Molecular behavior of mutant Lewis enzymes in vivo

Shoko Nishihara, Tsuneo Hiraga, Yuzuru Ikehara, Hiroko Iwasaki, Takashi Kudo, Shin Yazawa1, Kyoei Morozumi2, Yasuo Suda3 and Hisashi Narimatsu4

Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, 1Japan Immunoresearch Laboratories Co., Ltd., 351, Nishiyokote-cho, Takasaki, Gunma 370-0021, 2Division of Surgery, Fussa Hospital, 1-6-1 Kamidaira, Fussa, Tokyo 197-0012, and 3Department of Chemistry, Graduate School of Science, Osaka University, 1-1, Machikaneyama, Toyonaka, Osaka 560-0043, Japan

Received on June 22, 1998; revised on August 8, 1998; accepted on August 24, 1998

The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type [alpha](1,3/1,4)-fucosyltransferase (Lewis enzyme) encoded by the Fuc-TIII gene (FUT3 gene; Lewis gene). We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele (Le) and three mutated alleles, i.e., le1, which has missense mutations T59G and G508A, le2, which has T59G and T1067A, and le3, which has only T59G. Here we carried out an extensive study on the biological properties of the three mutant Lewis enzymes, the le1, le2, and le3 enzymes, using native tissues and obtained the following results. (1) In in vivo and in vitro experiments, the le1 and le2 enzymes were found to be susceptible to protease digestion probably because the one missense mutation in the catalytic domains, i.e., Gly170 to Ser in the le1 enzyme and Ile356 to Lys in the le2 enzyme, makes the three-dimensional structures of the enzymesunstable, while the le3 and wild-type Lewis enzymes wereresistant to protease digestion. (2) The le1 and le2 enzymes cannot synthesize type 1 Lewis antigens on either glycolipids or mucins. The le3 enzyme cannot synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes, while it can synthesize Lewis antigens on mucins in normal and cancerous colon tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in the Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. (3) The Lewis gene dosage actually has effects in vivo on the amount of the Lewis enzyme, its activity, and finally the amounts of Lewis carbohydrate antigens. This is the first article that clearly demonstrates the gene dosage effects on the amount of the glycosyltransferase protein, its activity, and the amounts of carbohydrate products in vivo.

Key words: colon tissues/[alpha](1,3/1,4)fucosyltransferase/Lewis enzyme/Lewis antigens/mutant allele

Introduction

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. Comparison of the kinetics of the Le enzyme and le3 enzymes.
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          

Results

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 1). The epitope recognized by FTA1-16 was identified to be the peptide sequence in the catalytic domain of the human Lewis enzyme in our previous study (Kimura et al., 1995). The untreated enzyme was detected at ~45 kDa, and were not degraded at 5 µg/ml trypsin. This molecular weight corresponded to that expected for the full-length Lewis enzyme containing a cytoplasmic domain, a transmembrane domain, a stem region and a catalytic domain. At higher concentrations of trypsin, a band with an estimated mwt of 43 kDa was obtained for the Le enzyme and the le3 enzyme (Figure 1a,b). In one experiment, we observed that heat-denatured Le enzyme was completely degraded by trypsin digestion (data not shown). These results indicated that the catalytic domain of the Le and le3 enzymes are folded in a fashion that is resistant to trypsin digestion. The stability of the le3 enzyme was almost the same as that of the Le enzyme, indicating that the Leu20 to Arg amino acid substitution in the transmembrane domain did not influence the stability of the catalytic domain. In contrast, a 43 kDa band was not detected for the two chimera enzymes, Le-le1 and Le-le2, even upon treatment with a relatively low concentration, 50 µg/ml, of trypsin (Figure 1c,d). This clearly demonstrated that the single amino acid substitutions in the two mutant enzymes render them sensitive to trypsin digestion. The le1 and le2 enzymes, possessing the Leu20 to Arg substitution in addition to the single substitution in the catalytic domain, were also degraded by low concentrations (50 µg/ml) of trypsin (data not shown).


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 2, and the results for the 20 individuals are summarized in Table II. Samples from all individuals with at least one active Lewis allele and with a weakened secretor allele, i.e., sej allele (Kudo et al., 1996), homozygously gave strong bands of Lea-active glycolipids and a very weak band of Leb. The latter finding is consistent with the weak [alpha]1,2fucosyltransferase activity of the sej enzyme, ~3% that of the wild-type Se enzyme (Kudo et al., 1996). Le/- and sej/sej individuals are known to be Le(a+b+) RBC phenotype (Henry et al., 1996). Samples from all individuals with at least one active Le and Se alleles, Le/- and Se/-, gave bands of Leb-active glycolipids, but not Lea-active glycolipids. No bands of Lea- and Leb-active glycolipids were found for any of the le/le individuals, i.e., le1/le1, le1/le2 and le1/le3 individuals, irrespective of their secretor genotype (Table II), demonstrating that all three mutant Lewis enzymes (le) are unable to produce Lewis-active plasma glycolipids. In a previous study, we demonstrated that le1/le1, le1/le2, and le2/le2 individuals had an Le(a-b-) RBC phenotype (Nishihara et al., 1994). Regarding the le3 enzyme, the two le1/le3 patients examined in this study did not have Lea- and Leb-active glycolipids in their plasma and had an Le(a-b-) RBC phenotype in a hemagglutination test (data not shown). The le3 enzyme was shown to be incapable of producing Lea- and Leb-active plasma glycolipids even though it was resistant to trypsin degradation like the Le enzyme.


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. TLC immunostaining of Lea and Leb active glycolipids extracted from plasma of Japanese individuals showing the Lewis and secretor genotypes
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 - -

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 3a). The 43 kDa band corresponding to the molecular size observed in the trypsin-digestion experiment in the preceding section was detected in saliva samples from the Le/Le and Le/le1 individuals, as indicated by arrows in Figure 3a. The intensity of the 43 kDa band correlated well with [alpha]1,4Fuc-T activity measured on saliva samples (Figure 3c), showing that the 43 kDa molecule detected by FTA1-16 is most likely the active Lewis enzyme, derived from the wild type Le allele. In contrast, saliva samples from the le1/le1 and le1/le2 individuals did not produce a positive band on Western blot analysis (Figure 3a,b), and exhibited no [alpha]1,4Fuc-T activity (Figure 3c). Although we do not know whether the le1 and le2 alleles are fully transcribed and translated in the cells of salivary glands, like in those of colon tissues, as described in a later section, this result suggested that the le1 and le2 enzymes are probably degraded by some protease(s) in the epithelial cells of salivary glands or somewhere en route to secretion into the saliva.

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 4a. The 45 kDa band of the Lewis enzyme, corresponding to the full-length Lewis enzyme, was detected in all samples. The intensity of each band measured with a densitometer is depicted in the lower panel of Figure 4a. The sample from the Le/Le individual gave a 45 kDa band with very strong intensity, whereas the sample from the Le/le1 or Le/le2 heterozygote gave a band with approximately half the intensity of that of the Le/Le individual. The sample from the le1/le3 individual showed approximately one-fourth the intensity of that of the Le/Le individual, and the sample from the le1/le1 individual gave a barely detectable band. The le1 and le2 enzymes in colorectal tissue might be degraded by a protease(s) because they are unfolded, as suggested in the case of saliva samples.


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 4b). This result also demonstrated that the Lewis enzyme detected on Western blotting analysis in colon tissues samples was the functional Lewis enzyme. The samples from all seven heterozygotes, Le/le1 and Le/le2, were determined to contain about half the amount of the functional Lewis enzyme compared with the amount in the samples from the four Le/Le homozygotes. Thus, a Lewis gene dosage effect, depending on the Le genotype, was clearly observed on the amount of the functional Lewis enzyme expressed in colon tissue.

As shown in Figure 4c, a linear correlation was found in all Le/Le, Le/le1, and Le/le2 individuals between the amount of the Lewis enzyme expressed in a colon tissue sample determined on Western analysis and [alpha]1,4Fuc-T activity in the sample. In the sample from the le1/le1 individual, neither the Lewis enzyme nor [alpha]1,4Fuc-T activity was found. These results demonstrated again that almost all the Lewis enzyme detected on Western blotting are functional and therefore able to exert [alpha]1,4Fuc-T activity, and are directed by the wild-type Le allele. The [alpha]1,4Fuc-T activity in colon tissue was also found to be under control of the Lewis gene dosage, depending on the Lewis genotype, i.e., the average [alpha]1,4Fuc-T activity in the samples from the seven Le/le heterozygotes was approximately half that in the samples from the four Le/Le homozygotes.

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 4d show that in the sample from each individual, the amount of the Lea antigen carried by glycoproteins, as quantified by Western blotting with anti-Lea mAb, is proportional to the amount of the functional Lewis enzyme determined on Western blotting with FTA1-16. No Lea antigen was detected in the sample from the le1/le1 individual. The amounts of Lea antigen in the samples from the Le/Le homozygous individuals were almost twice those in the Le/le heterozygotes, indicating a Lewis gene dosage effect.

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 4e).

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 5a). This indicates that the le3 enzyme is retained in colon tissue at half the level of the Le enzyme, since the le3 allele is transcribed at the same level as the Le allele. Thus, the le3 transcripts might be fully translated because it is very hard to consider that only one base change, T59G, affects the translational efficiency. The ratio of the amount of the Lea antigen determined on Western blotting to the amounts of Lewis transcripts in the le1/le3 individuals was also about half that in the Le/le1 individuals (Figure 5b). From the amount of the Lewis enzyme and that of the Lea antigen in each sample determined in the experiments shown in Figure 5a,b, we determined the activity of the le3 enzyme in the production of the Lea antigen in colon tissue. As shown in Figure 5c, the ratio of the amount of the Lewis enzyme to the amount of the Lea antigen on proteins in the le1/le3 individuals was approximately the same as that in the Le/le1 individuals. This indicates that the le3 enzyme is functional in the synthesis of the Lea antigen on proteins, exhibiting almost the same activity as the wild-type Le enzyme. Taking all the results together, we conclude that the le3 allele is fully transcribed and may be fully translated, but that only about a half-amount of the translated le3 enzyme is retained in the cells of colon tissue, and that the le3 enzyme retained in the cells is as functional as the wild-type Le enzyme in the synthesis of the Lea antigen on proteins.


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 6, samples from two (lanes 2 and 3) of the three Le/le1 and one (lane 4) of the two le1/le3 patients apparently gave sLea-positive mucin-like bands, while none of the samples from the three le1/le1 patients did (lane 6, the data for the other two le1/le1 patients is not shown). Thus, it was demonstrated that the le3 enzyme was capable of producing the sLea epitope on mucins in cancer tissues, resulting in elevation of the serum CA19-9 level in the le1/le3 patients. In fact, the le1/le3 patient whose sample gave a sLea-positive band (lane 4) had a very high serum CA19-9 level, ~600 units/ml.


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 3). The lack of staining of the le1/le2 samples with FTA1-16 indicated that the le1 and le2 enzymes must have been degraded in the cells, probably due to incomplete folding. When we interpret the staining results for the Le/le1 and le1/le3 samples, we do not need to consider the product of the le1 allele. As can be seen in Figure 7a, strong staining of the Lewis enzyme with FTA1-16 was observed in the supranuclear region, probably in the Golgi region, of tall columnar cells and goblet cells in the entire crypt of normal colon tissue from the Le/le1 individual, indicating that a large amount of functional Lewis enzyme exists in the Golgi region. The Lewis enzyme probed with FTA1-16 was well colocalized with the Lea (Figure 7a), Leb, and sLea antigens (data not shown), i.e., the Lewis enzyme existed in the cells in which the Lea, Leb, sLea antigens were positively stained, regardless of whether it was normal or cancer tissue.


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 7b). The staining intensities of the Lea and Leb antigens in normal colon tissue from the le1/le3 patient were much reduced in comparison with that in the sample from the Le/le1 patient. These results suggested that the T59G missense mutation (Leu20 to Arg) in the transmembrane domain of the le3 enzyme reduces the ability of Golgi retention of the le3 enzyme, leading to a decrease in the apparent enzymatic activity in the cells. The immunohistochemical result for the le3 enzyme was consistent with the result in the preceding section, i.e., that the amount of the le3 enzyme in normal colon tissue was about half that of the wild-type Le enzyme (Figure 5a). In cancerous tissue from the le1/le3 individual, sLea antigens were positively stained, indicating a correlation of the le3 enzyme in sLea synthesis on proteins (Figure 7d). This was also consistent with the results of Western blotting analysis shown in Figure 6.

Discussion

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 3a,b). In addition, we found that the [alpha]1,4Fuc-T activity detected in the saliva was correlated well with the amount of the active 43 kDa fragment of the Le enzyme (Figure 3c), indicating that the Le enzyme must be digested at the stem region by protease(s), and that the 43 kDa fragment containing its catalytic domain is secreted into the saliva as the active enzyme.

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 4d), but this was not the case in the normal right hemicolon, in which Leb antigens were markedly expressed through the [alpha]1,2Fuc-T activity, probably the secretor enzyme activity (data not shown). We also observed a Lewis gene dosage effect on the amount of sLea antigens in colon cancer tissues from sej/sej individuals, who genetically lack the active secretor allele (Figure 4e), whereas this was not the case in the colon cancer tissues from Se/- individuals who possess the active Se enzyme (data not shown). In accordance with our results, the Lewis gene dosage effect on the amount of sLea antigens has also been reported as an effect on the serum CA19-9 levels in colon cancer patients (Ørntoft et al., 1996; Narimatsu et al., 1998). We observed a similar gene dosage effect in native tissues on other glycosyltransferases, i.e., the Se and H enzymes (unpublished observations).

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 5b,c), but not on glycolipids (Figure 2), resulting in the Le(a-b-) RBC phenotype. In the present study, the le3 enzyme was demonstrated to be capable of synthesizing the sLea antigen on mucins in cancerous colon tissue (Figure 6), which results in positive serum CA19-9 levels in colon cancer patients. The finding that the le3 enzyme can produce CA19-9 antigens but not Lewis-active glycolipids as RBC antigens is considered as one of the reasons why the Lewis-negative RBC phenotype (Le(a-b-)) and positive serum CA19-9 levels are observed in some cancer patients, although the frequency of the occurrence of the le3 allele in Japanese people is very low (0.5%).

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.

Materials and methods

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.

Acknowledgments

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.

Abbreviations

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.

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4To whom all correspondence should be addressed


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