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Analysis of Lewis Fucosyltransferase Genes From the Human Gastric Mucosa
of Lewis-Positive and -Negative Individuals
By Yoshiro Koda, Hiroshi Kimura, and Eisuke Mekada
The expression of Lewis fucosyltransferase (FT) mRNA
was examined in gastric mucosa from two Lewis-positive
[Le( )] and t w o Lewis-negative [Le( )] individuals.
Northern blot analysis demonstrated that levels of mRNA
were similar in both Le( ) and Le( ) gastric mucosa. We
isolated the protein-coding region of the Lewis FT cDNA
from Le( ) and Le( - ) gastric mucosa by polymerase
chain reaction (PCR) amplification. The sequence of cDNA
from the Le( ) gastric mucosa shows two single-base
substitutions of G for T at position 59 and of A for G at
position 508 from the A of the initiation codon of cDNA.
These substitutions may be the cause of changes in t w o
amino acid residues, Arg for Leu at position 20 and Ser for
Gly at position 170 from the N-terminal. To determine
whether either or both of these base substitutions is responsible for the Le( ) gene, w e constructed chimera
cDNAs and expressed them in COS cells. Those COS cells
transfected with a chimera cDNA containing a mutation of
the 508th nucleotide did not express Lewis antigen,
whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G t o A at
position 508 is responsible for the Le( ) gene. The G to A
transition at position 508 created a new site for h u l l endonuclease. The digestion by h u l l endonuclease of PCR
products between the 386th and 612th nucleotides of
Lewis FT cDNA from one of the Le( ) individuals proved t o
be homozygous for the h u l l site. However, the other
Le( ) individual was heterozygous for the h u l l site, suggesting the presence of other Le( ) allele(s). Thus, w e isolated one of the silent Lewis genes (le).
0 1993 by The American Society of Hematology.
L
gawa, Department of Surgery, Kurume University School of Medicine.
RNA preparation and cDNA construction. Total RNA of gastric
mucosa was prepared by the guanidinium thiocyanate method.'*
The poly(A)+fraction was selected by oligo(dT) cellulose column
chromatography. Double-strand cDNA of gastric mucosa was constructed using an Amersham cDNA synthesis kit (Amersham Japan, Tokyo). Single-strand cDNA was constructed by Superscript
reverse transcriptase (BRL, Gaithersburg, MD).
Northern blot hybridization. Poly(A)+ RNAs (2 pg) were electrophoresed through a denaturing formaldehyde agarose gel and
transferred onto nylon membrane (Hybond N+; Amersham, Japan). According to the manufacturer's recommended method, the
filter was prehybridized and then hybridized with a 32Prandom
primed-labeled probetgfrom a 1.7-kb XholI-XbaI fragment of the
insert in pCDM7-a(1,3/1,4)FT (kindly donated by Dr J.B. Lowe,
Howard Hughes Medical Institute, University of Michigan)." The
membrane was then washed and subjected to an Image Analyzer
(Fuji film, Tokyo, Japan).
PCR amplification of Lewis gene product. The polymerase
chain reaction (PCR)*Owas performed using two pairs of synthetic
oligonucleotides as primers (-23 to -4 sense and 1 153 to I 174
antisense nucleotides of Lewis FT cDNA in the case of amplification of cDNA containing the protein-coding region (1.2 kb) of the
Lewis gene for DNA sequencing and expression study, and 386 to
407 sense and 592 to 612 antisense nucleotides of the catalytic domain of Lewis gene cDNA for restriction fragment analysis (230
+
-
+
-
+
-
-
EWIS ANTIGENS are oligosaccharides and are composed of Le" and Leb antigens. Lea antigen is formed
by the action of Lewis gene-encoded a( I ,4)fucosyltransferase (IT
from
) type 1 precursor, while Leb antigen is formed
by the action of Lewis gene-encoded a( 1,4)FT and Se geneencoded a( 1,2)FT from type 1 precursor in tissues such as
salivary gland, digestive mucosa, and respiratory mucosa.''*
Lewis F T also contains a( 1,3)FT activity, which catalyzes
the formation of Le" and LeYantigens from type 2 precursor
and H type 2, respectively, in ~ i t r o .Unlike
~ - ~ AB0 antigens,
which are synthesized in red blood cells (RBC), Lewis antigens on RBC are secondarily acquired from plasma.6 In
addition, the Lewis phenotype on RBC has been reported to
change with various conditions, such as pregnancy,' alcoholic pancreatitis and liver cirrhosis,' hydatid cyst,' and
various carcinomas.'0,'' It has been generally believed that
the Lewis-positive [Le(+)] phenotype results from the action of a( 1,3/1,4)FT encoded by an active allele Le, while
the Lewis-negative [Le(-)] phenotype results from the homozygous presence of the silent allele le." However, Lewis
antigens have been detected in the normal small inte~tine,'~
colonic mucosa,14 urotheliuml* and various cancer tisof Le(-) individuals.
Recently, Kukowska-Latallo et all' isolated cDNA of the
Lewis gene-encoded a( 1,3/ 1,4)FT from the cDNA library of
A43 1 cells by a gene transfer technique. However, the Le-)
mechanism has not yet been examined. An analysis of the
silent allele le from Le(-) individuals is important for understanding the Lewis gene and aberrant tissue expression
of these antigens in Le-) individuals. In the present study,
we examined levels of Lewis gene mRNA obtained from
gastric mucosa from Le(+) and Le(-) individuals. Because
the mRNA levels were found to be similar, we isolated and
analyzed Lewis gene cDNA of Le(+) and Le(-) individuals.
MATERIALS AND METHODS
Distant uninvolved gastric mucosa from gastrectomy samples of
patients with gastric cancer were kindly donated by Dr T. Kake-
Blood, Vol82, No 9 (November 1). 1993: pp 2915-2919
-
-
-
-
~~
From the Department of Legal Medicine, School of Medicine,
and Division of Cell Biology, Institute of Life Science, Kurume University, Kurume, Japan.
Submitted May 4, 1993; accepted July 2, 1993.
Address reprint requests to Hiroshi Kimura, MD, PhD, Department of Legal Medicine, Kurume University School of Medicine,
Kurume, Fukuoka 830, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hemutology.
0006-4971/93/8209-0033$3.00/0
2915
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2916
KODA, KIMURA, AND MEKADA
bp)" with Tth DNA polymerase (Toyobo, Osaka, Japan). Thirty
cycles of denaturation (94OC, I minute), annealing (55 to 60°C, I
minute), and DNA polymerization (72"C, 1 to 2 minutes) were
performed. Double-strand cDNAs from Le(+) and Le(-) gastric
mucosa were separately subjected to PCR, and then 1.2-kb Lewis
gene cDNAs were ligated to a synthetic BstXl adapter and constructed into P C D M ~ . PCR
~ ' products of the catalytic domain of
Lewis FT cDNA (230 bp) amplified from single-strand cDNAs
were digested with 20 U of Awl1 endonucleaseand analyzed by 2%
agarose gel.
Construction of Le(-)/Le(+) chimeras. pCDM8 containing
Lewis gene cDNA from Lewis-positive [Le(+) cDNA] or -negative
[Le(-) cDNA] gastric mucosa was digested with EcoRV and Mliil,
resulting in the formation of I .O-kband 4.5-kb fragments. Ligations
of a 1.0-kb fragment from Le(-) cDNA with a 4.5-kb fragment
from Le(+)cDNA and ofa I .O-kb fragment from Le(+) cDNA with
a 4.5-kb fragment from Le(-) cDNA produced chimera cDNAs
containing mutations of the 59th and 508th nucleotides, respectively.
DNA sequencing. Dideoxynucleotide termination sequencing
reactionz2 was performed with a double-strand plasmid DNA
'7
C
L
R
C
5'
3
3'
Fig 2. Nucleotide sequence of portions of PCR-products from
Le( ) and Le( ) gastric mucosa. The coding sequence is shown
aligned with the sequence ladders. 'Sies of nucleotide substitutions in the Le( - ) gene. The encoded amino acid residues are
shown.
+
Fig 1. Northern blot analysis
of gastric mucosa mRNA.
mRNAs (2 pg) from gastric mucosa of Le( ) and Le( ) individuals were subjected to Northern blot analysis. The blots
were probed with a radiolabeledXhol-Xhl fragment of the
insert in pCDMTI-a(1,3/1,4)FT.
The positions of 28s and 18s
ribosomal RNAs are indicated
by arrows.
+
-
-
(pCDM8 and pBluescript; Stratagene, San Diego, CA) insert. T7
promoter primer, MI3 universal primer, and several synthetic
primers were used. DNA sequencing was performed using Sequenase DNA polymerase (United States Biochemical,Cleveland, OH).
Detection of Lewis antigen. Formalin-fixed, paraffin-embedded 3-pm sections of human gastric mucosa were deparaffinized,
rehydrated, and stained with anti-Le' and -Leb antibodies using a
DAKO quick staining kit (DAKO, Carpinteria, CA).
COS cells were transfected with plasmid DNAs using a DEAEdextran method as described previously?' The cells were harvested
after an expression period of48 hours and incubated for 30 minutes
on ice with either anti-Le', anti-Leb (diluted 1:IOO in phosphatebuffered saline [PBS]; Immucor, Norcross, GA), or anti-Le" monoclonal antibody (diluted 1: 100 in PBS; Signet, Dedham, MA). The
cells were then stained with fluorexein-conjugated goat antimouse
IgM antibody (20 pg/mL).
RESULTS
We obtained gastric mucosa from four unrelated individuals. Two of the four individuals were k(a-b-) as judged
by a hemagglutination test and an immunohistochemical
study of gastric mucosa using anti-le" and -Lebantibodies
(not shown).
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2917
LEWIS-NEGATIVE GENE
I
-
Fig 3. Lewis antigen expression in COS cells after transfection of Lewis gene cDNA. Plasmid DNA (10 rg) was transfected into 5 X 1Oe
COS cells by a DEAE-dextran method. After transfection of plasmids for 48 hours, the cells were harvested and subjected to indirect
immunofluorescence staining. COS cells were transfected with pCDM8 containing Lewis gene cDNA from (A) Le(a b ) gastric mucosa,
(B) Le(a b - ) gastric mucosa, (C) chimera cDNA substituted G for T at position 59, and (D) chimera cDNA substituted A for G at position
508.
-
Northern blot hybridization. To detect the Lewis fl
mRNA, Northern blot hybridization was performed. As
shown in Fig 1, the hybridization with Lewis FT cDNA
demonstrated 2.4-kb bands in poly(A)+ RNAs from both
Le(+) and Le(-) gastric mucosa. The intensity of the bands
normalized by the amount of actin mRNA was similar (not
shown). Therefore, the structural, rather than expression,
failure of the Lewis FT gene is responsible for its inability to
express Lewis antigen.
Direct cloning of Lewis gene cDNA by PCR amplification. To investigate the detailed molecular mechanism,
we isolated the protein-coding region of Lewis FT cDNA by
PCR amplification from both Le(+) and Le(-) individuals.
The 1.2-kb specific bands were gel-purified and added to a
BsfXI adapter and then ligated into pCDM8 for DNA sequencing and for an expression study.
DNA sequencing. To identify the structural failure of
the Le(-) gene, DNA sequencing of the protein-coding regions of Le(+) cDNA and Le(-) cDNA was performed. The
sequence of the Le(+) cDNA was identical to that reported
- +
by Kukowska-Latallo et al.'7 However, the sequence of one
of the Le(-) cDNAs contained two base changes from T to
G at position 59 and from G to A at position 508 (Fig 2).
These nucleotide substitutions may result in the replacement of leucine by arginine at position 20 and of glycine by
serine at position 170 from the N-terminal of Lewis FT.
Expression of Lewis gene cDNA in COS cells. Kukowska-Latallo et a1 isolated Lewis FT cDNA from A431
cells by a gene transfer method using pCDM7 as mammalian expression vector and COS cells.'7 We used the same
systems, but used pCDM8 instead of pCDM7. The only
difference between pCDM7 and pCDM8 is that pCDM7
lacks the polyoma sequences present in pCDM8F4 Those
COS cells transfected with Le(+) cDNA expressed Le" (Fig
3A), but not Leb antigen. Neither Lea nor Leb antigen was
expressed in those COS cells transfected with Le(-) cDNA
(Fig 3B). Lewis FT contains two enzymatic activities; one is
a( 1,4)FT activity that produces Lewis antigens and the
other is a(1,3)FT activity that produces Le" and Ley antigens. Those COS cells transfected with Le(+) cDNA ex-
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KODA, KIMURA, AND MEKADA
2918
1
2
4
3
5
6
7
8
9
found in one of the alleles by sequencing of the 230-bp PCR
product of heterozygote for the PvuII site. To identify any
other mutation(s) responsible for Le(-), we attempted to
amplify the protein-coding region (1.2 kb) of Lewis FT
cDNA from heterozygote for the PvuII site; however, we
failed to amplify it, probably due to degradation of mRNA
as judged by Northern blot analysis.
DISCUSSION
Fig 4. Restriction endonuclease digestion of Lewis gene PCR
product. A PCR product of Lewis FT cDNA between the 386th and
612th nucleotideswas digested with 20 U of h u l l restrictionendonucleasefor 1 hour and subjected to 2%agarose gel. Lane 1 : molecular weight marker (Hpell-digested pUCl8); lanes 2 and 6: PCR
products from Le( ) gastric mucosa cDNA; lanes 4 and 8: PCR
products from Le( - ) gastric mucosa cDNA; lanes 3, 5, 7, and 9:
Arull-digested PCR products of lanes 2,4, 6, and 8.
+
pressed Le" antigen, whereas those COS cells transfected
with Le(-) cDNA did not (not shown).
To determine whether either or both of the nucleotide
substitutions (of G for T at position 59, and of A for G at
position 508) contributes to the failure of the expression of
Lewis antigen in COS cells, we constructed chimera
cDNAs. The chimera cDNA containing a base change from
T to G at position 59 (Leu to Arg) was able to express Le"
antigen (Fig 3C), whereas the chimera cDNA containing a
base change from G to A at position 508 (Gly to Ser) failed
to express Le" antigen (Fig 3D). Our results suggest that the
replacement of Gly by Ser at position 170 is responsible for
the inability to express Lewis antigen in COS cells.
Pvull endonuclease digestion. As shown in Fig 2, the G
to A transition at position 508 creates a new site for endonuclease PvuII. To investigate the presence of a mutation at
position 508 in other individuals, the endonuclease digestion of 230-bp PCR products between the 386th and 612th
nucleotides of Lewis FT cDNA was performed (Fig 4).
While the PCR products of the two Le(+) individuals (lanes
3 and 7) were not cleaved by PvuII as expected, the PCR
product of one of the Le(-) individuals was cleaved by
PvuII (lane 5). However, the PCR product of the other
Le(-) individual was demonstrated to be heterozygous for
the Pvull site (lane 9). A mutation at position 508 was not
In this report, we determined two individuals to be Le(-)
by RBC and immunostaining of gastric mucosa and isolated
mRNA from gastric mucosa of these two individuals. Those
COS cells transfected with Le(-) cDNA did not express Le
antigens, suggesting the presence of mutation($ in Le(-)
cDNA making Lewis FT activity extremely low or absent.
We found two single-base changes at positions 59 and 508
from one of the Le(-) cDNAs. From studies on transfections of chimera cDNAs, a single-base change from G to A
at position 508, which results in an amino acid substitution
of Ser for Gly at position 170 from the N-terminal of Lewis
FT,was found to be responsible for the defective Lewis FT.
That only a single-base change found in the Lewis gene
causes it to be Le(-) may not be incompatible with the
results of 0mtoft et
who found 5% to 16% of a(I ,4)FT
activity in Le(-) colonal mucosa compared with that of
Le(+) mucosa, since a single amino acid substitution would
result not in the complete absence of the activity, but rather
in a low activity of the enzyme. We need to characterize the
nature of the enzyme (activity, stability, affinity for substrates, etc) produced by the Le(-) cDNA after overexpression.
Recently, Lowe et a12s27 isolated three a(1,3)FT genes
that were different from Lewis FT using Lewis FT cDNA as
a probe. Another group also isolated cDNA for ELAM-I
ligand FT?* Its sequence showed that 133 of 231 amino
acids are identical at corresponding positions within the catalytic domain of the Lewis FT.25The other two a( I ,3)FT
genes showed approximately 90% homology with Lewis
FT.26,27
It is of interest that the 170th Gly of the Lewis gene,
which was found to be replaced by Ser in the Le(-) FT,
resulting in its inability to express Lewis antigen (Fig 3B), is
conserved between Lewis FT and these three a(1,3)FTs, and
located within the catalytic domains ofthese FTs,'~*~'*~
suggesting the importance of the 170th Gly for the activity of
the four different a(I,3)FTs.
In the present study, we found another Le(-) allele that
did not contain a substitution of G for A at position 508 by
PvuII digestion ofthe catalytic domain (230 bp) ofthe Lewis
FT gene. Unfortunately, we failed to amplify the proteincoding region (1.2 kb) of cDNA by PCR, probably due to
degradation of mRNA. Therefore, other functional or expression failure may be the cause of the Le(-) gene. However, this is the first report in which we have identified a
mutation of a nucleotide at position 508 in the Le(-) gene.
ACKNOWLEDGMENT
We thank Edward Moran, Fordham University, for linguistic
advice.
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2919
LEWIS-NEGATIVE GENE
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1993 82: 2915-2919
Analysis of Lewis fucosyltransferase genes from the human gastric
mucosa of Lewis-positive and -negative individuals
Y Koda, H Kimura and E Mekada
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