human tumors express fucosylated glycoconjugates that are absent in corresponding normal tissues, suggesting the presence of tumor associated {alpha}1,3-L-fucosyltransferases in human cancers (Alhadeff, 1989Go; Hakomori, 1989Go). Immunohistochemical staining with a monoclonal antibody, FTA 1-16, against human {alpha}1,3/4 fucosyltransferase revealed an enhanced expression of this enzyme in cancer cells in comparison to normal cells (Kimura et al., 1995Go). An inverse correlation was shown to exist between the survival of patients with primary lung cancer and the positive immunohistochemical staining of lung tumor by the monoclonal antibody M1A 15-5, which defines H/Ley/Lea antigens (Miyake et al., 1992Go). An indicator of poor prognosis in lung cancer was shown to be the expression of {alpha}1,3-L-fucosyltransferases Fuc T IV and Fuc T VII, which participate in the biosynthesis of sialyl Lewis x (Ogawa et al., 1996Go). The frequency of sialyl Lewis a expression was identified as 69% in lung cancer (Ogawa et al., 1994Go), whereas only 9% of lung tumors expressed Fuc T III. Such a discrepancy may be caused by the existence of a yet unknown {alpha}1,4-L-fucosyltransferase in human lung cancer (Ogawa et al., 1996Go). It was reported that the {alpha}1,4-L-fucosyltransferase activity in human serum and saliva did not correlate with Lewis antigen status in erythrocytes (DeBose-Boyd et al., 1996Go). The reason for this may be the fact that mutation in the catalytic domain of the Lewis gene would cause loss of enzyme activity, whereas that in the transmembrane domain may not affect the activity but affect the targeting of the enzyme to the Golgi apparatus (Nishihara et al., 1993Go; Machamer, 1993Go).

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, 1973Go; Oriol et al., 1986Go). The coexistence of {alpha}1,3- and {alpha}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 x–related structures. The {alpha}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., 1993Go). 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, 1999Go). The present article reports the partial purification, acceptor specificities, and kinetic properties of two molecular forms of {alpha}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 {alpha}1,2-L-fucosylating activity.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Purification of the {alpha}1,4-L-fucosyltransferase
The enzyme has been purified from 290 g of human lung tumor tissue, about 2000-fold as compared to the activity in Triton X-100 solubilized extract, by using (NH4)2SO4 fractionation followed by chromatography on an Affinity Gel-GDP (Table I). When this purified enzyme preparation was subjected to SDS–polyacrylamide gel electrophoresis (PAGE), a single sharp band at ~60 kDa was revealed on silver staining.


View this table:
[in this window]
[in a new window]
 
Table I. Purification of {alpha}1,4-L-fucosyltransferase from human lung tumor tissue (non–small cell poorly differentiated mucinous lung adenocarcinoma)
 
Isolation of two molecular forms of {alpha}1,4-L-fucosyltransferase from human lung tumor
When the Affinity Gel–GDP purified enzyme preparation was subjected to chromatography on a Sepharose S100 HR column, a larger molecular form containing ~15% activity slightly less than 100,000 Da (it emerges quite adjacent to Vo), in addition to the major peak of activity at ~65,000 Da (as evident from the MW marker) could be seen (Figure 1). The patterns of inhibition by N-ethyl maleimide on these two molecular forms of activity were observed to be the same, and Ki was 0.25 mM in both cases (data not shown). Both enzyme forms, FTA (minor) and FTB (major), were examined for their acceptor specificities using a battery of synthetic compounds (see Table II). Both forms lacked the typical {alpha}1,2-L-fucosyltransferase activity because Galß-O-Bn did not act as their acceptor. Both FTA and FTB exhibited almost the same extent of activity (when expressed as a percent of the activity toward 2-O-MeGalß1,3GlcNAcß-O-Bn) toward the same acceptor. The only significant difference between the two forms was their activity toward 3'-sialyl LacNAc in mucin core 2, namely, NeuAc{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3)GalNAc{alpha}-O-Me (activities: 24.2% versus 40.8%).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Chromatography of the Affinity Gel–GDP purified {alpha}1,4/3-L-fucosyltransferase on Sephacryl S-100 HR column.

 

View this table:
[in this window]
[in a new window]
 
Table II. The activity of the two molecular forms of human lung tumor {alpha}1,4-L-fucosyltransferase toward synthetic acceptors
 
A comparison of the activities among the various acceptors revealed the following (see Table II): The 3-O-unsubstituted LacNAc type 1 acceptor as compared to the corresponding LacNAc type 2 acceptors are 15–25 times more active with these enzymes. NeuAc{alpha} 2,3Galß1,3 GlcNAcß-O-Bn is ~ ninefold as active as NeuAc{alpha}2,3Galß1,4GlcNAcß-O-Bn, whereas the latter as a part of mucin core 2 structure is nearly as active as the former (FTB: 64.4% and 40.8%, respectively). In fact, among the mucin core 2 acceptors tested, 3'-sialyl LacNAc in mucin core 2 structure was the best acceptor for these two molecular forms (see Table II). Both forms showed considerable activity toward allyl glycosides containing substitution on C-4 OH or both C-4 and C-6 OH groups of the GlcNAc moiety, indicating the presence of novel {alpha}1,2-L- fucosylating activity. This is in accordance with our earlier report that this novel activity is associated with Lewis type {alpha}1,3/4-L-fucosyltransferase (Chandrasekaran et al., 1995bGo). It is interesting to note that the Lewis a determinant attached to a Gal moiety, namely, Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß-O-Me, became a good acceptor for this novel {alpha}1,2-L-fucosylating activity in contrast to the nonreactivity of Galß1,3(Fuc{alpha}1,4)GlcNAcß-O-Me (see Table II). The Fuc residue being added to the nonreducing terminal Gal moiety in Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß-O-Me is supported by the following findings: (a) Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al is a better acceptor than Galß1,3(Fuc-{alpha}1,4)GlcNAcß1,3 Galß-O-Me (activities: FTA-> 59.7% and 46.5% ; FTB-> 78.4% and 56.5%, respectively); (b) an analogue of Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß-O-Me, namely, Galß1,4(Fuc{alpha}1,3)GlcNAcß1,3Galß-O-Me is inactive as an acceptor for FTA and FTB (data not shown); (c) 3-O-substitution on terminal Gal abolishes {alpha}1,2-L-fucosylation (Chandrasekaran et al., unpublished results); and (d) as to our knowledge the occurrence of internal {alpha}1,2-L-fucosyl Gal in carbohydrate chains has not been reported in the literature.

Characterization of the product arising from Galß1,3(Fuc{alpha}1,4)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(Fuc{alpha}1,4)GlcNAcß1,3Galß-O-Me in two different solvent systems (Figure 2, panels I and II). The [14C] fucosylated product arising from Galß1,3(Fuc{alpha}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 {alpha}1,2-fucosidase (Prozyme), almost complete release of [14C] Fuc from the [14C] fucosylated product of Galß1,3(Fuc{alpha}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{alpha}1,4)GlcNAcß1,3Galß-O-Me (Figure 3A) and the Km values were FTA, 2.5 mM and FTB, 1.0 mM (Figure 3B).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 2. Thin-layer chromatography. (A) Galß1,3GlcNAcß1,3Galß-O-Me; (B) Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß-O-Me; (C) [14C]fucosylated product arising from A by the action of FTB; (D) [14C]fucosylated product arising from B by the action of FTB; (E) Treatment of D with {alpha}1,2-L-fucosidase (Prozyme, CA). Solvent systems: panel I, 1-butanol/acetic acid/water (3:2:1) developed twice; panel II, 1-propanol/NH4OH/water (12:2:5) developed twice; panel III, 1-propanol/NH4OH/water (12:2:5) developed once—autoradiography; panel IV, 1-butanol/acetic acid/water (3:2:1) developed once.

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Activity of Sephacryl S-100 fractions FTA and FTB toward Galß1,3(Fuc{alpha}1,4) GlcNAcß1,3Galß-O-Me. (A) Solid circles, FTA activity; open circles, FTB activity. (B) Determination of Km by Lineweaver-Burke plot. Solid circles, FTA activity; open circles, FTB activity.

 
Activity of human lung tumor {alpha}1,4-L-fucosyltransferase toward glycoproteins and glycopeptides
FTB was used as the enzyme source for this study. The enzyme was found to be more active with sialylated glycoproteins than asialo glycoproteins (activities: fetuin 2.25, asialo fetuin 0.59; ancrod 40.33, and asialo ancrod 22.21; see Table III). The best glycoprotein acceptor was ancrod, which contained several units of 3-sialylGalß1,3GlcNAcß- in the asparagine-linked carbohydrate chains. It is interesting to note that fetuin triantennary glycopeptide containing mainly 3-sialylGalß1,4GlcNAcß- units in asparagine linked chain was considerably less active than the corresponding asialo glycopeptide.


View this table:
[in this window]
[in a new window]
 
Table III. Human lung tumor molecular form B {alpha}-1,4-L-fucosyltransferase activity toward glycoproteins and glycopeptides
 
A comparison of the kinetics of FTA and FTB toward various synthetic acceptors
The major form (FTB) had maximum activity at 0.1 mM concentration of 3-O-SulfoGalß1,3GlcNAcß-O-Al, whereas the minor form (FTA) showed an increase in activity even beyond this concentration (Km FTA, 100 µM and FTB, 47 µM; Figure 4). On the other hand, the pattern of their activities toward 2-O-MeGalß1,3GlcNAcß-O-Bn appears to be the same (Km FTA, 400 µM and FTB, 160 µM; see Figure 4A).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4. Activity of Sephacryl S-100 HR fractions FTA and FTB toward LacNAc type 1 acceptors. (A) Open symbols, FTA activity; solid symbols, FTB activity; circles, acceptor: 3-O-SulfoGalß1,3GlcNAcß-O-Al; triangles, acceptor: 2-O-MeGalß1,3GlcNAcß-O-Bn. (B) Determination of Km by Lineweaver-Burke plot (symbols same as above).

 
The pattern of activities toward the sialylated acceptors, namely, NeuAc{alpha}2,3Galß1,3GlcNAcß-O-Bn and NeuAc-{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3)GalNAc{alpha}-O-Me, were quite similar between FTA and FTB (Figure 5A). The Km values were FTA, 2.50 mM and FTB, 0.67 mM for the former acceptor and FTA, 3.33 mM and FTB, 0.77 mM for the latter (see Figure 5B).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Activity of Sephacryl S-100 HR fractions FTA and FTB toward 3'-sialyl LacNAc type 1 acceptor and 3'-sialyl LacNAc type 2 as mucin core 2 structure. (A) Activity at varying concentration of acceptors; solid symbols, FTA activity; open symbols, FTB activity; circles, acceptor: NeuAc{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3)GalNAc{alpha}-O-Me; triangles, acceptor: NeuAc{alpha}2,3Galß1,3GlcNAcß-O-Bn. (B) Determination of Km by Lineweaver-Burke plot (symbols same as above).

 
Further examination of the novel {alpha}1,2-L-fucosylating activity as well as the {alpha}1,3-L-fucosylating activity toward 2'-fucosyl lactose, using FTB
Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al served as the best acceptor for the novel {alpha}1,2-L-fucosylating activity followed by Galß1,3(4-O-Me)GlcNAcß-O-Al. Even the well-established activity of Lewis type, that is, the {alpha}1,3-L-fucosylating activity on 2'-fucosyl lactose was considerably lower than the transfer of Fuc to Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al (Figure 6A).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6. Association of {alpha}1,3-L-fucosylating activity toward 2'-fucosyllactose, as well as the novel {alpha}1,2-L-fucosylating activity, with lung tumor {alpha}1,4/3-L-fucosyltransferase. (A) Open circles, Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al; solid circles, Galß1,3(4-O-Me)GlcNAcß-O-Al; solid triangles, Fuc{alpha}1,2Galß1,4Glc. (B) Open circles, Galß1,3(4-O-Me)GlcNAcß-O-Bn; solid circles, Galß1,3(Fuc{alpha}1,4)GlcNAcß-O-Al; solid triangles, Galß1,3(4,6-di-O-Me)GlcNAcß-O-Bn. (C) Determination of Km by Lineweaver-Burke plot (symbols same as in part A). (D) Determination of Km by Lineweaver-Burke plot (symbols same as part B).

 
Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al, Galß1,3(4-O-Me)Glc-NAcß-O-Al, Galß1,3(4,6-di-O-Me)GlcNAcß-O-Bn, Galß1,3(4-O-Me)GlcNAcß-O-Bn, Galß1,3(Fuc{alpha}1,4)GlcNAcß-O-Al, and Fuc{alpha}1,2Galß1,4Glc gave, respectively, the following Km values: 0.60 mM, 2.63 mM, 6.00 mM, 10.00 mM, 7.10 mM, and 1.52 mM (see Figure 6B).

Association of novel {alpha}1,2-L-fucosylating activity with human lung adenocarcinoma {alpha}1,4-L-fucosyltransferase
FTB was used as the enzyme source. When {alpha}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 {alpha}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 {alpha}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).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. Demonstration of the association of novel {alpha}1,2-L-fucosylating activity with human lung adenocarcinoma {alpha}1,4-L-fucosytransferase. (A) Inhibition of {alpha}1,4-L-fucosylating activity (acceptor: 3-O-sulfoGalß1,3GlcNAcß-O-Al) by Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al. Transfer of [14C]fucose into 3-O-sulfoGalß1,3GlcNAcß-O-Al in absence (open circles) and in presence of Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al (solid circles); transfer of [14C]fucose into Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al (open triangles). (B) Inhibition of novel {alpha}1,2-L-fucosylating activity (acceptor: Galß1,3(4,6-di-O-Me) GlcNAcß-O-Al) by 3-O-sulfoGalß1,3GlcNAcß-O-Al, transfer of [14C]fucose into Galß1,3(4,6-di-O-Me)GlcNAcß-O-Al in absence (open circles) and presence (solid circles) of 3-O-sulfoGalß1,3GlcNAcß-O-Al; transfer of [14C]fucose into 3-O-sulfoGalß1,3GlcNAcß-O-Al (open triangles).

 
Macromolecular substrate activity analysis of human lung tumor {alpha}1,4-L-fucosyltransferase
Ancrod showed saturating activity at 80 µg (0.114 mM), whereas asialo ancrod required >100 µg (0.133 mM) to reach maximum activity. Fetuin triantennary asialo glycopeptide showed maximum activity at 80 µg (1.0 mM), and the corresponding sialoglycopeptide needed 200 µg for maximum activity. The Km values (Figure 8B) were as follows: ancrod 45 µM; asialo ancrod 167 µM; fetuin triantennary glycopeptide 1.43 mM; and the corresponding asialo glycopeptide 0.63 mM.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8. Activity of Sephacryl S-100 HR fraction FTB toward ancrod, fetuin triantennary glycopeptide and their asialo derivatives. (A) Activity at varying concentration of acceptors: solid circles, ancrod; open circles, asialo ancrod; solid triangles, fetuin triantennary glycopeptide; open triangles, fetuin triantennary asialoglycopeptide. Determination of Km by Lineweaver-Burke plot: (B) ancrod, (C) asialo ancrod, (D) fetuin triantennary glycopeptide, (E) fetuin triantennary asialoglycopeptide

 
Further evidence for the existence of two molecular forms of {alpha}1,4-L-fucosyltransferase in human lung tumor
When FTA and FTB were subjected to native PAGE, we were able to locate the enzyme activity on the gel after slicing and eluting the gel slices. During this operation, the loss of enzyme activity was low with FTB as compared to FTA. From Figure 9 it is evident that the mobility of FTA activity on the gel was higher than that of FTB, suggesting an association of higher anionic charge with FTA.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 9. Native PAGE of Sephacryl S-100 HR fractions FTA and FTB. (A) FTB activity. (B) FTA activity.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
A considerable degree of {alpha}1,3/4-L-fucosyltransferase purification from a limited source, such as human lung tumor, has been achieved. The occurrence of two molecular forms of this enzyme has been demonstrated by molecular sieve chromatography on Sephacryl S-100 HR column and also by showing the difference in their mobilities by following their activity profiles after native PAGE. Further we have given an indication that native PAGE under the experimental conditions used could be a method for further purification of a small amount of {alpha}1,3/4-L-fucosyltransferase, or probably any other glycosyltransferase obtained, after a considerable extent of purification from a limited source.

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 {alpha}1,3/4-fucosyltransferases purified from human milk (Johnson et al., 1992Go) and the culture media of human A431 epidermoid carcinoma cell line (Johnson et al., 1993Go), 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 {alpha}1,3/4-FTs showed considerably reduced activity (46%) when sialic acid is linked to {alpha}2,3 to the terminal ß-galactosyl residue of type 1 structure, whereas FTA and FTA exhibited almost the same extent of activity with {alpha}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 {alpha}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 {alpha}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{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3)GalNAc{alpha}-O-Me, is four to five times active as NeuAc{alpha}2,3Galß1,4GlcNAcß-O-Bn, and the acceptor efficiency is 43% and 63% that of NeuAc{alpha}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., 1995bGo) we have shown an association of a novel {alpha}1,2-L-fucosylating activity, which leads to the expression of blood group Lewis b determinant from Lewis a,with the Lewis type {alpha}1,3/4-L-fucosyltransferase. In the present study we have shown that FTA and FTB can fucosylate the acceptors Galß1,3(Fuc{alpha}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{alpha}1,4)GlcNAcß1,3Galß-O-Me. The efficiencies of accepting Fuc by Galß1,3(Fuc-{alpha}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)Go found Galß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß1,4Glc as equally good as Galß1,3GlcNAc as an acceptor for A431 carcinoma cell line {alpha}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, NeuAc{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3)GlcNAc{alpha}-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)Go used carbohydrate acceptors containing no aglycans in their study on cell line A431 {alpha}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 {alpha}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., 1976Go), is a glycoprotein (36% carbohydrate by weight) of 35 kDa containing complex type N-glycans (di-, tri-, and tetraantennary carrying NeuAc{alpha}2,3Galß1,3GlcNAcß- in a molar ratio [mol/100 mol] 23:40:12) (Pfeiffer et al., 1992Go). Thus, clusters of oligosaccharides containing NeuAc-{alpha}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-{alpha}2,3Galß1,4GlcNAcß- in two antennae and a combination of NeuAc{alpha}2,3Galß1,4GlcNAcß- and NeuAc{alpha}2,3Galß1,3GlcNAcß- on the third (Townsend et al., 1986Go), was less efficient than the corresponding asialo acceptor. This would suggest that NeuAc{alpha}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 {alpha}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., 1999Go) 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 b–encoded {alpha}1,3-L-fucosyltransferase from type 2 activity to type 1 (Depuy et al., 1999Go). 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 103–153) (Legault et al., 1995Go). 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., 1998Go). 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., 1997Go). It is interesting to note that the {alpha}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)Go 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., 1997Go) reported the occurrence of the carbohydrate chain 3-O-SulfoGalß1,3(Fuc{alpha}1,4)GlcNAcß1,3Galß1,3GalNAc{alpha}- 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) {alpha}1,3/4-L-fucosyltransferase.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Purification of {alpha}1,4-L-fucosyltransferase from human lung tumor (non–small cell poorly differentiated mucinous adenocarcinoma in upper lobe of right lung)
The tumor was obtained during autopsy of a 58-year-old Caucasian female patient at Roswell Park Cancer Institute and stored at –70°C until processing. The tumor (291.5 g) was cut into small pieces in the cold room and homogenized with 1460 ml of ice-cold buffer (0.1 M Tris maleate, pH 6.3, containing 2% Triton X-100, 5 mM Mn acetate, 35 µm PMSF, and 0.1% NaN3) using Kinematica on an ice bath. The homogenate was centrifuged at 10,000 x g for 1 h at 5°C. The fat-free supernatant was adjusted to 20% saturation of (NH4)2SO4, stirred in the cold room for 0.5 h, and centrifuged as above. The supernatant was adjusted to 20–60% saturation of (NH4)2SO4 and centrifuged as above. The precipitate was dissolved in 450 ml of the above buffer and dialyzed against the same buffer for 24 h in the cold room with four changes. The dialyzed 20–60% (NH4)2SO4 fraction (510 ml) was subjected to affinity chromatography on an Affinity Gel-GDP (Calbiochem; 10 µm GDP/ml gel slurry) column (2.5 x 4.0 cm) (20 ml bed volume) in batches of 100 ml. The column had been equilibrated with the same buffer. After the entry of the sample (100 ml), the column was washed with 100 ml of the same buffer. The affinity column was then eluted with 60 ml of the same buffer containing 0.5 mM GDP. Then the column was washed with 100 ml of the same buffer containing 2 M NaCl and then equilibrated with the buffer for the fractionation of the second batch of 100 ml as above. The GDP eluates from the five batches of operation were pooled, concentrated by Amicon ultra filtration using PM 10 membrane, then dialyzed exhaustively against 50 mM Tris maleate, pH 6.3, containing 2% Triton X-100 and 0.1% NaN3. The {alpha}1,4-fucosyltransferase activity of this preparation (4 ml) was stable for at least 6 months when stored at –20°C.

Glycoproteins and glycopeptides
Fetuin, asialo fetuin, bovine {alpha}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., 1983Go, 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., 1976Go; Pfeiffer et al., 1992Go) 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., 1993Go, 1994, 1998; Chandrasekaran et al., 1995aGo, 1997). The chemical synthesis of NeuAc{alpha}2,3Galß1,4GlcNAcß1,6(Galß1,3) GalNAc{alpha}-O-Me will be reported elsewhere.

Assay of {alpha}1,4-fucosyltransferase
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 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., 1992Go). 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 Gel–GDP purified {alpha}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 {alpha}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 SDS–PAGE. Bio-Rad Mini-Protean II Electrophoresis Cell was used. Native PAGE was performed in the cold room with Tris–glycine 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 HEPES–NaOH, 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 {alpha}1,4-fucosyltransferase activity by using the acceptor 2-O-MeGalß1,3GlcNAcß-O-Bn. SDS–PAGE 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(Fuc{alpha}1,4)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(Fuc{alpha}1,4)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{alpha}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).


    Acknowledgment
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
This work was supported by Grant #CA35329 awarded by the National Cancer Institute.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Al, allyl; Bn, benzyl; FT, fucosyltransferase; LacNAc type 1, Galß1,3GlcNAcß-; LacNAc type 2, Galß1,4GlcNAcß-; Me, methyl; Naph, naphthyl; PAGE, polyacrylamide gel electrophoresis.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgment
 Abbreviations
 References
 
Alhadeff, J.A. (1989) Malignant cell glycoproteins and glycolipids. CRC Crit. Rev. Oncol./Hematol., 9, 37–107.

Chandrasekaran, E.V., Davila, M., Nixon, D., Goldfarb, M., and Mendicino, J. (1983) Isolation and structures of the oligosaccharide units of carcinoembryonic antigen. J. Biol. Chem., 258, 7213–7222.[Free Full Text]

Chandrasekaran, E.V., Jain, R.K., and Matta, K.L. (1992) Ovarian cancer {alpha}1, 3-L-fucosyltransferase: differentiation of distinct catalytic species with the unique substrate, 3'-Sulfo-N-Acetyllactosamine in conjunction with other synthetic acceptors. J. Biol. Chem., 267, 23806–23814.[Abstract/Free Full Text]

Chandrasekaran, E.V., Jain, R.K., Larsen, R.D., Wlasichuk, K., and Matta, K.L. (1995a) Selectin-ligands and tumor associated carbohydrate structures: specificities of {alpha}2, 3-sialyltransferases in the assembly of 3'-sialyl, 6-sulfo/sialyl Lewis a and x, 3'-sialyl, 6'-sulfo Lewis x and 3'-sialyl, 6-sialyl/sulfo blood group T-hapten. Biochemistry, 34, 2925–2936.[ISI][Medline]

Chandrasekaran, E.V., Jain, R.K., Rhodes, J.M., Srnka, C.A., and Matta, K.L. (1995b) Expression of blood group Lewis b determinant from Lewis a: association of this novel {alpha}(1, 2)-L-fucosylating activity with the Lewis type {alpha}(1, 3/4)-L-fucosyltransferases. Biochemistry, 34, 4748–4756.[ISI][Medline]

Chandrasekaran, E.V., Jain, R.K., Vig, R., and Matta, K.L. (1997) The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal. Glycobiology, 7, 753–768.[Abstract]

DeBose-Boyd, R.A., Nayame, A.K., Smith, D.F., and Cummings, R.D. (1996) {alpha}1, 4-fucosyltransferase activity in human serum and saliva. Arch. Biochem. Biophys., 335, 109–117.[Medline]

Depuy, F., Petit, J-M., Millicone, R., Oriol, R., Julien, R., and Maftah, A. (1999) A single amino acid in the hypervariable stem domain of vertebrate {alpha}1, 3/1, 4-fucosyltransferases determines the type 1/type 2 transfer: characterization of acceptor substrate specificity of the Lewis enzyme by site-directed mutagenesis. J. Biol. Chem., 274, 12257–12262.[Abstract/Free Full Text]

Hakomori, S. (1989) Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res., 52, 259–331.

Jain, R.K., Piskorz, C.F., and Matta, K.L. (1993) A convenient synthesis of N-acetyllactosamine-linked oligosaccharides from phenyl 3, 6, 2(, 4(, 6(-hex{alpha}-O-acetyl-2-deoxy-2-phthalimido-1-thio-ß-lactopyranoside. Carbohydr. Res., 243, 385–391.[ISI][Medline]

Jain, R.K., Vig, R., Rampal, R., Chandrasekaran, E.V., and Matta, K.L. (1994) Total synthesis of 3(-O-sialyl-6(-O-sulfo Lewisx, NeuAc{alpha}2, 3(6-O-Sulfo)Galß1, 4 (Fuc{alpha}1, 3)GlcNAcß-OMe: a major capping group of GlyCAM-1. J. Am. Chem. Soc., 116, 11123–11124.

Jain, R.K., Piskorz, C.F., Chandrasekaran, E.V., and Matta, K.L. (1998) Synthesis of Galß1, 4GlcNAcß1, 6(Galß1, 3)GalNAc{alpha}-O-Bn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal:3-O-sulfotransferases. Glycoconj. J., 15, 951–959.[ISI][Medline]

Johnson, P.H., Donald, A.S.R., Feeney, J., and Watkins, W.M. (1992) Reassessment of the acceptor specificity and general properties of the Lewis blood group gene associated {alpha}1, 3/4-fucosyltransferase purified from human milk. Glycoconj. J., 9, 251–264.[ISI][Medline]

Johnson, P.H., Donald, A.S.R., and Watkins, W.M. (1993) Purification and properties of the {alpha}-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line. Glycoconj. J., 10, 152–164.[ISI][Medline]

Kimura, H., Kudo, T., Nishihara, S., Iwasaki, H., Shinya, N., Watanabe, R., Hond, H., Takemura, F., and Narimatsu, H. (1995) Murine monoclonal antibody recognizing human {alpha}(1, 3/1, 4)-fucosyltransferase. Glycoconj. J., 12, 802–812.[ISI][Medline]

Legault, D.J., Kelly, R.J., Natsuka, T., and Lowe, J.B. (1995) Human {alpha}1, 3/4 fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J. Biol. Chem., 270, 20987–20996.[Abstract/Free Full Text]

Lo-Guidice, J.-M., Perini, J.-M., Lafitte, J.-J., Ducourouble, M.-P., Royssel, P., and Lamblin, G. (1995) Characterization of a sulfotransferase from human airways responsible for the 3-O-sulfation of terminal galactose in N-acetyllactosamine-containing carbohydrate chains. J. Biol. Chem., 270, 27544–27550.[Abstract/Free Full Text]

Lo-Guidice, J.-M., Herz, H., Lamblin, G., Planke, Y., Roussel, P., and Lhermitte, M. (1997) Structures of sulfated oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b–) patient suffering from chronic bronchitis. Glycoconj. J., 14, 113–125.[ISI][Medline]

Machamer, C.E. (1993) Targeting and retention of Golgi membrane proteins. Curr. Opin. Cell Biol., 5, 606–612.[Medline]

Miyake, M., Taki, T., Hitomi, S., and Hakomori, S. (1992) Correlation of expression of H/Ley/Leb antigens with survival in patients with carcinoma of the lung. N. Eng. J. Med., 327, 14–18.[Abstract]

Nguyen, A.T., Holmes, E.H., Whitaker, J.M., Ho, S., Shetterly, S., and Macher, B.A. (1998) Human {alpha}1, 3/4-Fucosyltransferases: Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis. J. Biol. Chem., 273, 25244–25249.[Abstract/Free Full Text]

Nishihara, S., Yazawa, S., Iwasaki, H., Nakazato, M., Kudo, T., Ando, T., and Narimatsu, H. (1993) {alpha}(1, 3/1, 4)-fucosyltransferase (Fuc-T III) gene is inactivated by a single amino acid substitution in Lewis histo-blood type negative individuals. Biochem. Biophys. Res. Commun., 196, 624–631.[ISI][Medline]

Nolan, C., Hall, L.S., and Barlow, G.H. (1976) Ancrod, the coagulating enzyme from Malayan pit viper (Agkistrondon rhodostoma) venom. Meth. Enzymol., 45, 205–213.[Medline]

Ogawa, J., Sano, A., Koide, S., and Shohtsu, A. (1994) Relation between recurrence and expression of proliferating cell nuclear antigen, sialyl Lewisx and sialyl Lewisa in lung cancer. J. Thorac. Cardiovasc. Surg., 108, 329–336.[Abstract/Free Full Text]

Ogawa, J., Inoue, H., and Koide, S. (1996) Expression of {alpha}1, 3-fucosyltransferase type IV and VII genes is related to poor prognosis in lung cancer. Cancer Res., 56, 325–329.[Abstract]

Oriol, R., La Pendu, J., and Mollicone, R. (1986) Genetics of ABO, H, Lewis x and related antigens. Vox Lang., 51, 161–171.

Oulmouden, A., Wierinckx, A., Petit, J-M., Costache, M., Palcic, M.M., Mollicone, R., Oriol, R., and Julien, R. (1997) Molecular cloning and expression of a bovine {alpha}1, 3 fucosyltransferase gene homologous to a putative ancestor gene of the human FUT3-FUT5-FUT6 cluster. J. Biol. Chem., 272, 8764–8773.[Abstract/Free Full Text]

Pfeiffer, G., Dabrowski, V., Dabrowski, J., Stirm, S., Strube, K-H., and Geyer, R. (1992) Carbohydrate structure of a thrombin-like serine propase from Agkistrodon rhodostoma: Structural elucidation of oligonucleotides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance. Eur. J. Biochem., 205, 961–978.[Abstract]

Sherwood, A.L., and Holmes, E.H. (1999) Analysis of the expression and enzymatic properties of {alpha}1, 3-fucosyltransferase from human lung carcinoma NCI-H69 and PC9 cells. Glycobiology, 9, 637–643.[Abstract/Free Full Text]

Szulman, A.E., and Marcus, D.M. (1973) The histologic distribution of the blood group substances in man as disclosed by immunofluorescence VI. The Lea and Leb antigens during fetal development. Lab. Invest., 28, 565–574.[ISI][Medline]

Townsend, R.R., Hardy, M.R., Wong, T.C., and Lee, Y.C. (1986) Binding of N-linked bovine Fetuin glycopeptides to isolated rabbit hepatocytes: Gal/GalNAc hepatic lectin discrimination between Galß1, 4GlcNAc and Galß1, 3GlcNAc in a triantennary structure. Biochemistry, 25, 5716–5725.[ISI][Medline]