The expression of Lewis histo-blood group antigens on erythrocytes (Koda et al., 1993; Nishihara et al., 1993b, 1994; Mollicone et al., 1994), including the CA19-9 tumor marker in digestive organs (Narimatsu et al., 1996), is determined by a Lewis-type [alpha](1,3/1,4)fucosyltransferase (Fuc-TIII; FUT3; Lewis enzyme), which is one of five homologous [alpha]1,3fucosyltransferases ([alpha]1,3Fuc-Ts; Goelz et al., 1990; Kukowska-Latallo et al., 1990; Lowe et al., 1991; Koszdin and Bowen, 1992; Weston et al., 1992a,b; Nishihara et al., 1993a; Natsuka et al., 1994; Sasaki et al., 1994).
We demonstrated in a previous study (Narimatsu et al., 1998) that the Lewis alleles encoding the Lewis enzyme (Fuc-TIII) in the Japanese population can be classified into four types, the wild-type allele, Le, and three mutant alleles, le1, le2, and le3. The frequency of occurrence of each Lewis allele in the Japanese population was determined by means of a random sampling study on more than 400 Japanese people. The results showed the following distribution: Le, 68.9%; le1, 24.8%; le2, 5.8%; and le3, 0.5%. The le1 and le2 alleles are null ones having two missense mutations, i.e., le1 possesses T59G (Leu20 to Arg) in the transmembrane domain and G508A (Gly170 to Ser) in the catalytic domain, and le2 possesses T59G and T1067A (Ile367 to Lys) in the catalytic domain. In vitro enzyme assays, using chimera enzymes, demonstrated that the single amino acid substitutions in the catalytic domains of the le1 and le2 enzymes, Gly170 to Ser and Ile367 to Lys, respectively, cause inactivation of the mutant enzymes (Nishihara et al., 1993b, 1994; Mollicone et al., 1994). But it remains unclear how such a single amino acid substitution in the catalytic domain results in an inactive enzyme. In addition, a study on the chimera enzyme indicated that the T59G mutation in the transmembrane domain does not cause enzyme inactivation (Nishihara et al., 1994). The le3 allele, which is very rare, 0.5%, in the Japanese population, has only the T59G (leu20 to Arg) mutation, and the same allele has been reported in Indonesian people (Mollicone et al., 1994). Mollicone et al. suggested, based on the weak le3 enzyme activity determined in vitro, that the Leu 20 to Arg substitution in the transmembrane domain might impair proper anchoring of the enzyme to the Golgi membrane. However, it remains uncertain whether this one amino acid substitution in the transmembrane domain actually affects the Golgi retention of the enzyme in vivo.
Point mutations inactivating the Le gene seem to be ethnic group specific, since the mutations frequently found inthe Scandinavian population (Ørntoft et al., 1996; Elmgrenet al., 1997), i.e. T202C and C314T, have not been found in the Le(a-b-) individuals in the Japanese population (Narimatsu et al., 1998).
In the present study, we extensively examined various aspects of the functions and characteristics of the mutated le1, le2, and le3 enzymes as well as the Le enzyme (the wild-type Lewis enzyme) in vitro and in vivo. We describe the molecular behavior of the mutant Lewis enzymes in relation to the Lewis antigen products. This is the first study demonstrating that glycosyltransferase gene dosage actually determines not only the amount of enzyme expression and their activity, but also the amount of carbohydrate products expressed in native tissues.
Table I.
Substrate | Le enzyme | le3 enzyme | Relative Vmax of le3 enzyme compared to Le enzyme | ||
Km | Vmax | Km | Vmax | ||
µM | nmol/mg/h | µM | nmol/mg/h | % | |
GDP-fucose | 161 | - | 160 | - | - |
2[prime]OMeLacto- | 1110 | 40 | 1200 | 52 | 132 ± 18 |
N-Biose I [beta]Bn |
In vitro activity of the enzyme directed by the le3 allele
In a previous study (Nishihara et al., 1994), we analyzed the level of the [alpha]1,3- and [alpha]1,4Fuc-T activity directed by the le1 and le2 alleles in COS-1 cell lysates obtained from cells in which each allele was transiently transfected. The results of this study indicated that the le1 and le2 enzymes were almost completely inactive in vitro.
Recently, we identified two colorectal cancer patients with the le1/le3 genotype. The le3 allele from the patient was subcloned into the pCDM8 vector, and transiently transfected into COS-1 cells. A lysate of the transfected cells was used for determination of the kinetics of the le3 enzyme activity. As shown in Table I, the le3 enzyme exhibited the same Km and Vmax values, for both the donor substrate and the acceptor substrate, as those of the Le enzyme, indicating that the T59G mutation in the transmembrane domain does not affect the net enzyme activity.
Degrees of protease resistance of Lewis mutant enzymes
In general, incompletely folded proteins, e.g., proteins in the denatured form, are much more sensitive to protease digestion than a native protein (Vestweber and Schatz, 1988). We analyzed the protease sensitivity of the Le enzyme, the le3 enzyme, and two chimera Lewis enzymes, Le-le1 and Le-le2, possessing a single mutation in the catalytic domain, Gly170 to Ser and Ile356 to Lys, respectively. A lysate prepared from COS-1 cells expressing each enzyme was treated with various concentrations of trypsin, and then fragments resistant to trypsin digestion were determined by Western blotting analysis using the anti-Lewis enzyme monoclonal antibody (mAb), FTA1-16 (Kimura et al., 1995; Figure
Figure 1. Degrees of protease sensitivity of the Le enzyme (a), le3 enzyme (b), and chimera enzymes, Le-le1 (c), and Le-le2 (d). A lysate of COS-1 cells transfected with each allele was subjected to trypsin digestion at the concentrations indicated at the bottom of each figure. The bands obtained after trypsin digestion were detected by Western blotting analysis using FTA1-16 as a probe.
Lewis a (Lea)- or Lewis b (Leb) -active glycolipids in plasma of individuals with different Lewis and secretor alleles
The Lea and Leb antigens on RBCs (Hakomori and Strycharz, 1968) and those in plasma (Tilley et al., 1975; Hanfland, 1978; Rohr et al., 1980) are primarily glycosphingolipids and not glycoproteins. The adsorption of antigens to RBCs is known to be frequently inhibited in certain biological conditions such as pregnancy (Hammer et al., 1981) and cancer (Yazawa et al., 1988, 1995). Thus, the Lewis RBC phenotype does not reflect the production of Lewis-active glycolipid antigens, whereas the amount of Lewis-active glycolipid antigens in the plasma is not influenced by these biological conditions. Thus, to determine the level of the Le, le1, le2, and le3 enzyme activity relative to the amount of Lewis-active antigens on glycolipids in vivo, we extracted glycolipids from the plasma of 20 Japanese individuals, including two le1/le3 patients with colorectal cancer, and analyzed them by thin layer chromatography (TLC)-immunostaining with anti-Lea and anti-Leb mAbs. Representative TLC patterns are shown in Figure
Figure 2. TLC immunostaining of Lea- and Leb-active glycolipids extracted from plasma of Japanese individuals with various Lewis and secretor genotypes. The amount of Leb-active glycolipids in the plasma of thesej/sej individuals was very small, but was detectable, as indicated by arrows.
Table II. Lewis enzymes in saliva of individuals with different Lewis genotypes
The 45 kDa band of the full-length Lewis enzyme was detected on Western blotting with FTA1-16 (anti-human Lewis enzyme) of a cell lysate of Namalwa cells, which are stable transformant cells with the Le allele (the leftmost lane in Figure Quantitative analysis of the Le, le1, le2, and le3 transcripts, Lewis enzyme, Lea antigen, and sialyl Lewis a (sLea) antigenin colon tissues
Normal and cancerous colorectal tissues were obtained from 14 patients by means of surgical resection. Their Lewis genotypes were determined. There were four Le/Le patients, seven Le/le1 or Le/le2 patients, two le1/le3 patients, and one le1/le1 patient. Their normal colon epithelial tissues were subjected to five different assays, i.e., (1) reverse transcribed-polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) to determine the transcriptional level of each Lewis allele, in particular, to determine whether the mutated Lewis alleles, i.e. le1, le2 and le3, are transcribed or not; (2) Western blotting analysis with FTA1-16 to determine the amount of the Lewis enzyme; (3) competitive RT-PCR to quantitatively measure the transcripts of the Lewis gene; (4) measurement of [alpha]1,4Fuc-T activity; and (5) Western blotting analysis with anti-Lea mAb to determine the amounts of the Lea epitope carried by glycoproteins.
Figure 3. Detection of the Lewis enzyme in saliva of Japanese individualswith various Lewis genotypes. (a) Western blotting analysis of saliva using FTA1-16. The apparent positive bands corresponding to the solubilized form of the Lewis enzyme are indicated by arrows. (b) Relative intensities of the positive bands detected on Western blotting analysis. (c) [alpha]1,4Fuc-T activity detected in saliva.
We first carried out RT-PCR-RFLP, which revealed the amount of the transcript derived from each allele. The method of RT-PCR-RFLP was described in detail in our previous paper (Kudo et al., 1998). All three mutated Lewis alleles, i.e., le1, le2, and le3, of all nine patients were fully transcribed as well as the wild type Le allele (data not shown).
Representative patterns obtained on Western blotting analysis with FTA1-16 of normal colon tissues are shown in the upper panel of Figure
Figure 4. Relative amounts of Lewis gene transcripts, Lewis enzyme, [alpha]1,4Fuc-T activity, Lea antigen and sLea antigen expressed in colon tissues. (a) Western blotting analysis of normal colon tissues with FTA1-16. The representative Western blotting patterns are shown in the upper panel, and the actual amounts of the Lewis enzyme determined with a densitometer are presented in the lower panel. (b) Correlation between the amount of functional Le transcripts derived from the wild-type Le allele and the amount of the Lewis enzyme detected on Western blotting analysis in normal colon tissues. The amount of Lewis transcripts and the Lewis enzyme were determined by competitive RT-PCR, and Western blotting analysis with FTA1-16, respectively. Solid squares, Le/Le individuals; solid triangles, Le/le1 or Le/le2 individuals. (c) Correlation between the amount of the Lewis enzyme and [alpha]1,4Fuc-T activity in normal colon tissues. Solid squares, Le/Le individuals; solid triangles, Le/le1 or Le/le2 individuals; open circle, le1/le1 individual. (d) Correlation between the amount of the Lewis enzyme and the amount of the Lea antigen on glycoproteins in normal left hemicolon tissues. The amount of Lea antigens on glycoproteins was determined by Western blotting analysis with anti-Lea mAb. Solid squares, Le/Le individuals; solid triangles, Le/le1 or Le/le2 individuals; open cycle, le1/le1 individual. (e) Correlation between the amount of the Lewis enzyme and the amount of the sLea antigen in cancerous colon tissues from the sej/sej individuals. solid square, Le/Le individual; solid circles, Le/le1 individuals; open circle, le1/le1 individual.
The transcripts of various Lewis genes in each sample were quantitatively measured by competitive RT-PCR (Kudo et al., 1998). Although this method was not able to distinguish the transcripts of the mutant alleles from those of the Le allele, the results of RT-PCR-RFLP analysis clearly showed that the le allele of all Le/le heterozygotes was fully transcribed at a level equal to that of the Le allele. So the Lewis genotype of each individual was taken into consideration to express the amounts of Lewis transcripts, that is, for the samples from Le/Le individuals, the values obtained on competitive RT-PCR were regarded as the actual amount of the functional Lewis transcripts, whereas for the samples from Le/le1 or Le/le2 heterozygotes half the values obtained on competitive RT-PCR were regarded as the amount of the functional Lewis transcripts. Although the total amounts of the transcripts varied depending on the individual, when the amount of the functional Lewis transcripts in the sample from each individual thus calculated were plotted against the amount of Lewis enzyme in the sample determined on Western blotting, a straight line was obtained, demonstrating a direct correlation between the amount of functional Lewis transcripts and the amount of the Lewis enzyme (Figure
As shown in Figure
Next, we examined the correlation between the amount of the Lewis enzyme and the degree of expression of the Lea antigen in colon tissues. For this purpose, we chose left hemicolon tissues as test samples, since the right hemicolon is known to express a large amount of Leb antigen through the action of strongly expressed [alpha]1,2fucosyltransferases ([alpha]1,2Fuc-Ts), and this would affect the expression of the Lea antigen, whereas the left hemicolon is known to express very little Leb antigen because of the low expression of [alpha]1,2Fuc-Ts (Ernst et al., 1984; Yuan et al., 1985; Itzkowitz et al., 1986; Ørntoft et al., 1991; Jass and Roberton, 1994), and would not affect the expression of the Lea antigen. The results in Figure
The correlation between the amount of the Lewis enzyme and the degree of expression of the sLea antigen in cancerous tissues was determined in sej/sej (nonsecretor) colorectal cancer patients, because we recently found that the serum CA19-9 level is markedly influenced by the secretor status of the cancer patient, suggesting competition between the Se enzyme and [alpha]2,3sialyltransferase for the substrate in both [alpha]1,2fucosylation and [alpha]2,3sialylation (Narimatsu et al., 1998). In each sej/sej patient, the amount of the sLea antigen in cancer tissue, as quantified by Western blotting with anti-sLea mAb, correlated well with the amount of the functional Lewis enzyme in the tissue determined on Western blotting with FTA1-16 (Figure Lewis enzyme and expression of the Lea antigen in normal colon tissue as well as expression of the sLea antigen in cancerous colon tissue of le1/le3 patients
As mentioned in the preceding sections, the le3 enzyme can not produce Lea- and Leb-active glycolipids in plasma, resulting in an Le(a-b-) RBC phenotype, however, the le3 enzyme retains an active catalytic domain. To elucidate the mechanism underlying the impaired activity of the le3 enzyme in the in vivo synthesis of Lewis antigens, we examined the levels of transcripts and protein derived from the le3 allele, and the levels of Lea and sLea antigens produced by the le3 allele in colorectal tissue from the le1/le3 patients.
As described above, the le1 enzyme is nonfunctional for the synthesis of any type 1 Lewis antigens, and a protein band of the le1 enzyme was barely detected for colorectal tissue on Western blotting with FTA1-16. Therefore, we do not need to consider the effects of the le1 allele on the expression of type 1 Lewis antigens and the Lewis enzyme protein when the results for the le1/le3 individuals are interpreted. We compared the amount of the Lewis enzyme detected on Western blotting analysis with the amount of transcripts of the Lewis gene determined by competitive RT-PCR of normal left hemicolorectal tissues from three Le/le1 and two le1/le3 patients. The ratio of the amount of the Lewis enzyme to the amount of the Lewis transcripts in the le1/le3 individuals was approximately half that in the Le/le1 individuals (Figure
Figure 5. Expression of the Lewis enzyme and Lea antigen in relation to Lewis transcripts in normal left hemicolon tissues from le1/le3 patients. Normal left hemicolon tissues from three Le/le1 and two le1/le3 patients were examined, and the results for each genotype are presented as mean values with the standard deviation. (a) Ratio of the amount of the Lewis enzyme detected on Western blotting analysis with FTA1-16 to Lewis transcripts determined by competitive RT-PCR. (b) Ratio of the amount of the Lea antigen on proteins determined on Western blotting analysis with anti-Lea mAb to the amounts of Lewis transcripts. (c) Ratio of the amount of the Lea antigen on protein and the amount of the Lewis enzyme, both of which were determined by Western blotting analysis.
We also determined the expression of the sLea antigen in cancerous colon tissues from the le1/le3 patients by Western blotting with anti-sLea mAb. As shown in Figure
Figure 6. Detection of the sLea antigen in colon cancer tissues on Western blotting analysis. Western blotting analysis with anti-sLea mAb was performed on colon cancer tissues from patients with various Lewis genotypes. Lanes 1-3, Le/le1 patients; lanes 4 and 5, le1/le3 patients;lane 6, le1/le1 patient. Immunohistochemical staining of normal and cancerous colorectal tissues of Le/le1, le1/le2, and le1/le3 individuals
Immunohistochemical staining with FTA1-16, and anti-Lea, anti-sLea, and anti-Leb mAbs was performed on sequential sections of normal and cancerous colon tissue specimens from Le/le1, le1/le2, and le1/le3 individuals. No staining with any of the mAbs was observed in the specimens from the le1/le2 individual, regardless of whether it was normal or cancerous tissue (data not shown). This was consistent with our previous immunohistochemical results (Narimatsu et al., 1996) and the results in the preceding sections, i.e., no staining of the Lewis enzyme on Western blotting and no [alpha]1,4Fuc-T activity in the sample from the le1/le2 individual (Figure
Figure 7. Photomicrographs of immunohistochemical staining of normaland cancerous colon tissues from Le/le1 and le1/le3 individuals. Immunohistochemical staining was carried out on sequential sections of the tissues. The antibodies used for immunostaining were indicated to the right of the photomicrographs. Scale bar, 20 µm. (a) Normal colon tissue from a Le/le1 individual. (b) Normal colon tissue from a le1/le3 individual. (c) Cancerous colon tissue from a Le/le1 individual. (d) Cancerous colon tissue from a le1/le3 individual. The micrographs in the second row (a and b) were magnified, as shown in the top row. In these magnified photographs, localization of the Lewis enzyme and the difference in the amount of the Lewis enzyme between Le/le1 and le1/le3 individuals can be clearly seen.
Regarding the le3 enzyme of the le1/le3 patients, a weak signal for the Lewis enzyme stained with FTA1-16 was observed for the supranuclear region of tall columnar cells, but no signal was observed for goblet cells in the normal colon (Figure
In the present study, we examined various aspects of the molecular behavior of Lewis enzymes encoded by the mutated Lewis alleles, le1, le2, and le3, in native human tissues.
Determination of the in vitro protease resistance of the mutated Lewis enzymes in the present study suggested that (1) the catalytic domain of the Le and le3 enzymes is contained in one large domain with a folded structure, (2) the Leu20 to Arg substitution in the transmembrane domain does not affect the folding of the catalytic domain, and (3) the Gly170 to Ser or Ile356 to Lys substitution in the catalytic domain affects the folding of the catalytic domain. In accordance with these findings in an in vitro experiment, Western blotting with FTA1-16 and the [alpha]1,4Fuc-T activity in saliva samples indicated that the le1 and le2 enzymes were completely digested by protease(s) in the salivary gland in vivo before secretion, while the wild type Le enzymes were secreted into the saliva as a 43 kDa fragment (Figure
A glycosyltransferase gene dosage effect on the amount of carbohydrate product cannot be easily expected. The amount of acceptor substrate and the competition among glycosyltransferases for the acceptor substrates must be taken into consideration. In particular, it is difficult to expect a gene dosage effect in native tissues. However, we found in this study, for the first time, that the Lewis gene dosage actually correlates in vivo not only with the quantity of the Lewis enzyme and their activity, but also with the amount of Lewis carbohydrate antigens. The levels of transcripts of the le1, le2, and le3 alleles were found to be equal to that of the active Le allele (data not shown). This showed that the upstream regions of these alleles are functional and therefore able to correctly regulate gene expression, and that the transcripts of the le1, le2, and le3 alleles are fully translated. This indicates that inactivation of the le1 and le2 alleles as to coding for the functional enzymes was caused only by the missense mutation in the ORF, i.e., not by transcriptional regulation. It was also found that the translational efficiency of the Le allele is constant in all individuals, although the level of the transcripts vary depending on the individual. These two facts enabled us to observe the Lewis gene dosage effects on the carbohydrate products, Lea and sLea antigens, unless competition for a substrate occurs between the Lewis enzyme and other glycosyltransferases, such as [alpha]1,2Fuc-Ts. In fact, we observed a Lewis gene dosage effect on the amount of Lea antigens on proteins in the normal left hemicolon, in which Leb and sLea antigens were barely detected on proteins (Figure
Mollicone et al. reported that 9 of 10 Indonesian le2/le3 individuals showed the Lewis-negative RBC phenotype (Le(a-b-)), but a Lewis-positive saliva phenotype, the remaining individual showed both RBC and saliva Lewis-negative phenotypes (Mollicone et al., 1994). The Lewis antigens of RBCs are on glycolipids, while the antigens in saliva are usually detected on mucins. Therefore, the phenotypes of the Indonesian le2/le3 individuals can be completely explained by our finding that the le3 enzyme is capable of synthesizing Lewis antigens on mucins in colon tissue (Figure
The kinetics of the activity of the le3 enzyme, which was prepared from transiently le3 gene-transfected COS-1 cells in our study, were not consistent with those reported by Mollicone et al. (Mollicone et al., 1994). Our results showed that both the Vmax and Km values for the le3 enzyme were similar to the respective values for the wild-type Le enzyme (Table I), whereas they reported that the Vmax value for the le3 enzyme was markedly reduced compared with that for the Le enzyme, while the two enzymes exhibited similar Km values. Although the discrepancy regarding the Vmax value for the le3 enzyme might be explained by the different acceptor substrates used for the assays, the discrepancy could be explained as follows. We determined the amount of the Lewis enzyme in a homogenate of Le gene- or le3 gene-transfected COS-1 cells by Western blotting with FTA1-16. In the kinetic study, we adjusted the amount of the Le and le3 enzymes used in the experiment to be the same based on the intensities of the bands observed on Western blotting. However, they did not adjust the amount of the Le enzyme for measurement of the kinetics of the enzyme activity. We conjecture that the COS-7 cells used by Mollicone et al. might have contained a reduced amount of the le3 enzyme compared to that of the wild type Le enzyme, due to a lower level of Golgi retention. We did not detect impaired Golgi retention of the le3 enzyme in the COS-1 cells overexpressing the le3 enzyme (data not shown), probably due to that the large amount of the le3 enzyme was enough to overcome the impaired ability. In the cells of native tissues, however, the le3 enzyme was apparently far less retained in the Golgi region than the wild type Le enzyme, as demonstrated by Western blot analysis and an immunohistochemical study.
Regarding the mechanism underlying Golgi retention, Colley (Colley, 1997) reviewed two models. The first model suggests that the shorter transmembrane domain of Golgi-residing proteins prevents the proteins from entering cholesterol-rich transport vesicles which are destined for the plasma membrane, and thus the proteins are retained in the Golgi (Bretscher and Munro, 1993; Masibay et al., 1993). The second is the oligomerization/kin recognition model of Golgi retention, which proposes that the formation of insoluble proteins as homo-oligomers or very large hetero-oligomers prevents the proteins from entering transport vesicles (Machamer, 1991; Nilsson et al., 1994). As an additional mechanism of Golgi retention of proteins, the interaction between the cytoplasmic region of glycosyltransferase and some cytoskeleton matrix components was recently reported to play a role in Golgi retention (Weisz et al., 1993; Slusarewicz et al., 1994; Yamaguchi and Fukuda, 1995). In these studies, it was observed that [beta]1,4 galactosyltransferase expressed in COS cells was dimerized through interaction with proteins in the cytoskeleton matrix. With respect to this, we did not find a dimerized form of the Le enzyme in native tissues, i.e., normal and cancerous colon tissues, whereas we were able to detect a dimerized form of the Le enzyme overexpressed in COS-1 cells (unpublished observations). Still, we do not know which mechanism proposed above is actually involved in Golgi retention of the Le enzyme. We determined the structures of the transmembrane domains of the Le and le3 enzymes by calculation of the cvff force field using Discover and Insight II (Molecular Simulation/Biosym; Albrand et al., 1995). The amino acid substitution (Leu20 to Arg) broke the coiled structure observed in the transmembrane domain of the Le enzyme. Regarding the weakened ability of the le3 enzyme as to Golgi retention, we suppose that the drastic substitution of a hydrophilic residue (Arg) for a hydrophobic one (Leu) in the le3 enzyme decreases the stabilizing energy for its hydrophobic interaction with the Golgi membrane, which results in a reduction in its ability as to Golgi retention. Samples
Plasma and saliva samples were obtained from randomly chosen healthy volunteers, and from the patients with colorectal cancer undergoing surgical resection at the Department of Surgery, Fussa Hospital (Tokyo, Japan). Normal colon tissues, which comprised the tunica mucosa with the muscularis mucosae, but devoid of the muscularis propria histologically, and cancerous colon tissues were also obtained from the patients through surgical resection. Lewis and secretor genotyping
The Lewis genotype was assigned based on the detection of three missense mutations, T59G, G508A, and T1067A, in the Lewis gene. The secretor genotype was assigned based on the detection of two missense mutations, C357T and A385T, in the secretor gene. The PCR-RFLP methods for Lewis and secretor genotyping were described in detail in our previous papers (Nishihara et al., 1994; Kudo et al., 1996). Transfection and expression of the Le gene and the chimera Lewis genes
The Le gene and two chimera Lewis genes, i.e., a Le-le1 gene possessing only the G508A mutation and a Le-le2 gene possessing only the T1067A mutation, subcloned into the pCDM8 vector (Seed, 1987) have been reported in previous studies (Nishihara et al., 1993b, 1994). The le3 allele obtained from the le1/le3 patient was subcloned into the pCDM8 vector in this study. Transient transfection of each gene into COS-1 cells was performed by the same method as described previously (Nishihara et al., 1994). One microgram of the [beta]-actin promoter-driven luciferase expression vector was cotransfected into COS-1 cells as an indicator of the transfection efficiency. Assaying of [alpha]1,4Fuc-T activity
We used two acceptor substrates to assay [alpha]1,4Fuc-T activity. The first assay used 2[prime]O-MeGal[beta]1,3GlcNAc[beta]Bn (2[prime]OMeLacto-N-biose I [beta] Bn), which was kindly provided by Dr. K.L.Matta (Roswell Park Memorial Institute, Buffalo, NY), and has been described in detail in a previous paper (Nishihara et al., 1994).
Another acceptor substrate, lacto-N-tetraose (LNT), was used for the measurement of [alpha]1,4Fuc-T activity in colon tissues. The tissues solubilized in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100 were assayed for [alpha]1,4Fuc-T activity in 0.1 M cacodylate buffer (pH6.8), 5 mM ATP, 10 mM L-fucose, 75 mM GDP-fucose (GDP-Fuc), 25 mM MnCl2, and 25 mM pyridylaminated LNT, as an acceptor substrate. After incubation at 37°C for 2 h, the enzyme reactions were terminated by boiling for 3 min, followed by dilution with water. After centrifugation of the reaction mixtures at 15,000 r.p.m. for 5 min, 10 µl of each supernatant was subjected to high performance liquid chromatography (HPLC) analysis on a TSK-gel ODS-80TS column (4.6 × 300 mm; Tosoh, Tokyo, Japan). The reaction products were eluted with 20 mM ammonium acetate buffer (pH 4.0) at the flow rate of 1.0 ml/min at 35°C and monitored with a fluorescence spectrophotometer (JASCO FP-920, Tokyo, Japan). Protease resistance assaying of the Le enzyme, le3 enzyme, and two chimera enzymes, Le-le1 and Le-le2
COS-1 cells transfected with Le, le3, or a chimera (Le-le1 or Le-le2) DNA were sonicated in 20 mM HEPES buffer (pH 7.2) containing 1% Triton X-100. Each cell lysate, containing 100 µg protein, was incubated with various concentrations of sequencing-grade modified trypsin (Promega, Madison, WI) on ice for 10 min. The reaction was terminated by the addition of 10 mM phenylmethanesulfonyl fluoride. Each reaction sample was immediately suspended in Laemmli's sample buffer, boiled for 5 min, and then subjected to Western blotting analysis with FTA1-16 (Kimura et al., 1995). TLC-immunoblotting analysis of glycolipids in plasma
For the extraction of glycolipids from plasma, 1 ml of plasma was mixed with 3.3 ml of methanol and 3.3 ml of chloroform, followed by brief sonication and standing for 3 h. This extraction procedure was repeated twice. After centrifugation at 3000 r.p.m. for 10 min. at 4°C, the supernatant was dried, cooled on ice, washed with cold acetone twice, and then used as a sample for analysis of the Lewis-active glycolipids. One-twentieth of each sample was applied on a TLC plate (Merck, HPTLC silica gel 60 pre-coated plate, Germany), which was developed with chloroform-methanol-0.2% CaCl2 (60:37.5:8.5). The plate was coated with polyisobutylmethacrylate, blocked with phosphate-buffered saline, 1% bovine serum albumin (PBS-BSA), and then incubated with the first antibody, i.e., the anti-Lea mAb (7LE) (Torrado et al., 1992) or anti-Leb mAb (anti-Leb antibody neokokusai (Narimatsu et al., 1996)), overnight at 4°C. After washing five times with PBS-BSA, the plate was incubated with a biotinylated second antibody in PBS-BSA, followed by incubation with a mixture of streptavidin and horseradish peroxidase (HRP)-conjugated biotin from a Vecta-stain kit (Vector). Color development was carried out with a Konica staining kit (Konica, Tokyo, Japan). Western blotting analysis
Cell pellets or tissues were solubilized in 20 mM HEPES buffer (pH 7.2) containing 2% Triton X-100 by brief sonication. Proteins (40 µg) separated on 10% or 6% SDS-PAGE were transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA) in a Transblot SD cell (Bio-Rad, Richmond, CA). The membrane was blocked with PBS containing 5% skim milk at 4°C overnight and then incubated with 10 µg/ml of FTA1-16, 7LE, or anti-sLea mAb (1116 NS19-9; which is used for detection of tumor marker CA19-9; Magnani, 1982). The membrane was stained according to the manual with the ECL Western blotting detection reagents (Amersham, UK). A densitometer (Shimadzu, Japan) was used to determine the intensities of positive bands on the exposed film (Hyper film TM-ECL, Amersham). The experimental conditions were improved for accurate quantitative analysis. Quantitative analysis of Lewis transcripts in normal colorectal tissues by means of a competitive RT-PCR
Competitive RT-PCR for Lewis transcripts was performed by the method described in detail in our previous paper (Kudo et al., 1998). Immunohistochemical staining
Normal and cancerous colon tissues were fixed in 10% formaldehyde and then embedded in paraffin. Deparaffinized 4 µm sections of the tissues were washed in PBS three times, and then treated with 0.3% (v/v) H2O2 in methanol for 15 min to block endogenous peroxidase. They were washed in PBS three times, and incubated for 20 min with 0.5% normal swine serum in PBS at room temperature to block nonspecific staining. Antigen detection was carried out by applying the primary mAb at a concentration of 10 µg/ml for 12 h at room temperature, followed by the streptavidin-biotin complex method (Elite ABC Kit, Vector). The mAbs used in this study were FTA1-16, 7LE, anti-Leb antibody neokokusai, and 1116 NS19-9. The specimens were washed with PBS between each step, and 0.1 mg/ml of DAB4HCl (Dojin, Kumamoto, Japan) in 0.1 M Tris-HCl buffer (pH7.6) was used for the peroxidase reaction. Nuclei were counterstained with Mayer's hematoxylin.
We gratefully acknowledge the great help of Prof. K. Saito in critical reading and editing of the manuscript. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, No.10178104, from the Ministry of Education, Science and Culture of Japan, and by Special Coordination Funds for the Promotion of Science and Technology from the Science and Technology Agency of Japan.
CA19-9, carbohydrate antigen 19-9; Fuc-T, fucosyltransferase; GDP-Fuc, guanosine diphosphate-fucose; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; Le, Lewis; Lea, Lewis a; Leb, Lewis b; Lewis enzyme, Lewis type [alpha](1,3/1,4)fucosyltransferase; LNT, lacto-N-tetraose; mAb, monoclonal antibody; 2[prime]OMeLacto-N-biose I [beta] Bn, 2[prime]O-MeGal[beta]1,3GlcNAc[beta]Bn; TLC, thin-layer chromatography; PAGE, polyacrylamide gel electrophoresis; PBS-BSA, phosphate-buffered saline, 1% bovine serum albumin; RBCs, red blood cells; RT-PCR-RFLP, reverse transcribed polymerase chain reaction-restriction fragment length polymorphism; sLea, sialyl Lewis a.
4To whom all correspondence should be addressed
Genotype
Lea glycolipids
Leb glycolipids
Lewis
Secretor
Le/-
Se/-
-
++
sej/sej
++
+
le1/le1
Se/-
-
-
sej/sej
-
-
le/le
le1/le2
Se/-
-
-
sej/sej
-
-
le1/le3
Se2/Se2
-
-
Se2/sej
-
-
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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