Identification of a missense mutation G329A; Arg110 Gln ... · spondyloarthrosis and Sjögren´s syndrome but had no history of recurrent bacterial infections 2. or leukocytosis

Identification of a missense mutation (G329A; Arg110→Gln)

in the human FUT7 gene

Per Bengtson1, Cecilia Larson2, Arne Lundblad1, Göran Larson2 and Peter Påhlsson1

1Department of Biomedicine and Surgery, Division of Clinical Chemistry, Linköping

University, SE-581 85 Linköping, Sweden; 2Institute of Laboratory Medicine, Department of

Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, SE-413 45

Göteborg, Sweden.

Corresponding author: Göran Larson, Institute of Laboratory Medicine, Department of

Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, SE-413 45

Göteborg, Sweden.

Tel +46-31-3421330

Fax +46-31-828458

E-mail [email protected]

Running title: FUT 7 mutation

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Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII) which is

involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The

FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients

with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns

by Western blot analysis using monoclonal antibodies directed against SLex and SLex-

related epitopes. One individual showed lower levels of SLex expression and an elevated

expression of CD65s compared to controls. The coding regions of the FUT7 gene from this

individual were cloned and a G329A point mutation (Arg 110→Gln) was found in one allele,

whereas the other FUT7 allele was of wild type. No Fuc-TVII enzyme activity was detected

in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg110 is

conserved in all previously cloned vertebrate α1,3-fucosyltransferases. PCR followed by

restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A

mutation and a frequency of d1% for this mutation was found (3 heterozygotes). Genetic

characterization of the family members of one of the additional heterozygotes identified one

individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of

this homozygously mutated individual showed a lowered expression of SLex and an elevated

expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous

individual was diagnosed with ulcer disease, non-insulin dependant diabetes, osteoporosis,

spondyloarthrosis and Sjögren´s syndrome but had no history of recurrent bacterial infections

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or leukocytosis.

Introduction

Recruitment of leukocytes to sites of inflammation or infection is initiated by interaction of

leukocytes with activated vessel-wall endothelium leading to “rolling” of leukocytes along

endothelial cell surfaces. This interaction subsequently leads to extravasation of leukocytes

into the surrounding infected or inflamed tissue (1). E- and P-selectins, which are expressed

on activated endothelial cells, are involved in this interaction (2-4). The third member of the

selectin family, L-selectin, is involved when lymphocytes extravasate into secondary

peripheral lymphoid organs, where it interacts with counter-receptors on the post-capillary

high endothelial venules (HEV2) (5).

All three selectins recognize glycoprotein counter-receptors that must be properly

glycosylated for binding to occur. All glycans that have been described for efficient

recognition by selectins are modified by α2,3-sialylation and α1,3-fucosylation, and the

minimal common epitope for all selectins is the sialyl Lewis x (SLex , NeuAcα2-3Galß1-

4[Fucα1-3]GlcNAcß1-3-) epitope (6).

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The final step in the biosynthesis of the SLex antigen involves the action of an α1,3-

fucosyltransferase (7). Of the six human α1,3-fucosyltransferases cloned so far, only three

are expressed in leukocytes; Fuc-TIV (8-10), Fuc-TVII (11,12) and the recently cloned Fuc-

TIX (13). The expression level of Fuc-TIX in human leukocytes is however significantly

lower compared to Fuc-TIV and Fuc-TVII (13).

Fuc-TIV has a wide acceptor specificity for GlcNAc in polylactosamines and sialylated

polylactosamines forming for example the Lewis x (Lex, Galß1-4[Fucα1-3]GlcNAcß1-3)

and CD65s (NeuAcα2-3Galß1-4GlcNAcß1-3Galß1-4[Fucα1-3]GlcNAcß1-3-) antigens.

The Fuc-TVII acceptor specificity is restricted to the distal GlcNAc on α2,3-sialylated

lactosamines forming the SLex antigen (14,15). Although Fuc-TIV can synthesize SLex in

vitro (14) Fuc-TVII has been proved to be crucial for the synthesis of SLex and selectin

ligands on leukocytes (16,17). In addition, Fuc-TVII expression in peripheral lymph HEV has

been correlated with expression of L-selectin ligands (5,18). Transfection of human lymphoid

cell lines by antisense cDNA to selectively down-regulate Fuc-TVII suppressed SLex

expression and E-selectin mediated binding (19). Furthermore, mice made deficient in the

Fuc-TVII enzyme showed blood leukocytosis, deficiency in expression of selectin ligand

activity, impaired neutrophil trafficking in inflammation and defects in lymphocyte

recirculation, strongly establishing a role for Fuc-TVII in selectin ligand synthesis (20).

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Several of the cloned α1,3-fucosyltransferases are highly polymorphic in humans. Point

mutations inactivating or disrupting Fuc-TIII (α1,3/1,4-fucosyltransferase, Lewis enzyme)

give rise to Lewis negative phenotypes (21-24). Inactivating mutations have also been found

in the FUT6 gene coding for the plasma α1,3-fucosyltransferase, Fuc-TVI (25-28).

However, there have been no reports on genetic polymorphism in the genes encoding for the

α1,3-fucosyltransferases expressed in human leukocytes.

In this paper we describe for the first time a missense mutation of the FUT7 gene associated

with an altered expression of SLex and CD65s (VIM-2) epitopes on human

polymorphonuclear leukocytes.

Materials and methods

Patients and controls

Fifteen patients with ulcerative colitis (n=13) or proctitis (n=2) were examined. Twelve

healthy controls were also studied. Restriction endonuclease analysis was performed on DNA

samples from 106 plasma donors in Göteborg (29) and 258 unselected adult individuals from

the Linköping area. A pedigree study of FUT7 genetics was performed in one Swedish

family. The study was approved by local ethical committees in Göteborg and Linköping.

Isolation of polymorphonuclear leukocytes

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Human polymorphonuclear leukocytes (PMN) were isolated from 10 ml of freshly drawn

EDTA anticoagulated blood using density gradient centrifugation (Polymorphprep, Nycomed,

UK). The cell preparations had a purity of >90% as determined by analyses on an automatic

cell counter (H3 instrument, Bayer Diagnostics, Germany).

Antibodies

Primary antibodies used were KM93 and CSLEX-1 directed against sialyl Lewis x (SLex),

(Serotech Ltd., Oxford, UK, and Becton Dickinson, San José, CA); VIM-2 directed against

CD65s (kindly provided by Prof. W. Knapp, University of Vienna, Vienna); 911-F11

directed against Lewis x (Lex) and 9001/1H10 directed against sialyl Lewis a (SLea)

(BioCarb AB, Lund, Sweden). For flow cytometry FITC-conjugated primary mouse antibody

against CD15 (Le x, Leu-M1, Becton-Dickinson No 347423) and control FITC-conjugated

mouse IgG1 antibody (X0927, Dako A/S Glostrup, Denmark) were used. FITC-conjugated

F(ab´)2 fragment of rabbit anti-mouse immunoglobulins (F0313, Dako A/S) were used as

secondary antibody. For immunoflourescence analyses the secondary antibody used was

fluorescein conjugated rat anti mouse Ig F261, and for Western blot analyses secondary

peroxidase conjugated rat anti mouse Ig P161 and peroxidase conjugated goat anti rabbit Ig

P448 (Dako A/S, Glostrup, Denmark) antibodies were used. Antigen purified rabbit anti-

mouse Fuc-TVII antiserum was kindly provided by Prof. J. B. Lowe (University of Michigan,

MI).

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Immunoblot analysis

Cell lysates were prepared by solubilizing the purified PMN in 400 µl of lysis buffer (50 mM

Tris-HCl pH 8.0; 150mM NaCl; 5 mM EDTA, 1% IGEPAL CA-630 (Sigma Inc., St. Louis,

MO) containing 0.12 µM PMSF; 2.3 µM Leupeptin and 1.5 µM Pepstatin. Protein

concentration in the supernatant was determined by the 4,4´-Dicarboxy-2,2´biquinoline,

Bicinchonicic Acid (BCA) method (30) (PIERCE, Rockford, IL). An aliquot of the

supernatant corresponding to 20 µg protein was separated by 10% SDS-PAGE (31) and

transferred to an Immobilon-P polyvinylidene fluoride microporous membrane (Millipore,

Bedford, MA, USA) (32). The membrane was blocked with Tris buffered saline (TBS, 50

mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 5% Tween 20 and 5 % defatted milk

powder (Semper, Stockholm, Sweden) at room temperature for 1 hour. The membrane was

then washed twice with TBS, incubated overnight at 4°C with primary antibody in TBS, 1%

BSA, washed three times with TBS containing 0.3% Tween 20 and incubated with

peroxidase-conjugated secondary antibody in TBS containing 1% milk powder and 0.1%

Tween 20 for 1 hour at room temperature. The membrane was rinsed three times with TBS

containing 0.3% Tween 20 and positive bands were visualized using ECL Western blotting

reagents (Amersham Pharmacia, UK). For Western blot analysis of COS-7 cells the

membranes were blocked in 10% BSA in PBS containing 0.2% Tween-20 (PBS-T) over

night at 4°C. PBS-T was used for washing and PBS-T containing 3% of BSA was used for

incubations with primary and secondary antibodies.

For calculation of molecular size, prestained molecular mass standards (Bio-Rad, Hercules,

CA) were used. Digital images of the blots were analyzed using a CCD camera (512 x 512

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pixels) in combination with a computerized imaging 8-bit system (Visage 4.6 from

BioImage, Ann Arbor, MI). The quantitative expression of each epitope in a lane was

assessed as background-corrected optical density, integrated over all pixels in the lane and

expressed as integrated optical density.

Molecular cloning of FUT7 cDNA

Buffy coat was isolated from freshly drawn EDTA anticoagulated blood from one

heterozygously mutated patient (M.N.). Total RNA was isolated using SV Total RNA

isolation system (Promega Corp. Madison, WI) and cDNA was prepared using oligo (dT)15

primers and Reverse Transcription System A3500 according to the manufacturers protocol

(Promega Corp.). PCR was used to amplify the coding regions and immediately adjacent 5´-

and 3´-flanking regions of FUT7 using 0.25 µg of cDNA as template. The PCR-program

used an initial temperature of 8°C for 10 min followed by 40 amplification cycles run for 15 s

at 95°C, 15 s at 59°C and 3 min at 68°C. The last extension step was kept for 10 min at 68°C.

The sense primer (25 pmol), VII-3s, 5´-gctagcgaattcCTGATCCTGGGAGACTGTGG-3´ is

complementary to nucleotides -20 to -1 and contains additional nucleotides (lowercase) at its

5´end, including an EcoRI (underlined) restriction site. The antisense primer (25 pmol) VII-

4as, 5´- gtcgactctagaGTAAGGGCCGGATGCCTGGT-3´ anneals to nucleotides 1107-1088,

and contains additional nucleotides (lowercase) at its 5´ end, including an XbaI (underlined)

restriction site. The 1151-bp PCR product was ligated into the pCR2.1 TA-cloning vector

(Invitrogen Corp., San Diego, CA). Transformation of InVαF bacteria, was performed

according to the manufacturers protocol (Invitrogen Corp.). Positive clones were identified by

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blue-white screening, preparation of plasmids and cleavage with XbaI and EcoRI (Life

Technologies, Paisley, UK.). Twenty-four clones were analyzed with PCR-RFLP (PCR-

Restriction Fragment Length Polymorphism) to identify TA clones with wild type or mutated

alleles. Primer VII-15s (5´-CATCGCCCGCTGCCACCTGAGT-3´) corresponding to

nucleotides 216-237, and VII-5as (5´-GCTGCCGCTCCTGGAAGTTGCTGAC-3´)

corresponding to nucleotides 529-554, were used to amplify a 338 bp fragment of FUT7

cDNA. The G329A mutation abolishes the restriction site GC↓GGCCGC for NotI (Life

Technologies). When the 338 bp PCR product was treated with NotI and analyzed by gel

electrophoresis the wild type allele was digested into two products of 247 and 91 bp, whereas

the mutant allele remained intact. Complete sequencing of 9 TA clones was done on an Alf

II-express using the Cy5-dye terminator kit (Amersham Pharmacia Biotech Inc.). One wild

type clone and one clone containing the G329A point mutation without PCR-induced errors

were chosen for subcloning of the FUT7 insert into the pSI mammalian expression vector

(Promega Corp.). The resulting plasmid containing the FUT7 wild type was called pSI-wt

and the plasmid containing the mutated construct was called pSI-329.

Cloning of the FUT7 gene from genomic DNA

PCR was used to amplify the two coding regions and the 253 bp intron (33) as well as the

immediately adjacent 5´- and 3´- flanking regions of the FUT7 gene from DNA prepared

from whole blood. The PCR-program used an initial temperature of 85 °C for 10 min

followed by 30 amplification cycles run for 15 s at 60 °C, 15 s at 59 °C and 3 min at 68 °C.

The last extension step was kept for 10 min at 68 °C. The VII-3s sense primer (30 pmol), and

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the VII-4as antisense primer (30 pmol) were used. The 1404-bp PCR product was ligated

into the pCR2.1 TA-cloning vector as above and sequenced with the AmpliTaq DNA

polymerase FS kit (Perkin-Elmer, Foster City, CA) on an Applied Biosystems 373A DNA

sequencer (Perkin-Elmer).

Transfection

COS-7 cells (~106 cells) cultured in Dulbecco´s modified Eagle’s medium supplemented

with 10% heat-inactivated fetal calf serum were transfected with 10 µg of expression vector

constructs using the DEAE-dextran method (34). The cells were transfected with pSI without

insert as a negative control or with the two pSI-FUT7 constructs (pSI-wt and pSI-329). The

medium was changed 24 hours post transfection. Transfected cells were harvested after a 96 h

growth period post transfection. Transfection efficiency was controlled using quantitative

PCR analysis. Total RNA was isolated from transfected cells according to the manufacturer’s

instructions using a Total RNA kit (Promega Corp.) including treatment with DNAse. A

reverse transcription kit (Promega Corp.) was used according to the manufacturer’s

instructions to transcribe 1 µg of total RNA.

Quantitative PCR analysis was performed using the TaqMan PCR Core Reagent Kit (PE

Biosystems, CA). Reactions for FUT7 quantification were performed in 30 µL with 0.2 µg of

cDNA; 3 µL of 10 × TaqMan Buffer A (500 mM of KCl; 100 mM of Tris-HCl, pH 8.3);

5 mM MgCl2; 200 µM each of dATP, dCTP, dGTP; 400µM dUTP; 0.3 U uracil-N-

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glucosidase (UNG); 0.75 U of AmpliTaq Gold DNA polymerase; 50 nM of FUT7-probe;

and 100 nM of FUT7 sense and anti sense primers. The following FUT7 consensus primers

and probe were used: r1s, 5’-CTTGGCTGACTGACTCTGG-3’ (nucleotides –29 to -11);

r2as, 5’-CCTCGCAGCCTCCG-3’ (nucleotides 28 to 41) and FUT7-probe, 5’-

CCGTGCCCAAGCATTATTCATCCA-3’ (nucleotides –3 to 20). The FUT7-probe was

designed to cover the sequence over the splice site in FUT7 cDNA to avoid amplification of

contaminating genomic DNA sequences. As an additional control for contaminating DNA,

quantitative PCR was also performed leaving out the first RT-PCR step. This did not generate

any product. The PCR-program used an initial temperature of at 50°C for 2 min and then

95°C for 10 min, followed by 40 amplification cycles run for 15 s at 95°C and 1 min at

60°C. The amplifications were performed on an ABI Prism 7700 sequence detector equipped

with a 96-well thermal cycler. Data were collected and analyzed with Sequence Detector

v1.6.3 software (PE Biosystems). Reactions for quantifying ß-actin were performed exactly

as described above except for using 3.5 mM MgCl2 and 300 nM sense primer 5’-

TCACCCACACTGTGCCCATCTACGA-3’, 300 nM anti sense primer 5’-

CAGCGGAACCGCTCATTGCCAATGG-3’, and 200 nM ß-actin probe 5’-

ATGCCCCCCCCATGCCATCCTGCGT-3’ (PE Biosystems). All analyses were performed

in triplicates and with probes labeled with 6-carboxyfluorescein (FAM)- and 6-

carboxytetramethylrhodamine (TAMRA).

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Immunofluorescence analysis of Lewis antigen expression on the surface of transfected COS-

7 cells

The transfected cells were trypsinized, washed, and incubated with primary antibodies against

CD65s, SLea, SLex and Lex. After 30 min incubation with the primary antibody, the cells

were washed with phosphate-buffered saline (PBS) without Ca2+ and Mg2+ and incubated

another 30 min with fluorescein conjugated rat anti mouse IgG secondary antibody. After

incubation with the secondary antibody the cells were washed with PBS without Ca2+ and

Mg2+. The cell pellets were then fixed with Mowiol (Hoechst, Frankfurt am Main, Germany)/

paraformaldehyde (4%, pH 7.3) 1/3 (v/v) and mounted on glass. The cells were observed

under a Leitz SM-LUX epifluorescence microscope. Immunofluorescence studies were also

conducted on adherent cells in 8-well tissue culture chamber slides (Nunc Inc, Naperville, IL)

without using trypsin treatment.

Fucosyltransferase assay

Enzyme activity was analyzed by measuring the incorporation of GDP-[14C] fucose, 300

mCi/mmol (Amersham Pharmacia Biotech Inc.), to a sialylated type 2 acceptor substrate,

NeuAcα2-3Galβ1-4GlcNAcβ1-sp-biotin or a sialylated type 1 acceptor substrate,

NeuAcα2-3Galβ1-3GlcNAcβ1-sp-biotin (Syntesome, Munich, Germany) COS-7 cells transfected

with pSI, pSI-wt or pSI-329 were lysed in 50 mM MOPS buffer (pH 7.5) containing 1%

Triton X-100. Apparent Km for GDP-Fuc was determined using Lineweaver-Burk plots

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with GDP-Fuc concentrations between 2 and 10 µM and an acceptor concentration of 10 mM.

Apparent Km for the sialylated type 2 acceptor was determined with acceptor concentrations

between 0.25 and 10 mM and a GDP-Fuc concentration of 100 µM. The assay was initiated

with the addition of cell-lysate (45 µg protein) to a reaction mixture containing GDP-Fuc,

acceptor, 10 mM α-L-Fucose and 10 mM MnCl2 in 50 mM MOPS buffer (pH 7.5). The

mixture was incubated at 37 °C for 2 h. The product was purified by the Sep-PAK C18

isolation procedure (35), and analyzed by liquid scintillation counting. Product formation was

also measured at 0.5, 1 and 2 h and found to be linear in this time range.

Mutation screening by restriction-endonuclease analysis

Genomic DNA isolated from 5 ml EDTA anticoagulated blood according to (36), was

amplified by primers VII-15s and VII-5as. The 338 bp product was used without prior

purification for restriction-enzyme analysis by Not I and electrophoresis on a 1.75 %

SeaKem-agarose gel (FMC), followed by ethidium bromide staining. For Not I restriction-

endonuclease analysis of selected family members the primer pair VII-3s/VII-4as was used

for amplification (generating a 1404 bp product) with 791 and 613 bp cleavage products.

Flow cytometry

Flow cytometric analyses were performed on a FACScan instrument (Becton Dickinson)

operating with CELLQuest software and calibrated with 6 µm CaliBRITE beads with the

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AutoCOMP program (Becton Dickinson). One ml of EDTA-anticoagulated peripheral blood

was diluted into 50 ml lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 90 mM Titriplex III

(Merck p.a.) pH 7.3), allowed to stand in room temperature for 7 min, centrifuged and washed

once with 50 ml PBS, pH 7.2. Leukocytes were resuspended in PBS with 0.1% bovine serum

albumin (BSA, Sigma) to a final concentration of 5-10x106 cells/ ml. Fifty µl of cell

suspensions were incubated with 5 µl of primary antibody (Leu-M1 diluted 1:5; VIM-2

diluted 1:100; KM93 diluted 1:40, CSLEX-1 diluted 1:50) and incubated for 15 min at RT.

Cells were then washed with 2 ml of PBS, resuspended in 55 µl of FITC-conjugated F(ab´)2

fragment of rabbit anti-mouse immunoglobulins diluted 1:10 in PBS and incubated for

another 15 min at room temperature. The cells were washed in PBS and fixed in 200 µl of 1%

paraformaldehyde. Mouse FITC-conjugated IgG1 antibodies were used as negative controls.

Of 5000 cells counted only data on the gated granulocyte population is presented.

Results

Identification of a patient with an abnormal expression of SLex and CD65s on her PMN

PMN lysates were analyzed by Western blot analysis to detect differences in expression of

SLex and CD65s. All PMN samples analyzed from healthy volunteers expressed a similar set

of SLex carrying glycoproteins with most intensely stained bands in the molecular weight

region around 90-115 kDa. A representative sample is shown in Fig. 1, lane B. VIM-2

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antibody directed against the CD65s epitope weakly stained glycoproteins with molecular

weights around 60-70 kDa (Fig. 1, lane D). Western blot analyses of PMN lysates from

patients with ulcerative colitis showed staining patterns comparable to the healthy population.

However one of the patients (M.N.) exhibited a different staining pattern. The Western blot

analysis of PMN lysate from this individual showed a significant reduction in the staining of

SLex-bearing glycoproteins. The staining intensity was about 60% compared to control

samples for identical amounts of total protein (Table I, Fig. 1, lane A). In addition, staining of

one band in the 100 kDa region was selectively lost. This pattern was seen using two different

antibodies (KM-93, CSLEX-1) both known to react with SLex, albeit with somewhat

different binding properties ((37)data not shown). Western blot analysis of PMN lysates from

this patient (M.N.) using the VIM-2 antibody directed against the CD65s epitope, showed an

increased staining (480%) compared to control samples (Fig. 1, Table I). This patient was

analyzed both at the time of active disease and in clinical remission at several occasions

during a two year period. The reduced expression of SLex and elevated expression of CD65s

remained constant during this time.

Lowered SLex expression correlates with a G329A mutation in the gene coding for

Fucosyltransferase VII

The lowered SLex expression in PMN of patient M.N. indicated a potential defect in the Fuc-

TVII enzyme. The gene coding for Fuc-TVII, FUT7, was amplified from genomic DNA by

PCR and TA-cloning. Plasmids were isolated from 13 bacterial clones and sequenced over

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the FUT7 insert. The two exons and the 253 bp intron present in the FUT7 gene were

sequenced in both directions. There were no differences in the intron sequences between the

two alleles from this individual. However, a G329A missense mutation was found in 7 out of

the 13 bacterial clones in exon 2 indicating that M.N. carried this mutation heterozygously in

one allele. The G329A nucleotide change leads to an amino acid shift from arginine to

glutamine at position 110. Sequence alignment (38) showed that FUT7-Arg110 is conserved

in all the 16 α1,3-fucosyltransferases cloned so far from vertebrate species (13,39-41).

Screening for G329A by restriction-endonuclease analysis

A restriction fragment length polymorphism (RFLP) assay was used to screen for the G329A

mutation in DNA preparations from 106 plasma donors in Göteborg and 258 unselected adults

in the Linköping area. In this population three additional individuals carrying the G329A

mutation heterozygously were identified. The overall frequency of the G329A mutation in the

analyzed populations was 0.82%.

Identification of an individual homozygous for the G329A mutation in FUT7

DNA from another of the identified heterozygotes (M.L.) was cloned and sequenced. This

confirmed the presence of the G239A mutation in one allele and no other mutations or

alterations in the coding sequences or in the intron sequence. Not I restriction endonuclease

analysis of M.L. and five of her family members are summarized in Figure 2. Apart from the

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heterozygous individual M.L., her brother (R.J.) and both of her daughters (A.L. and L.L.)

also showed a cleavage pattern consistent with heterozygous expression of the G329A

mutation. Her husband did not carry the G329A mutation. However, the PCR product

obtained from the mother of M.L. (S.J.) was not digested at all, which indicated a

homozygous expression of the G329A mutation (Figure 2). The two FUT7 exons and the

intron were completely sequenced in both directions from 14 clones obtained from this

individual. All clones contained the G329A mutation. No other nucleotide changes were

found. This individual thus carried the isolated mutation in both of her FUT7 alleles. When

PMN lysates prepared from this individual were analyzed by Western blot using antibody

KM93 directed against SLex there was an almost complete lack of expression of SLex

binding glycoproteins compared to control samples (Table I, Figure 3, lanes A and B). When

the same samples were analyzed using the VIM-2 antibody directed against CD65s a marked

increase in the expression of this epitope was found for this individual (Figure 3, lane C). The

increased staining intensity was 980% compared to control samples (Table I, Figure 3, lane D)

and 205% compared to individual M.N.

Flow cytometry analysis of PMN from the individual homozygous for the G329A mutation in

FUT7

The expression of SLex on PMN from individuals with or without the G329A mutation was

investigated using flow cytometry. Most of the PMN from the homozygous individual (S.J.)

showed a KM93 staining just above background. However, a sub-population of cells from

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this individual showed an intermediate staining with this antibody. Antibody KM93 reacted

strongly with PMN from an individual lacking the G329A mutation (Fig. 4a). Staining of

PMN with the anti-SLex antibody CSLEX-1 showed the same pattern with lower expression

for the homozygous individual and a higher expression for an individual lacking the G329A

mutation (Fig. 4b). In contrast to the results obtained by Western blot there was no major

differences between these individuals in staining of PMN with the anti-CD65s antibody

VIM-2 (Fig. 4c). As expected PMN from all analyzed individuals expressed a high level of

Lex (Fig. 4d). Sialidase treatment of PMN prior to flow cytometry analysis reduced binding

of KM93, CSLEX-1 and VIM-2 antibodies to background levels (data not shown).

Cell surface expression of SLex is not detected on COS-7 cells transfected with FUT7

G329A cDNA

The Western blot and flow cytometry analyses of PMN from hetero- and homozygously

mutated individuals indicated that the Arg 110→Gln substitution affects Fuc-TVII activity.

To confirm this, COS-7 cells were transiently transfected with plasmids containing either the

mutated or the wild type FUT7 cDNA sequence (pSI-329 and pSI-wt, respectively). Mock

transfectants using vector only (pSI) was used as negative controls. After transfection the

expression of SLex, CD65s, Lex and SLea were analyzed by immunofluorescence staining.

Cells transfected with pSI-wt were clearly stained with anti-SLex antibody (Fig. 5A),

whereas there was no staining of cells transfected with pSI-329 with this antibody (Fig. 5B).

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The same pattern was seen using both KM-93 and CSLEX-1 antibodies (data not shown).

This indicated that the G329A mutation significantly reduces the activity of Fuc-TVII in

transfected COS-7 cells. Neither of the transfectants was stained with antibodies directed

against SLea, CD65s or Lex.

Fucosyltransferase VII activity is not detected in COS-7 cells transfected with FUT7 G329A

cDNA.

Fuc-TVII activity was analyzed using sialylated type 1 and 2 acceptors and whole cell lysates

of COS-7 cells transfected with pSI-wt, pSI-329 or pSI. The pSI-wt construct produced an

active enzyme whereas there was no detectable enzyme activity in cells transfected with the

mutated construct or vector only (Table II), in accordance with the immunofluorescence

results. When a sialylated type 1 chain acceptor was used, no activity was detected in either of

the transfectants. As a measure of transfection efficiency RNA was isolated from the

transfected cells and a fragment of the FUT7 transcript was amplified using quantitative real-

time RT-PCR analysis. There were no quantitative differences in FUT7 mRNA between pSI-

wt and pSI-329 transfected cells (Table III). This pattern was seen for all transfection

experiments used for Fuc-TVII activity measurements. All values were corrected for

differences in total mRNA content using amplification of β-actin mRNA as an internal

control in each experiment (Table III).

Expression of the Fuc-TVII enzyme in transfected COS-7 cells

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COS-7 cells transfected with pSI-wt, pSI-329, or pSI were analyzed by Western blot using

purified antiserum against the Fuc-TVII enzyme. The antiserum stained a band with a

molecular weight of 39 kDa with similar intensity in cells transfected with pSI-wt and pSI-

329, whereas this band was not detected in mock-transfected (pSI) cells (Fig 6). The

molecular weight of the stained band corresponds to the molecular weight previously reported

for FucT-VII (12). In addition a specific band with an apparent molecular weight of 55 kDa

was stained in pSI-wt transfected cells. This band was not detected in either pSI-329 or pSI

transfected cells (Fig. 6).

Discussion

When analyzing the expression of SLex and SLex-related antigens on PMN from patients

with ulcerative colitis one patient with decreased expression of SLex was identified. The

FUT7 gene of this individual was cloned and sequenced and a single point mutation, G329A,

was found in one of the alleles. This mutation gives an amino acid shift from an arginine to a

glutamine at position 110 in Fuc-TVII (Arg110→Gln). When the G329A mutation was

screened for in two small Swedish populations 3 out of 364 individuals were found

heterozygous for this mutation (Fuc-TVII R/Q). Although a larger population needs to be

examined to ascertain the exact overall frequency of this mutation, this indicates that it might

be carried by approximately 1% of the population. FUT7 should thus be considered to be a

polymorphic gene; especially since the G329A allele might be only one of several mutated

alleles to be found in various populations around the world. Genetic analysis of the family

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members of one of the identified heterozygotes revealed an individual carrying the G329A

mutation in both alleles (Fuc-TVII Q/Q). The two exons and the 253 bp intron of FUT7 (33)

were fully sequenced in two of the identified heterozygotes (M.N. and M.L.) and in the

homozygote. Apart from the G329A mutation there was no other structural alteration

compared to the wild type FUT7 sequence.

Western blot analysis of PMN lysates and flow cytometry of PMN showed that individuals

carrying the G329A mutation had a lowered expression of SLex, which is consistent with that

the G329A mutation affects Fuc-TVII activity. Western blot analysis of PMN lysates from

individuals carrying the G329A mutation also showed an increased staining of CD65s. Flow

cytometry analysis of PMN from the Fuc-TVII Q/Q individual also indicated an increased

surface expression of CD65s compared to Fuc-TVII R/R individuals. However the increase

was not as pronounced as seen in the Western blot analysis. Since there is a possible

competition between Fuc-TVII, Fuc-TIV and Fuc-TIX for the same sialylated

polylactosamine acceptor substrate a lowered activity of Fuc-TVII would theoretically

increase the substrate availability for Fuc-TIV and Fuc-TIX, which would explain the

observed increase in CD65s expression (Fig. 7). Surprisingly, the major increase in CD65s

antigens was detected on proteins in the 60-70 kDa region whereas the expression of SLex

was mainly detected on proteins migrating in the 90-115 kDa region. This would suggest

that the observed phenotypic changes are not only explained by substrate availability.

Previous studies have shown a reciprocal expression of Fuc-TVII and Fuc-TIV during

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differentiation of HL60 cells and in HL60 cells deficient in FUT7 expression (42,43),

indicating a linked transcriptional regulation of these enzymes. The possible effect of the

FUT7 mutation on the transcriptional levels of fucosyltransferases in PMN needs to be further

studied. Furthermore, there is always a possibility that the analyzed individuals in this study

may have other differences in glycosyltransferase activity in addition to the lowered activity

of Fuc-TVII, which would affect the glycoprotein profiles obtained in the Western blot

analysis.

The phenotypic changes observed in individuals carrying the G329A mutation suggested a

decreased activity of the Fuc-TVII enzyme. To study the effect of the G329A mutation in

more detail COS-7 cells were transfected with wild type and mutated FUT7 constructs.

COS-7 cells transfected with the FUT7 gene containing the G329A mutation did not express

SLex on the cell surface in contrast to cells transfected with the wild type FUT7 construct. In

addition there was no detectable α1,3-fucosyltransferase activity in whole cell lysate of

COS-7 cells transfected with the mutated construct when an α2,3-sialylated lactosamine

acceptor was used as substrate. The reported Km values of Fuc-TVII is in the low millimolar

range when Neu5Acα2-3Galβ1-4GlcNAc is used as acceptor (44,45). The obtained Km

value for the sialylated type 2 acceptor used in the present study was 6 mM. Km for GDP-

Fuc was 5 µM. An acceptor concentration of 10 mM and a GDP-Fuc concentration of 100

µM would ensure an individual reaction rate at saturating acceptor concentrations but still

there was no detectable activity in the cells transfected with the pSI-329 construct. Even

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when incubation with the cell lysate was prolonged to 18 h the activity in the COS-7 cell

transfected with the mutant construct gave the same incorporation as the mock-transfected

COS-7 cells, indicating that the Arg 110→Gln substitution inactivates the Fuc-TVII enzyme.

There was an over-expression of FUT7 transcripts in both cells transfected with the wild

type and mutated constructs and the levels of transcripts were similar for both constructs. This

indicates that the decrease in enzymatic activity in the COS-7 cells was not an effect of

reduced transcription efficiency for the mutated FUT7 construct. The lack of activity when a

α2,3-sialylated type 1 chain was used as acceptor was to be expected as only Fuc-TVII was

overexpressed in the COS-7 cells and this enzyme specifically recognizes only the sialylated

type 2 chain acceptor (11,12,15,44).

Western blots using the polyclonal anti Fuc-TVII antiserum positively identified the expected

39 kDa band in pSI-wt and pSI-329 transfected cells in about equal quantities. Interestingly,

the cells transfected with pSI-wt, but not those transfected with pSI-329, showed an

additional specific band at 55 kDa. This heavier band might correlate to a hetero-dimer or a

highly glycosylated form of the enzyme and imposes an interesting question on the structural

and functional consequences of the G329A mutation. The molecular explanation for the lack

of this band is now under focus and will be the subject of a separate publication.

Sequence alignment showed that Fuc-TVII-Arg110 is conserved in all the 16 α1,3-

fucosyltransferases cloned so far from vertebrate species (18,39,40). This amino acid is found

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just in-between the hypervariable regions of α1,3-fucosyltransferases considered to be

responsible for the acceptor binding domain and the peptide motifs presumed to be involved

in the GDP-fucose binding (41). This arginine residue has not before been directly linked to

enzymatic activity or specificity. It remains to be studied whether the Arg110→Gln

substitution directly affects enzyme activity or if the substitution affects other functions of the

enzyme such as ER or Golgi retention and degradation. One of the naturally occurring

mutations found to inactivate the Lewis enzyme (Fuc-TIII) has been found to induce

susceptibility to protease digestion rather than directly affecting enzymatic-binding sites (46).

The role of Fuc-TVII in the synthesis of selectin ligands has been demonstrated in vitro using

antisense oligonucleotides (19). The role of Fuc-TVII in vivo has also been clearly indicated

by the generation of mice completely deficient in this enzyme (20). These mice showed blood

leukocytosis, non existent binding of leukocytes to E- and P-selectin, impaired neutrophil

trafficking in inflammation, and defects in lymphocyte recirculation. However Fuc-TVII

deficient mice did not develop a phenotype as severe as mice deficient in E- and P-selectin.

E/P-selectin deficient mice exhibit extreme leukocytosis, systemic infections and plasma cell

proliferation (47) implying that lack of Fuc-TVII would not completely abolish all functional

selectin ligands. The role of Fuc-TIV in generating selectin ligands has been debated.

However recent studies on mice deficient in Fuc-TVII and/or Fuc-TIV support a role for

Fuc-TIV in selectin dependent adhesion of leukocytes (48). Although Fuc-TVII seems to

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play the major role in generating selectin ligands it is clear that inactivation of both Fuc-TVII

and Fuc-TIV is needed to completely inhibit leukocyte adhesion to activated endothelium. In

addition several studies have shown that specific cell lines can synthesize selectin ligands

upon transfection with only Fuc-TIV (49-52). Studies using HL60 cells have also shown that

CD65s can act as a ligand for E-selectin in in vitro flow systems (53). We are currently

analyzing PMN from Fuc-TVII Q/Q and Fuc-TVII R/Q individuals in a selectin adhesion

assay under dynamic flow conditions to address these questions.

The phenotype that can be related to neutrophil dysfunction in the Fuc-TVII deficient mice is

similar to some of the clinical symptoms of patients with the disease called leukocyte

adhesion deficiency type II (LAD II). LAD II patients and Fuc-TVII deficient mice both have

raised leukocyte counts and impaired neutrophil rolling on E- and P-selectins. But in contrast

to Fuc-TVII deficient mice, LAD II patients also suffer from an increased incidence of

bacterial infections in early infancy. In addition, LAD II patients exhibit growth and mental

retardation (54,55). The LAD II deficiency affects all fucosylated glycoconjugates including

the selectin ligands. Recently, a mutation in a GDP-fucose transporter has been implied as

responsible for the LAD II phenotype (56). Thus the clinical symptoms in LAD II patients

cannot be attributed to a specific deficiency in selectin ligand synthesis alone but reflects a

general deficiency of fucose metabolism and transport. The individual homozygously mutated

in FUT7 and presented in this paper is diagnosed with ulcer disease, non-insulin dependant

diabetes, osteoporosis, spondyloarthrosis and Sjögren´s syndrome. The latter diagnosis was

confirmed by signs of keratoconjunctivitis sicca, sialoadenitis and a positive titer for anti-

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nuclear antibodies. There was however no history of recurrent bacterial infections and the

white blood cell count was repeatedly within the reference range thus excluding a phenotype

similar to LAD II for this patient. Nor have any consistent medical conditions associated with

impaired neutrophil function been reported for the heterozygous members of this patient’s

family.

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Footnotes

1This study was supported by grants from the Swedish Medical Research Council (MFR 0002

and MFR 8266), University Hospital Governmental grants and grants from the Swedish

Foundation for Strategic Research (A.L. and G.L.).

2Abbreviations: HEV, high endothelial venules; SLex, sialyl Lewis x; SLea, sialyl Lewis a;

Lex, Lewis x; Fuc-T, fucosyltransferase; PMN, polymorphonuclear leukocytes; LAD II.

Leukocyte adhesion deficiency type II.

Acknowledgement

We thank Dr. Sven Almer and Professor Peter Söderkvist for providing patient and DNA

samples. We also thank Ammi Grahn and Anna-Kristina Granath for excellent technical

assistance and Dr. Anders Elmgren for helpful discussions.

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Figure legends

Figure 1

Western blot analysis of lysed PMN isolated from a patient with ulcerative colitis (M.N.)

(lane A and C) and a control sample (lane B and D). Each lane was loaded with 25 µg of

protein. The blots were probed with antibodies against SLex (KM93, lanes A and B) and

CD65s (VIM-2, lanes C and D). Molecular weight standards are indicated to the left.

Figure 2

Restriction endonuclease analyses of individual M. L. and her family. Squares denote males

and circles denote females. The completely filled circle denotes the Fuc-TVII Q/Q individual

(S.J.). Half-filled symbols denote Fuc-TVII R/Q individuals and the open square denotes a

Fuc-TVII R/R individual. A slash across the symbol indicates that the person is deceased.

The agarose gel electrophoresis pattern of each individual after Not I digestion of the 1404 bp

product is shown below each symbol. The G329A mutated allele is not digested, whereas the

wild type allele is digested into two fragments of 791 and 613 bp.

Figure 3

Western blot analysis of lysed PMN isolated from the Fuc-TVII Q/Q individual (S.J.) (lane A

and C) and a control sample (lane B and D). Each lane was loaded with 20 µg of protein.

The blots were probed with antibodies against SLex (KM93, lanes A and B) and CD65s

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(VIM-2, lanes C and D). Molecular weight standards are indicated to the left.

Figure 4

Flow cytometry analysis of isolated PMN from the FucT-VII Q/Q individual (S.J.) (gray

histograms) and a FucT-VII R/R individual (white histograms). Negative control is shown as

black histograms. A, KM93 antibody; B, CSLEX-1 antibody; C, VIM-2 antibody; D, Anti-

Lex antibody.

Figure 5

Immunostaning of COS-7 cells transfected with pSI-wt (A) and pSI-329 (B). Transfected

cells were incubated with primary antibody against SLex (KM-93) and fluorescein

conjugated rat anti mouse IgG secondary antibody.

Figure 6

Western blot analysis of transfected COS-7 cell lysates using antiserum directed against the

Fuc-TVII enzyme. Cells transfected with pSI-wt (Lane A), pSI-329 (Lane B) and pSI alone

(Lane C). Molecular weight standards are indicated to the left.

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Figure 7

Biosynthetic pathway for the SLex and CD65s epitopes from a sialylated polylactosamine

precursor (15,57).

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Table I

Densitometry analysis of Western blots (integrated optical density)PMN lysates SLex (KM93) CD65s (VIM-2)

M.N. 100A 100A

S.J. 0.2 205.0

Controls(n=10, mean ± SD)

169.3±22.2 20.9±17.5

A Value set to 100

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Table II

Analysis of Fucosyltransferase VII activity Specific Activity

(pmol/(min*mg prot))Km acceptora

(mM)

Km GDP-Fuc

(µM)

pSI-wt 10.5 ± 1.3 6.0 ±0.8 5.0 ±0.4

pSI-329 ndb - -

pSI nd - -

COS-7 cells transfected with pSI-wt, pSI-329 or pSI were lysed and aliquots

corresponding to 45 µg protein were used to measure the fucosyltransferase

activity by analyzing the incorporation of GDP-[14C] fucose to the acceptor

substrate. The values are mean values of three experiments ± SD.

a NeuAcα2-3Galβ1-4GlcNAc-O-biotin (10 mM)

b nd=not detectable

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Table III

Analysis of mRNA content, number of cycles before ∆Rn reaches the threshold*

Data from two different transfections are shown.

FUT7 ß-actin

mean SD mean SD

pSI 40.00 0.00 18.16 0.54

pSI-wt 30.37 0.05 17.32 0.09

pSI-329 29.80 0.50 17.62 0.13

pSI 40.00 0.00 17.64 0.04

PSI-wt 31.22 0.29 17.86 0.11

pSI-329 30.06 0.70 18.23 0.05

*Threshold (∆Rn) set to 0.05

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Per Bengtson, Cecilia Larson, Arne Lundblad, Göran Larson and Peter PåhlssonIdentification of a missense mutation (G329A; Arg110-Gln) in the human FUT7 gene

published online June 12, 2001J. Biol. Chem.

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