©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
1,3-

L

-Fucosyltransferase Expression in Developing Human Myeloid Cells

ANTIGENIC, ENZYMATIC, AND mRNA ANALYSES (*)

(Received for publication, January 17, 1996)

Julia L. Clarke Winifred M. Watkins (§)

From the Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London, W12 ONN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In an attempt to correlate the cell surface expression of Le^x and sialyl-Le^x structures in immature and mature myeloid cells with the genes expressing alpha1,3-fucosyltransferase(s) we have examined: 1) the properties of the cellular alpha1,3-fucosyltransferases and the mRNA transcripts corresponding to the five cloned genes, Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, and Fuc-TVII, in mature granulocytes and in the myeloid cell line HL-60, before and after dimethyl sulfoxide-induced differentiation and 2) the properties of the alpha1,3-fucosyltransferases expressed in COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNAs. The previously shown increase in cell surface expression of sialyl-Le^x on differentiation of HL-60 cells (Skacel P. O., Edwards A. J., Harrison C. T., and Watkins W. M.(1991) Blood 78, 1452-1460) is accompanied by a sharp fall in expression of Fuc-TIV mRNA and a persistence of expression of Fuc-TVII mRNA. The properties of the alpha1,3-fucosyltransferase expressed in COS-7 cells transfected with Fuc-TIV are consistent with this being the major gene responsible for the expression of Le^x in the immature myeloid cells. In Northern blot analyses, no transcripts of Fuc-TIII, Fuc-TV, or Fuc-TVI were detected in total RNA from mature granulocytes or mRNA from HL-60 cells before or after differentiation. In total RNA from mature granulocytes, Fuc-TIV transcripts were only faintly visible, whereas Fuc-TVII transcripts were quite definitely expressed. The specificity properties of Fuc-TVII expressed in COS-7 cells are consistent with this gene being the major candidate alpha1,3-fucosyltransferase controlling the expression of sialyl-Le^x on mature cells. However, Le^x continues to be expressed on the surface of mature granulocytes and cell extracts retain the capacity to transfer fucose to non-sialylated acceptor substrates. The question therefore remains as to whether these properties result from the weakly expressed Fuc-TIV gene or whether another alpha1,3-fucosyltransferase gene remains to be identified.


INTRODUCTION

In the process of differentiation of hemeopoietic cells the capacity to express alpha-L-fucosyltransferases catalyzing the transfer of fucose to different positional linkages in the sugar acceptor molecules becomes restricted to certain cell lineages. alpha1,3-Fucosyltransferase (^1)activity is present in mature granulocytes, monocytes, and lymphocytes whereas neither alpha1,2- nor alpha1,4-fucosyltransferase activities are detectable in these cells(1, 2, 3) . Transfer of fucose to the O-3 position of the N-acetylglucosamine residue in a Type 2 chain ending (Galbeta1-4GlcNAc) results in the formation of Le^x when the terminal beta-galactosyl residue is unsubstituted and to the formation of sialyl-Le^x when the beta-galactosyl unit is substituted with an alpha2,3-linked sialic acid residue; once formed the Le^x sequence is not a substrate for the addition of sialic acid (reviewed in (4, 5, 6) ). The Le^x determinant is expressed on mature granulocytes and on all myeloid cells from the promyelocyte stage onwards(7, 8, 9, 10) . Sialyl-Le^x is expressed on myeloid progenitor cells but has been reported to be down-modulated at intermediate stages of granulocyte maturation and up-regulated to levels similar to those in progenitor cells after termination of proliferative activity (11) .

High levels of alpha1,3-fucosyltransferase activity are expressed in human leukemic myeloblasts (12, 13, 14) and the finding of even higher levels in normal bone marrow cells (15) suggests that the activity in the leukemic blasts is a reflection of the stage of maturation arrest of the cells. Earlier studies on partially purified preparations of alpha1,3-fucosyltransferases isolated from granulocytes from healthy individuals and from patients with chronic myeloid leukemia, indicated that two different species of this enzyme are expressed in myeloid cells since the preparation from the leukemic cells utilized substrates containing terminal 3`-sialyl-N-acetyllactosamine groupings to only a very limited extent, whereas the preparation from mature granulocytes had a much greater capacity to make sialyl-Le^x determinant sequences(16) .

The leukemia cell line HL-60 consists predominantly of promyelocytes which can be induced to differentiate into myelocytes, metamyelocytes, and granulocytes by the addition of dimethyl sulfoxide (Me(2)SO) to the growth medium(17) . The surface glycoprotein (18, 19) and glycolipid profiles (20, 21) of the cells specifically change during differentiation. Therefore, although granulocytic maturation of HL-60 cells has been shown to be defective in some respects(20, 22, 23, 24, 25) , this cell line offers a useful model for studying the enzymatic basis of changes that take place in expression of carbohydrate cell surface structures during differentiation of myeloid cells. Our previous studies demonstrated that Me(2)SO-induced differentiation of HL-60 cells resulted in a lowering of alpha1,3-fucosyltransferase and alpha2,6-sialyltransferase activities, an increase in cell surface expression of sialyl-Le^x, and a loss of the marked preference for non-sialylated acceptors shown by the alpha1,3-fucosyltransferase(s) in undifferentiated cells(14) .

Five human alpha1,3-fucosyltransferase genes had been cloned: Fuc-TIII (26) , Fuc-TIV(27, 28, 29) , Fuc-TV(30) , Fuc-TVI(31, 32) , and Fuc-TVII (33, 34) . Fuc-TIII, Fuc-TV, and Fuc-TVI are clustered on chromosome 19 and the catalytic domains share up to 95% nucleotide and amino acid sequence homology(26, 30, 31, 32) . Fuc-TIV, located on chromosome 11(35, 36) , is less closely homologous but still shares about 60% homology with the three chromosome 19 alpha1,3-fucosyltransferases(32) . This gene is expressed in HL-60 cells(27, 28, 29) , and in other myeloid leukemic cell lines, and by consensus was termed the ``myeloid'' alpha1,3-fucosyltransferase gene. However, when cDNA corresponding to this gene was transiently expressed in mammalian cells, there was lack of agreement as to whether the Fuc-TIV gene gave rise to cell surface expression of sialyl-Le^x(27, 28, 29) and when extracts of the transfected cells were tested for alpha1,3-fucosyltransferase activity the expressed enzyme failed to utilize alpha2,3-sialylated acceptors (27) . The most recently cloned gene, Fuc-TVII, which maps to chromosome 9, has about 40% homology with the chromosome 19 cluster and is reported to give rise to cell surface expression of sialyl-Le^x, but not Le^x, when transiently expressed in mammalian cells(33, 34) . Transcripts of this gene have been detected in mRNA from undifferentiated HL-60 cells(33, 34) and it is therefore a plausible candidate to be the major gene encoding an enzyme that leads to the expression of sialyl-Le^x in cells of the myeloid lineage.

The aim of the present study was to examine further the changes that take place in the specificity and general properties of alpha1,3-fucosyltransferase(s) in the course of maturation of myeloid cells and to determine whether any direct correlation can be shown between enzyme activity, mRNA expression of the five cloned alpha1,3-fucosyltransferase genes, and cell surface expression of Le^x and sialyl-Le^x.


EXPERIMENTAL PROCEDURES

Materials

GDP-L-[^14C]fucose (270 mCi/mol) and UDP-[^14C]-D-galactose (330 mCi/mol) were purchased from Amersham, United Kingdom. N-Acetyllactosamine, lacto-N-biose I, 2`-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, 3`-sialyllactose, 6`-sialyllactose, and 6`-sialyl-N-acetyllactosamine were the gifts of Dr. A. S. R. Donald (formerly of the MRC Clinical Research Centre, Harrow, U.K.). The structures and purities of all of these compounds had been checked by ^1H NMR spectroscopy(37) . 3`-Sialyl-N-acetyllactosamine was purchased from Dextra Laboratories Ltd., Reading, U.K. Lactose, phenyl-beta-D-galactoside, fetuin, alpha(1)-acid glycoprotein, transferrin, Dowex 1 times 8-200, Sephadex G-50 (Fine), N-ethylmaleimide, iodoacetamide, p-mercuribenzoate, and Me(2)SO were purchased from Sigma, U.K. The glycoproteins fetuin and alpha(1)-acid glycoprotein have O- and N-linked oligosaccharide chains but only the N-linked chains are believed to carry peripheral Type 2 (Galbeta1-4GlcNAc) core structures that would be potential acceptors for fucosyltransferases. A proportion of these chains are substituted with alpha2,3-linked sialic acid residues(38, 39) , whereas the N-linked chains in transferrin carry Type 2 structures substituted predominantly with alpha2,6-linked sialic acid (40) . Tamm-Horsfall urinary glycoprotein (41) was a gift of Professor W. T. J. Morgan (formerly of the MRC Clinical Research Centre, Harrow, U.K.); this glycoprotein has complex N-linked oligosaccharide chains, many of which terminate with alpha2,3-linked sialic acid residues(42) . A human precursor blood group glycoprotein number 484 was isolated as described (43) from an ovarian cyst fluid removed from a patient who was a non-secretor of ABH, Le^a, or Le^b(44) ; this glycoprotein has extended O-linked oligosaccharide chains terminating with both Type 1 (Galbeta1-3GlcNAc) and Type 2 peripheral core structures. Asialoglycoproteins, apart from asialofetuin, which was purchased from the Sigma, U.K., were prepared by treatment of a 1% solution of the glycoproteins with 0.05 M H(2)SO(4) for 1 h at 80 °C, followed by thorough dialysis against distilled water.

Cell Lines and Cell Culture

The human leukemic cell line, HL-60, was obtained from the European Collection of Animal Cell Cultures (PHLS Centre for Applied Microbiology and Research, Porton Down, U.K.). This cell line which contains predominantly promyelocytes (17) was grown in RPMI 1640 medium (Life Technologies, Inc. Ltd, U.K.), supplemented with 10% heat inactivated (56 °C) fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin, in a 5% CO(2) atmosphere. The cells were passaged twice weekly to maintain exponential growth. Granulocytic differentiation of HL-60 cells (45) was induced with Me(2)SO. Cells were seeded at an initial concentration of 2.5 times 10^5 cells/ml into duplicate flasks, one containing fresh medium alone and the other fresh medium plus Me(2)SO at a concentration of 1.3%. After periods of growth between 24 h and 4 days, cells were harvested from the culture medium by centrifugation at 800 times g for 5 min and examined for morphological evidence of differentiation(45) . The cells were washed with phosphate-buffered saline (PBS), pH 7.3 (containing in 1 liter: 8 g NaCl, 0.2 g KCl, 1.15 g Na(2)HPO(4), and 0.2 g KH(2)PO4), suspended in PBS and counted in a hemocytometer. Viability was judged by Trypan blue dye exclusion and was greater than 95% in all experiments.

The epidermoid carcinoma cell line, A431, was supplied by Dr. W. Gullick (Imperial Cancer Research Fund Unit, Hammersmith Hospital, London, U.K.) and COS-7 cells by Dr. P. H. Johnson (MRC Blood Group Unit, University College London, U.K.) and Dr. P. J. Mason (Royal Postgraduate Medical School, London, U.K.). These cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc. Ltd., U.K.), supplemented with 10% heat inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin, in a 5% CO(2) atmosphere. The cells were grown to confluence, the medium removed, the adherent cells washed with PBS and detached from the flask by treatment for 5 min with trypsin (0.5%), EDTA (0.2%). The detached cells were suspended in a small volume of Dulbecco's modified Eagle's medium, spun at 800 times g, washed with PBS, resuspended in PBS, and counted.

Isolation of Granulocytes from Peripheral Blood

Granulocytes from blood of normal volunteers were isolated from heparin anti-coagulated blood (200 µl of heparin/10 ml of blood). Hydroxyethyl starch (Hespan, Du Pont Pharmaceuticals, Letchworth Garden City, Hertfordshire, U.K.) (1 volume) was added to 10 volumes of whole blood and the erythrocytes were left to sediment for 1 h. The upper plasma and leukocyte layer (6 volumes) was then gently layered onto Lymphoprep (Nycomed Pharma, AS., Oslo, Norway) (4 volumes) and centrifuged at 400 times g for 30 min to deposit the granulocytes. Erythrocytes contaminating the granulocyte pellet were lysed by suspending the pellet in buffer, pH 7.4 (155 mM NH(4)Cl, 1.0 mM KHCO(3), 0.1 mM EDTA), for 5 min on ice. The granulocytes were then washed, resuspended in PBS, and counted.

Mouse Monoclonal Antibodies

LeuM1 (anti-Le^x) (46) was purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). CSLEX1 (anti-sialyl-Le^x) (47) supplied as affinity purified antibody at a concentration of 2.8 mg/ml, was a gift from Dr. P. Terasaki (UCLA Tissue Typing Laboratory, Los Angeles, CA). Antibody FH6 (anti-sialyl-dimeric-Le^x) (48) was supplied by Dr. S. Hakomori, Biomembrane Institute, Seattle, WA. Anti-CD11b (Clone 44, IgG1), anti-CD16 (Clone B-E16, IgG2a), anti-CDw65 (Clone VIM-2(49) , IgM) and anti-CD34 (Clone QBend/10, IgG1) were purchased from Serotec Ltd., Oxford, U.K.

Indirect Immunofluorescence

Antibody binding was assessed by indirect immunofluorescence on freshly harvested cells grown in culture, or freshly isolated granulocytes, fixed in 1% paraformaldehyde. The cell suspension (100 µl containing 0.5 times 10^6 cells) was incubated for 30 min at 4 °C with 10 µl of antibody at the appropriate dilution, the cells were washed with PBS containing 200 mg/liter sodium azide (PBS/azide) and then incubated for a further 30 min at 4 °C with 4 µl of R-phycoerythrin-conjugated goat (Fab`(2)) anti-mouse immunoglobulins (Dako Ltd, High Wycombe, U.K.). An irrelevant antibody was used as a negative control. Stained cells were analyzed by flow cytometry on a FACScan analyzer (Becton Dickinson Immunocytometry Systems).

Neuraminidase Treatment of Cells

Neuraminidase from Newcastle disease virus (Oxford Glycosystems, Oxford, U.K.) was used to treat differentiated HL-60 cells. Cells (5 times 10^6 in 100 µl of PBS) were incubated with 10 µl of neuraminidase (10 miliunits) at 37 °C for 30 min. The treated cells were washed with PBS/azide and resuspended in 1.0 ml of 1% paraformaldehyde for flow cytometric analysis.

Fucosyltransferase Assays

Immediately before the assays were carried out the cells were lysed by treatment of 1.5 ml of the cell suspension in PBS (containing 0.8 times 10^6 cells/15 µl) with 0.5 ml of 1% Triton X-100 for 60 min at 4 °C. The resultant extract was spun briefly (5-10 s) at 10,000 times g and the supernatant was used as the enzyme source. Standard reaction mixtures for alpha1,2-, alpha1,3-, and alpha1,4-fucosyltransferase assays were carried out in a total volume of 80 µl and contained 0.48 µM GDP-L-[^14C]fucose, (24,000 cpm), 12.5 mM MnCl(2), 63 mM sodium cacodylate buffer, pH 7.0, 3 mM ATP, 0.06% (v/v) Triton X-100, 20 µl of enzyme source, and either 3 mM low molecular weight acceptor substrate or 400 µg of glycoprotein acceptor. The reaction mixtures containing the low molecular weight oligosaccharide acceptors were incubated at 37 °C for 2 h and the radioactive product was separated on a Dowex-1 column (0.8 ml) in the formate form. Except for the reaction mixtures containing sialyl compounds as acceptors, the columns were eluted with 1.6 ml of H(2)O, the eluant was mixed with 4 ml of water-miscible scintillant (Pico-Fluor 40, Canberra Packard, Pangbourne, U.K.) and counted on a Beckman LS 6800 scintillation counter. The reaction mixtures containing products formed with low molecular weight sialyl oligosaccharides were eluted from the Dowex-1 columns with 1.6 ml of 40 mM pyridine/acetate. Control mixtures not containing acceptor substrates were included in all these experiments and any eluted counts representing breakdown of the labeled nucleotide sugar were subtracted from the product counts.

The reaction mixtures containing the glycoprotein acceptors were incubated at 37 °C for 16 h and the radioactive products were separated on columns (0.7 x 16 cm) of Sephadex G-50 by elution with 0.2 M NaCl. The first 2 ml of eluate were discarded and the next 2 ml, containing the product, were mixed with 5 ml of scintillant and counted as above. Control mixtures not containing the exogenous glycoprotein acceptors were included in all these experiments and radioactivity resulting from incorporation into endogenous macromolecular acceptors was subtracted from the product counts.

beta1,4-Galactosyltransferase activity was assayed with a reaction mixture containing in a total volume of 85 µl: 0.68 µM UDP-[^14C]-D-galactose (50,000 cpm), 21 mM MnCl(2), 52 mM Tris-HCl buffer, pH 7.2, 5.2 mM ATP, 0.05% Triton X-100, 20 µl of enzyme source, and 2.6 mMN-acetylglucosamine. The mixtures were incubated at 37 °C for 30 min and the radioactive N-acetyllactosamine product was separated on Dowex columns and counted as described for the fucosyltransferase assays.

Treatment with Sulfhydryl Binding Reagents

In most experiments NEM, p-mercuribenzoate, or iodoacetamide were added to the alpha1,3-fucosyltransferase assay mixtures immediately before incubation to give a range of final concentrations of the reagents from 0.01 to 10 mM. In a second set of experiments the cell extracts were mixed with different concentrations of NEM and incubated for 30 min at 37 °C before addition to the remainder of the assay mixtures. In a third set, NEM was added to aliquots of the enzyme extract to give a final concentration of 1 mM and incubated at 37 °C for 5, 10, and 30 min before an equal volume of 2 mM dithiothreitol was added to each to destroy excess NEM. The final mixtures were assayed for alpha1,3-fucosyltransferase activity by the standard procedures.

Heat Treatment of Cell Extracts

Triton X-100 extracts of cells, prepared as described for the preparation of the enzyme source used in fucosyltransferase assays, were aliquoted in 80-µl amounts and heated at 50 °C for different times between 0 and 30 min. The heated enzyme solutions (20 µl/assay) were then tested for alpha1,3-fucosyltransferase activity.

Northern Blot Analysis

Total RNA was isolated by the guanidinium-CsCl method as described by Wilkinson(50) . Poly(A) RNA was isolated from the total RNA by oligo(dT)-cellulose column chromatography as described by Maniatis et al.(51) . The concentration, and freedom from protein contamination, of the isolated RNA was determined from optical density measurements at 260 and 280 nm. Measured amounts of the poly(A) RNA, denatured in the presence of formaldehyde, were electrophoresed on a 1.25% agarose/formaldehyde gel for 3-4 h at 100 V. The RNA was transferred to a nylon membrane (Hybond N, Amersham, U.K.) by capillary blotting (52) and the membrane was baked for 2 h at 80 °C and prehybridized for 3-6 h at 42 °C in a freshly prepared solution of hybridization fluid (50% deionized formamide (Life Technologies, Inc. Ltd., U.K.), 1.0 M NaCl, 1% (w/v) SDS, 10% (w/v) dextran sulfate, and 200 µg/ml denatured salmon sperm DNA (Sigma)).

Five alpha1,3-fucosyltransferase probes were prepared from plasmids kindly provided by Dr. J. B. Lowe (Howard Hughes Medical Centre, Ann Arbor, MI). The probes were: the 1.7-kb XhoI-XbaI fragment isolated from the insert in plasmid pCDM7-alpha-(1,3/1,4)-fucosyltransferase for the detection of Fuc-TIII (26) , the 1.05-kb PvuII fragment from the insert in pcDNA1-alpha-(1,3)-fucosyltransferase for the detection of Fuc-TIV(27) , the 1.9-kb XbaI-EcoRI fragment from the insert in pcDNA1-Fuc-TV for the detection of Fuc-TV(30) , the 1.2-kb HindIII fragment from the insert in pcDNA1-Fuc-TVI for the detection of Fuc-TVI(32) , and the 2.0-kb XhoI-XbaI fragment from the insert in pCDM8-Fuc-TVII for the detection of Fuc-TVII(34) . A glucose-6-phosphate dehydrogenase (G6PD) cDNA probe, supplied by Dr. C. M. Corcoran (Royal Postgraduate Medical School, London), was used as a control for quality and even loading of the mRNA. P-Labeled cDNA probes were prepared by the random oligonucleotide priming method according to the manufacturers instructions (Megaprime, Amersham) to a specific activity equal to, or greater than, 1 times 10^8 cpm/µg DNA. The P-labeled cDNA probes (50 ng) were added to 5 ml of the hybridization solution and the membranes were then heated for 16 h at 42 °C. After hybridization the blots were washed once in 0.45 M NaCl, 0.045 M sodium citrate, 1% sodium dodecyl sulfate (SDS) for 15 min at room temperature, twice with 0.3 M NaCl, 0.03 M sodium citrate, 1% SDS for 30 min at 42 °C and once with 0.03 M NaCl, 0.003 M sodium citrate, 1% SDS for 30 min at 42 °C. The membranes were air dried and then exposed to Kodak X-Omat film with an enhancing screen at -70 °C.

Transfection of COS-7 Cells

COS-7 cells were transfected with 20 µg of expression vectors containing Fuc-TIV and Fuc-TVII coding sequences using the DEAE-dextran procedure(53) . Plasmid pcDNA1-Fuc-TIV (27) was used for Fuc-TIV expression. Initial attempts to transfect the plasmid pCDM8-Fuc-TVII (34) proved unsuccessful and a 2.0-kb insert containing the coding cDNA for Fuc-TVII was cut out by XbaI digestion and cloned into the XbaI site of the vector pSI (Promega). COS-7 cells were subjected to mock transfection by treatment with DEAE-dextran in the absence of the plasmids and with the pSI vector in the absence of the Fuc-TVII cDNA insert. The transfected cells were grown for 48 h in Dulbecco's modified Eagle's medium and harvested as described under ``Cell Culture.''


RESULTS

Flow Cytometric Analysis of HL-60 Cells Before and After Treatment with Me(2)SO

Me(2)SO-induced differentiation of HL-60 cells over a period of 4 days resulted in changes in the cell morphology with a decrease in the ratio of nucleus to cytoplasm and the appearance in a few cells of segmented nuclei. Coincident with the morphological maturation, marked changes were observed in the antigens expressed on the cell surface as measured by indirect immunofluorescence with monoclonal antibodies. The marker CD11b, part of the family of integrins, which is first expressed at the myelocytic stage of normal maturation (54) was detected at a very low level on untreated HL-60 cells but appeared more strongly after 2 days exposure to Me(2)SO and continued to increase in strength up to 4 days (Fig. 1). However, the differentiated cells showed very little activity with the antibody that detects CD16, an antigen which, in the course of normal differentiation, begins to appear at the metamyelocyte and band stages(11) . Therefore the majority of the differentiated HL-60 cells were expressing a marker of the myelocytic stage of maturation after 4 days Me(2)SO treatment, but were not expressing a marker of fully differentiated granulocytes. CD34, an antigen present on early myeloid progenitor cells (55) was not detected on either the undifferentiated or differentiated HL-60 cells (Fig. 1).


Figure 1: Flow cytometry analyses of mature granulocytes and HL-60 cells, before and after Me(2)SO (DMSO)-induced differentiation. HL-60 cells, grown with or without Me(2)SO (DMSO) in the standard growth medium for periods up to 4 days and granulocytes isolated from peripheral blood were washed, stained with monoclonal antibodies, and examined by indirect immunofluoresence as described under ``Experimental Procedures.'' The commercial antibodies were used at the following dilutions: anti-LeuM1 (anti-Le^x) 1:50, anti-CDw65 (anti-VIM2) 1:10, anti-CD11b undiluted, and anti-CD34 undiluted. CSLEX1 (anti-sialyl-Le^x) was diluted 1:50 for tests on HL-60 cells and 1:100 for tests on granulocytes. FH6 (anti-sialyl-dimeric Le^x) was used at the concentration supplied. The data are presented as the percentage of cells giving positive reactions with the antisera.



Both the differentiated and undifferentiated HL-60 cells reacted with anti-CD15 (anti-Le^x) and neither the percentage of positive cells, nor the mean fluorescence intensity (data not shown), changed significantly on Me(2)SO-induced maturation (Fig. 1). As observed earlier(14) , differentiation of HL-60 cells was accompanied by an up-regulation in the expression of sialyl-Le^x; the increase in the number of positive cells was apparent after 24 h and continued to develop up to 48 h (Fig. 1). Treatment with 10 milliunits of Newcastle disease virus neuraminidase decreased the number of cells reacting with anti-sialyl-Le^x by 89%, thus demonstrating that the antibody was reacting with newly formed structures terminating with sialic acid residues. The undifferentiated HL-60 cells reacted quite strongly with antibody CDw65 which detects the VIM2 epitope(49) , but differentiation led to a decrease in the number of cells positive with this antibody (Fig. 1). The antibody FH6, which is reported to react with sialyl-dimeric-Le^x but not sialyl-Le^x or the VIM2 epitope(48) , failed to react with untreated HL-60 cells and no significant change was detectable after 4 days exposure to Me(2)SO (Fig. 1).

Flow Cytometric Analysis of Mature Granulocytes

Mature granulocytes isolated from peripheral blood reacted more strongly than the differentiated HL-60 cells with anti-sialyl-Le^x (CSLEX 1) (Fig. 1) with an approximate 5-fold increase (432 to 2230) in the mean fluorescence intensity. Although virtually all granulocytes were positive with anti-Le^x, the mean fluorescence intensity was relatively much weaker than the activity with anti-sialyl-Le^x and was comparable with the activity found for the HL-60 cells (150). The antibody to VIM2 (CDw65) also reacted quite strongly with granulocytes, a result which contrasts with the loss of activity with this antibody found on differentiation of HL-60 cells (Fig. 1). The granulocytes reacted weakly with the antibody to sialyl-dimeric-Le^x structures, FH6, which failed to react with the differentiated HL-60 cells. As expected, the granulocytes gave positive reactions with antibodies to both of the late myeloid markers CD11b and CD16 and were negative with the antibody detecting the marker of early myeloid development, CD34 (Fig. 1).

Flow Cytometric Analysis of COS-7 Cells Transfected with Fuc-TIV and Fuc-TVII cDNAs

Two samples of untransfected COS-7 cells obtained from different laboratories each gave small but definite reactions in the flow cytometer with the sialyl-Le^x antibody, CSLEX1, but not with the other monoclonal antibodies used in this investigation. The percentage of positive cells and mean fluorescence intensity of the untransfected cells reacting with this antibody were subtracted from the values obtained with the transfected cells. COS-7 cells transfected with 20 µg of the plasmid containing the Fuc-TIV cDNA in the pCDNA1 vector, gave evidence of weak but definite cell surface expression of Le^x, as measured by the antibody LeuM1, and of VIM2 measured with anti-CDw65, after growth for 48 h (Fig. 2). The question of whether mammalian cells transfected with Fuc-TIV express sialyl-Le^x has been controversial (27, 28, 29) but in the present experiments, after subtraction of the activity found for the untransfected COS-7 cells with the antibody CSLEX1, the transfected cells appeared to have very low and doubtful activity with this reagent. COS-7 cells transfected with 20 µg of the plasmid containing the Fuc-TVII cDNA in the pSI vector on the other hand, had weak but definite activity with the anti-sialyl-Le^x but no detectable activity with anti-Le^x or anti-CDw65 (Fig. 2). The results for the expression of Fuc-TVII in COS-7 cells are in agreement with those reported by Natsuka et al.(34) . Increasing the amount of either of the plasmids from 20 to 50 µg, or the time of growth from 48 to 72 h, did not increase the cell surface expression of these antigens. COS-7 cells transfected with a mixture of both Fuc-TIV and Fuc-TVII plasmids (20 µg of each) reacted with antibodies to both Le^x and sialyl-Le^x but the level of expression of Le^x and VIM2 was decreased in comparison with the level on the cells transfected with the Fuc-TIV plasmid alone and the level of expression of sialyl-Le^x was decreased in comparison with that found on the cells transfected with Fuc-TVII alone.


Figure 2: Flow cytometry analyses of COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNA. COS-7 cells were transfected with either plasmid pcDNA1-Fuc-TIV (FT4) or pSI-Fuc-TVII (FT7), or with a mixture of the two plasmids (FT4 + 7), and the transfected cells were grown for 48 h in standard medium and then harvested, washed, stained with monoclonal antibodies directed to carbohydrate epitopes and examined by indirect immunofluoresence as described under ``Experimental Procedures.'' The antibodies anti-LeuM1 (anti-Le^x), CSLEX1 (anti-sialyl-Le^x), and anti-CDw65 (anti-VIM2) were used at the concentration supplied. The data are presented as: a, the mean fluorescence intensity with the antibodies; and b, the percentage of cells giving positive reactions with the antibodies.



Specificity of alpha1,3-Fucosyltransferase in Untreated and Me(2)SO-differentiated HL-60 Cells

Extracts of HL-60 cells assayed with low molecular weight acceptors showed incorporation of fucose into both N-acetyllactosamine and 3`-sialyl-N-acetyllactosamine but, as had been shown previously(14) , the activity with the non-sialylated disaccharide was more than 10 times higher than the activity with the sialylated trisaccharide ( Table 1and Fig. 3). The glycoprotein acceptors fetuin(38) , alpha(1)-acid glycoprotein (40) , and Tamm-Horsfall glycoprotein(41, 42) , that contain complex N-linked chains many of which terminate with NeuAcalpha2-3Galbeta1-4GlcNAc sequences, were more effective acceptors after removal of sialic acid residues (Table 1). The glycoprotein, transferrin, which is known to contain N-linked chains terminating in alpha2,6-linked sialic acid residues (39) and the ovarian cyst glycoprotein which carries O-linked chains, also believed to be largely substituted with alpha2,6-linked sialic acid(56) , were also better acceptors after removal of sialic acid. The levels of incorporation into both N-acetyllactosamine and asialoglycoproteins were very much higher than the levels found in mature granulocytes but the levels of incorporation into the untreated glycoproteins were closely similar (Table 2).




Figure 3: Acceptor specificity of alpha1,3-fucosyltransferase(s) expressed in HL-60 cell extracts with low molecular weight oligosaccharide and glycoprotein acceptors before and after growth of the cells in the presence of Me(2)SO. HL-60 cells were grown in the presence of Me(2)SO (DMSO) for periods up to 4 days and extracts were prepared and assayed for alpha1,3-fucosyltransferase activity with: a, N-acetyllactosamine (NAL) and 3`-sialyl-N-acetyllactosamine (SNAL); and b, fetuin and asialofetuin as described under ``Experimental Procedures.'' The percentage incorporation of [^14C]fucose represents the mean values of three different experiments.





On growth of the HL-60 cells in the presence of Me(2)SO, the activity of alpha1,3-fucosyltransferase measured with either N-acetyllactosamine or asialofetuin sharply decreased; the levels were reduced by 40-50% after exposure to Me(2)SO for 48 h (Fig. 3). In contrast, the low level of activity measured with the sialylated oligosaccharide acceptor 3`-sialyl-N-acetyllactosamine remained largely unchanged on differentiation of the cells while incorporation into the glycoprotein acceptor fetuin showed a gradual increase (Fig. 3): after 4 days exposure the ratio of incorporation of fucose into asialofetuin and fetuin was changed from approximately 3:1 to 1:1.

The level of beta1,4-galactosyltransferase measured with N-acetylglucosamine as acceptor remained virtually unchanged in HL-60 cells grown for up to 4 days in the presence of Me(2)SO (data not shown), indicating that the changes observed in the alpha1,3-fucosyltransferase activity were not attributable to a general down-regulation of glycosyltransferase activity.

Specificity of alpha1,3-Fucosyltransferase(s) in Mature Granulocytes

Extracts of granulocytes from three unrelated healthy donors each transferred fucose to both sialylated and non-sialylated oligosaccharide and glycoprotein acceptors (Table 2). The extracts were free from alpha1,2-fucosyltransferase acting on phenyl beta-D-galactoside and alpha1,4-fucosyltransferase acting on Type I chain acceptors, lacto-N-biose 1 or lacto-N-tetraose. All three granulocyte extracts gave higher incorporation into untreated fetuin or Tamm-Horsfall glycoprotein (39) than into these glycoproteins after the sialic acid had been removed. The alpha1,3-fucosyltransferases in the granulocytes thus exhibit a preference for the sialylated Type 2 structures in the glycoproteins but also have the capacity to utilize unsialylated acceptors. The precursor blood group substance purified from ovarian cyst fluid, believed to have alpha2,6-linked sialic acid residues(56) , was a better acceptor after removal of sialic acid. The preference for the sialylated acceptors shown with the glycoprotein acceptors was not reflected in the results obtained with the low molecular weight oligosaccharide acceptors (Table 2). The unsubstituted Type 2 substrates, N-acetyllactosamine and lacto-N-neotetraose, were the best acceptors of those tested and 3`-sialyl-N-acetyllactosamine had only about one-third to one-half of the activity of the unsustituted di- or tetrasaccharide.

Specificity of Enzymes Expressed in COS-7 Cells Transfected with Fuc-TIV or Fuc-TVII cDNAs

COS-7 cells transfected with the plasmid pCDNA1-Fuc-TIV (27) expressed relatively high levels of alpha1,3-fucosyltransferase acting on N-acetyllactosamine and lacto-N-neotetraose and gave only low levels of incorporation into 3`-sialyl-N-acetyllactosamine (Table 3). With the glycoprotein acceptors fetuin, Tamm-Horsfall glycoprotein, and alpha(1)-acid glycoprotein, incorporation of [^14C]fucose was low compared to the amount transferred to the glycoproteins after removal of sialic acid (Table 3). These results are therefore in agreement with other observations (29, 30) showing that Fuc-TIV has a preference for unsialylated acceptors but do not rule out the possibility that this enzyme, when overexpressed, could synthesize sialyl-Le^x structures. Activity with N-acetyllactosamine, but not 3`-sialyl-N-acetyllactosamine, was detectable in the culture medium from the cells transfected with the Fuc-TIV plasmid but no activity was found in the medium from mock transfected COS-7 cells (data not shown).



Extracts of COS-7 cells transfected with Fuc-TVII cDNA in the vector pSI, under the same conditions used for transfection of the Fuc-TIV plasmid, transferred fucose to 3`-sialyl-N-acetyllactosamine and had virtually no activity with the unsubstituted Type 2 disaccharide or any of the other oligosaccharides tested (Table 3). Similarly, incorporation into fetuin and Tamm-Horsfall glycoprotein took place into the intact sialylated glycoproteins and negligible transfer took place to the asialoglycoproteins. These results are thus in agreement with the earlier reports(33, 34) , that, based on specificity studies with low molecular weight oligosaccharides and cell surface expression of sialyl-Le^x, suggested that the Fuc-TVII gene-encoded enzyme could act only on sialylated acceptor substrates. No activity with either N-acetyllactosamine or 3`-sialyl-N-acetyllactosamine was detectable in the culture media of the cells transfected with the Fuc-TVII plasmid.

Co-transfection of the Fuc-TIV and Fuc-TVII plasmids into COS-7 cells led to a decrease in activity of the cellular alpha1,3-fucosyltransferase(s) measured with the asialo-substrates, N-acetyllactosamine and asialofetuin, and an increase in the activity with the sialylated substrates, 3`-sialyl-N-acetyllactosamine and fetuin, in comparison with the activities found in the extracts of the cells transfected with Fuc-TIV alone. The levels of incorporation of [^14C]fucose into the low molecular weight, or glycoprotein, sialylated substrates did not change from those observed when the Fuc-TVII plasmid was transfected on its own (Table 3).

Effect of Sulfhydryl Reagents on alpha1,3-Fucosyltransferase Activity in HL-60 Cells, Granulocytes, and Transfected COS-7 Cells

NEM, p-Mercuribenzoate, and Iodoacetamide Treatment of Enzymes in Extracts of HL-60 Cells Before and After Me(2)SO-induced Differentiation

Susceptibility to inactivation by sulfhydryl reagents is a property that varies among different fucosyltransferases (57, 58) . The alpha1,3-fucosyltransferase in human plasma is rapidly inactivated by N-ethylmaleimide (57, 58) and tests with Bombay O(h) plasma (which contains alpha1,3-fucosyltransferase free from alpha1,2-fucosyltransferase present in normal ABO blood groups (59) ), confirmed that addition of NEM to give a final concentration of 0.1 mM completely abolished transfer of fucose to N-acetyllactosamine (Fig. 4). In comparison, the alpha1,3-fucosyltransferases in HL-60 cells were resistant to treatment with NEM both before and after Me(2)SO-induced differentiation. The low molecular weight acceptors N-acetyllactosamine and 3`-sialyl-N-acetyllactosamine gave similar inactivation patterns with some 80% of the original activity remaining in the presence of 1 mM NEM and some 60-70% activity still detectable with 10 mM NEM. However, the profiles obtained with the glycoprotein acceptors differed according to whether or not the sialic acid had been removed. Activity with fetuin was gradually reduced and only about half remained after treatment of extracts of either the differentiated or undifferentiated cells with 10 mM NEM. In contrast, with asialofetuin as acceptor a distinct increase in alpha1,3-fucosyltransferase activity was observed for the enzyme in both differentiated and undifferentiated HL-60 cells on addition to the reaction mixtures of 1 mM NEM and full activity was displayed in the presence of 10 mM NEM (Fig. 4). The increase in activity with the asialoglycoprotein acceptor was found irrespective of whether the NEM was added directly to the fucosyltransferase assay mixtures or whether the cell extracts were first preincubated at 37 °C with the reagent. Similarly, activation was observed when the cell extracts were preincubated with the NEM at 37 °C and then treated with dithiothreitol to destroy excess NEM before incubation with the other components of the reaction mixture. The increase in activity therefore appears to result from the action of the sulfhydryl reagent on the alpha1,3-fucosyltransferase protein and not from its action on the glycoprotein substrate. Activation of a similar order to that found with asialofetuin was observed when asialo-alpha(1)-acid glycoprotein and asialo-ovarian cyst glycoprotein were tested as substrates for the alpha1,3-fucosyltransferase(s) in HL-60 cells (data not shown).


Figure 4: The effect of NEM on the activity of alpha1,3-fucosyltransferases expressed in HL-60 cells before and after Me(2)SO-induced differentiation, in COS-7 cells transfected with Fuc-TIV and FUC-TVII plasmids, in mature granulocytes, and in plasma. Different concentrations of NEM were added to the cell extracts and the mixtures were assayed for alpha1,3-fucosyltransferase activity with either asialofetuin (circle) or fetuin (bullet) as acceptors as described under ``Experimental Procedures.'' a, untreated HL-60 cells. b, HL-60 cells after 4 days exposure to Me(2)SO (DMSO). c, COS-7 cells transfected with a plasmid containing Fuc-TIV cDNA. d, COS-7 cells transfected with a plasmid containing Fuc-TVII cDNA. e, mature human granulocytes. f, human plasma from a blood group Bombay O(h) donor.



A similar pattern of activation of the alpha1,3-fucosyltransferase(s) transferring fucose to asialofetuin (130% increase), and inhibition of the transfer to fetuin (60% decrease) was found when extracts of HL-60 cells were treated with 0.1 mMp-mercuribenzoate, although with this reagent all activity was abolished with both substrates at 10 mM concentration. In contrast, iodoacetamide, at concentrations up to 10 mM induced no detectable change in the activity of the alpha1,3-fucosyltransferase(s) in HL-60 cells toward asialofetuin and the activity toward fetuin was reduced by only 30%.

The Effect of NEM on the alpha1,3-Fucosyltransferase(s) in Granulocyte Extracts

The alpha1,3-fucosyltransferase(s) in mature granulocytes acting on asialofetuin and fetuin was also resistant to inactivation by NEM (Fig. 4). The activity with both asialofetuin and fetuin showed a slight increase with 1 mM NEM and only minimal inactivation was apparent with 10 mM NEM (Fig. 4).

Treatment of Extracts of COS-7 Cells Transfected with Fuc-TIV and Fuc-TVII cDNAs with NEM

The activity profiles with the glycoprotein acceptors after treatment with NEM of detergent extracts of COS-7 cells transfected with the plasmid containing Fuc-TIV cDNA resembled those obtained with HL-60 cells (Fig. 4). The incorporation of fucose into asialofetuin was increased with 1 mM NEM and 100% of the original activity remained after exposure to 10 mM NEM, whereas with fetuin as acceptor increasing concentrations of NEM led to a gradual decrease of alpha1,3-fucosyltransferase activity with about 50% remaining after exposure to 10 mM NEM.

COS-7 cells transfected with Fuc-TVII did not show any alpha1,3-fucosyltransferase activity with asialofetuin but the activity in the cell extracts toward fetuin was resistant to NEM under the conditions tested, with virtually 100% activity remaining on exposure to 10 mM NEM (Fig. 4).

Effect of Heating on alpha1,3-Fucosyltransferase Activity in Extracts of HL-60 Cells, Granulocytes, and Transfected COS-7 Cells

Heat Treatment of Extracts of HL-60 Cells Before and After Differentiation

Susceptibility to heat is another property that varies among the different alpha1,3-fucosyltransferases. In agreement with the observations of others(58) , the alpha1,3-fucosyltransferase in plasma was found to be rapidly inactivated by heating at 50 °C (Fig. 5). In comparison the enzymes in extracts of HL-60 cells were more resistant to inactivation at this temperature but differences were observed in the activity of the heated extracts toward intact and asialoglycoprotein acceptors (Fig. 5). About 50% of the activity with fetuin disappeared in 7 min, whereas 30 min were required for the same enzyme preparation to show a similar degree of inactivation with asialofetuin. After Me(2)SO-induced differentiation the activity profiles of the heated enzyme preparation for the glycoprotein acceptors were essentially similar although inactivation appeared to proceed at a slightly faster rate (Fig. 5).


Figure 5: The effect of heat on the activity of alpha1,3-fucosyltransferases expressed in HL-60 cells before and after Me(2)SO-induced differentiation, in COS-7 cells transfected with Fuc-TIV and FUC-TVII plasmids, in mature granulocytes, and in plasma. Cell extracts were heated at 50 °C for various lengths of time and assayed for alpha1,3-fucosyltransferase activity with either fetuin (bullet) or asialofetuin (circle) as acceptors as described under ``Experimental Procedures.'' a, untreated HL-60 cells. b, HL-60 cells after 4 days exposure to Me(2)SO (DMSO). c, COS-7 cells transfected with a plasmid containing Fuc-TIV cDNA. d, COS-7 cells transfected with a plasmid containing Fuc-TVII cDNA. e, mature human granulocytes. f, human plasma from a blood group Bombay O(h) donor.



The difference in heat inactivation of the enzyme measured with the sialylated and non-sialylated glycoprotein acceptors was not found with the low molecular weight substrates. Inactivation proceeded at much the same rate with 50% of the activity toward both N-acetyllactosamine and 3-sialyl-N-acetyllactosamine being destroyed in 50 min.

Heat Treatment of Extracts of Mature Granulocytes

The alpha1,3-fucosyltransferase activity toward asialofetuin and fetuin in Triton X-100 extracts of mature granulocytes showed a similar pattern to the enzyme(s) in extracts of HL-60 cells inasmuch as the activity with asialofetuin was more heat resistant than the activity with fetuin. However, with both substrates less inactivation of the enzyme(s) occurred than was observed for the HL-60 cell extracts (Fig. 5) with 100% of the original activity with asialofetuin and 50% of the activity with fetuin remaining after 30 min heating at 50 °C.

Heat Treatment of Extracts of COS-7 Cells Transfected with Fuc-TIV and Fuc-TVII Plasmids

Extracts of COS-7 cells transfected with the plasmid containing Fuc-TIV cDNA gave inactivation profiles of alpha1,3-fucosyltransferase activity with asialofetuin and fetuin on heating at 50 °C for varying lengths of time which closely resembled the profiles obtained for the enzyme in HL-60 cells both before and after Me(2)SO differentiation.

The susceptibility to heat of the alpha1,3-fucosyltransferase acting on fetuin in COS-7 cells transfected with the plasmid containing Fuc-TVII cDNA was very similar to that of the enzyme in cells transfected with the plasmid containing Fuc-TIV (Fig. 5) and only about 20% of the original activity remained after 30 min at 50 °C.

Expression Levels of Five Cloned alpha1,3-Fucosyltransferases in HL-60 Cells Before and After Me(2)SO-induced Differentiation

In agreement with the finding of others(27, 28, 29) , Northern blot analyses of 15 µg of mRNA from untreated HL-60 cells with a P-labeled Fuc-TIV cDNA probe revealed three major transcripts, 2.3, 3.0, and 6.0 kb (Fig. 6), with the 6.0-kb band appearing as a doublet when smaller quantities of mRNA were examined. Me(2)SO-induced differentiation of the HL-60 cells brought about a sharp fall in the level of expression of the Fuc-TIV transcripts after exposure to Me(2)SO for 24 and 48 h (Fig. 6), but all three major transcripts continued to be manifested. Growth of the cells in the presence of Me(2)SO for 3 more days led to a further slight decrease in the level of response of the mRNA to the Fuc-TIV probe but all three transcripts were still discernible when the same amount of mRNA was analyzed (data not shown). The largest change in the level of expression of Fuc-TIV in the HL-60 cells was therefore the fall that occurred in the first 24 h after exposure to Me(2)SO. Northern blot analysis of 30 µg of HL-60 mRNA with P-labeled cDNA probes for Fuc-TIII, Fuc-TV, or Fuc-TVI revealed only very faint transcripts of these genes in the mRNA from the undifferentiated HL-60 cells and the signal strengths did not increase in mRNA from the cells treated for 96 h with Me(2)SO. mRNA from A431 cells, included as a control in these experiments, gave well defined 2.3-kb transcripts with each of the Fuc-TIII, Fuc-TV, and Fuc-TVI cDNA probes (Fig. 7).


Figure 6: Expression of Fuc-TIV transcripts in mRNA from HL-60 cells, before and after growth in the presence of Me(2)SO. Poly(A) RNA (15 µg) prepared from untreated HL-60 cells and cells grown in 1.3% Me(2)SO (DMSO) were electrophoresed, transferred to a membrane, and hybridized with a P-labeled Fuc-TIV probe as described under ``Experimental Procedures.'' The Fuc-TIV probe contained 4.1 times 10^8 cpm/µg DNA. A P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (4.7 times 10^8 cpm/µg DNA) was used to probe 4.5 µg of the same poly(A) RNA preparation. The blots were exposed to Kodak X-Omat film with an intensifying screen for 24 h at -70 °C. The figure shows transcripts in mRNA isolated from untreated HL-60 cells and cells cultured for 24 and 48 h in the presence of Me(2)SO.




Figure 7: Northern blot analyses of mRNA from HL-60 cells, before and after exposure to Me(2)SO, and from A431 cells, probed with P-labeled Fuc-TIII, Fuc-TV, and Fuc-TVI cDNAs. Poly(A) RNA aliquots (30 µg) prepared from untreated HL-60 cells and HL-60 cells grown for 96 h in the presence of 1.3% Me(2)SO were electrophoresed, transferred to a membrane, and hybridized with the P-labeled probes as described under ``Experimental Procedures.'' The Fuc-TIII, Fuc-TV, and Fuc-TVI probes contained, respectively, 2.4 times 10^8 cpm/µg DNA, 1.2 times 10^8 cpm/µg DNA, and 1.6 times 10^8 cpm/µg DNA. mRNA isolated from A431 cells (10 µg) was hybridized with the same three probes and a P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (4.8 times 10^8 cpm/µg DNA) was hybridized with 5 µg mRNA isolated from A431 cells and from HL-60 cells before and after growth in the presence of Me(2)SO. The membranes were exposed to X-Omat film for 72 h at -70 °C with an intensifying screen. First lane, mRNA from untreated HL-60 cells, second lane, mRNA from HL-60 cells treated for 96 h with Me(2)SO, third lane, mRNA from A431 cells.



Examination of mRNA from undifferentiated HL-60 cells in Northern blot analyses with the P-labeled Fuc-TVII cDNA probe revealed three transcripts at 2.0, 2.3, and 3.0 kb (Fig. 8) as reported by Natsuka et al.(34) . In contrast to the results observed with the Fuc-TIV probe there was no dramatic change in the level of expression of the Fuc-TVII mRNA transcripts after Me(2)SO-induced differentiation and although there was some reduction in the levels of the 2.3- and 3.0-kb bands, the major 2.0-kb band was strongly expressed after 4 days exposure to Me(2)SO.


Figure 8: Expression of Fuc-TVII transcripts in HL-60 cells before and after growth in the presence of Me(2)SO. Poly(A) RNA aliquots (15 µg) prepared from untreated HL-60 cells before and after growth in 1.3% Me(2)SO (DMSO) for 24 and 96 h were electrophoresed, transferred to a membrane, and hybridized to the P-labeled Fuc-TVII probe (5.6 times 10^8 cpm/µg DNA) as described under ``Experimental Procedures.'' The blots were exposed to X-Omat film with an intensifying screen for 72 h at -70 °C. The same mRNA was subsequently probed with a P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (1.9 times 10^8 cpm/µg DNA) and exposed to X-Omat film without an intensifying screen for 24 h at -70 °C.



Mature Granulocytes

Northern blot analysis of 22 µg of total RNA from mature granulocytes isolated from peripheral blood revealed only very weak signals with the P-labeled Fuc-TIV cDNA probe, although the three transcripts, 2.3, 3.0, and 6.0 kb were all faintly discernable (Fig. 9). The signals were, however, considerably weaker than those seen with a corresponding amount of total RNA from undifferentiated HL-60 cells. In contrast, the same blot hybridized with the P-labeled Fuc-TVII cDNA probe gave two distinct transcripts at 1.8 and 2.3 kb that were more strongly expressed than the same bands in the RNA from undifferentiated HL-60 cells. In both samples the fastest band (1.8 kb) was in a slightly different position from the 2.0-kb transcript seen in the mRNA from the HL-60 cells (Fig. 8) but this displacement was probably attributable to the ribosomal RNA present in the total RNA preparations. No transcripts were detectable when the same blot was tested with the P-labeled Fuc-TIII, Fuc-TV, or Fuc-TVI cDNA probes.


Figure 9: Expression of Fuc-TIV and Fuc-TVII transcripts in total RNA from mature granulocytes and HL-60 cells. Total RNA (22 µg) from untreated HL-60 cells and mature human granulocytes isolated from peripheral blood were electrophoresed, transferred to a membrane, and hybridized to a P-labeled Fuc-TIV probe (1.2 times 10^8 cpm/µg DNA) as described under ``Experimental Procedures.'' The blot was exposed to X-Omat film with an intensifying screen at -70 °C for 17 days. The same blot was subsequently reprobed, first with a P-labeled Fuc-TVII probe (5.6 times 10^8 cpm/µg DNA) and exposed to X-Omat film -70 °C for 11 days and second with a P-labeled glucose-6-phosphate dehydrogenase (G6PD) cDNA (4.8 times 10^8 cpm/µg DNA) and exposed to X-Omat film -70 °C for 3 days. First lane with each probe, total RNA from mature granulocytes; second lane, total RNA from untreated HL-60 cells.




DISCUSSION

The carbohydrate determinant, sialyl-Le^x, is well expressed on the surface of mature granulocytes and is believed to be a major ligand recognized by the adhesion molecules, E- and P-selectin, which are involved in the inflammatory response(60, 61, 62) . The fact that this antigen is expressed on myeloid progenitor cells but is down-modulated during the intermediate stages of granulocyte maturation (11) suggests that it may also have a function in specific recognition mechanisms in the course of myeloid development in the bone marrow. The human leukemic cell line, HL-60, contains cells arrested at the promyelocytic stage(17) , that is, cells which in normal maturation along the granulocytic pathway would undergo further differentiation through myelocyte, metamyelocyte, band, and polymorphonuclear stages before leaving the bone marrow as mature granulocytes(11) . Sialyl-Le^x and Le^x are carried as terminal sequences on oligosaccharide chains in both glycoproteins and glycolipids on the surface of myeloid cell lines and on mature granulocytes(47, 63, 64, 65) . Le^x determinants are strongly expressed on HL-60 cells but expression of sialyl-Le^x is very weak(14) . Both the glycoprotein and glycolipid profiles on the surface of HL-60 cells undergo changes when the cells are induced to differentiate(18, 19, 20, 65) ; the process of maturation from promyelocyte to granulocyte is accompanied by a great increase in the quantity and the degree of branching of the polylactosamine structures of the sialoglycoprotein, leukosialin(19, 65) , and in the amounts of sialic acid containing glycosphingolipids(20, 21) .

At the stage where the strongly active alpha1,3-fucosyltransferase, with a marked preference for the non-sialylated Type 2 acceptors (Table 1), is expressed in the undifferentiated HL-60 cells, transcripts of the Fuc-TIV gene are also readily detectable in mRNA from these cells (Fig. 6). Transfection of COS-7 cells with a plasmid containing Fuc-TIV cDNA gives rise to an alpha1,3-fucosyltransferase with specificity properties (Table 3) and responses to heat and sulfhydryl reagents that are very similar to those found for HL-60 cells ( Fig. 4and Fig. 5). The differences in the heat and NEM inactivation profiles of the alpha1,3-fucosyltransferase(s) in HL-60 cells when measured with the intact glycoproteins or the asialoglycoproteins were also found for the enzyme in COS-7 cells transfected with the plasmid containing Fuc-TIV cDNA; thus these differences do not, as we at first thought, arise from the presence of two distinct enzymes. Structural changes accompanying the cleavage of an -S-S- bond has been suggested as a cause of activation of an enzyme by sulfhydryl reagents (66, 67) and it could be postulated that a conformational change in the enzyme molecule induced by heat or by the cleavage of -S-S- bonds might enable the alpha1,3-fucosyltransferase to combine more readily with the unsubstituted Type 2 chain ending in a glycoprotein but be insufficient to enable increased binding to the bulkier sialylated-Type 2 structure.

Flow cytometric analysis of the HL-60 cells (Fig. 1) showed major reactivity with antibodies directed toward Le^x and VIM2 determinants and a similar pattern was exhibited by the COS-7 cells transfected with the plasmid containing Fuc-TIV cDNA (Fig. 2). The expression of antigens on the cell surface, together with evidence from enzyme specificity studies and susceptibility to inactivation by heat or sulfhydryl compounds is therefore consistent with the interpretation that Fuc-TIV is the major gene encoding the alpha1,3-fucosyltransferase expressed in the myeloid cells at the promyelocyte stage.

In an attempt to see whether the increased expression of sialyl-Le^x on differentiation of HL-60 cells, and the switch in specificity of the alpha1,3-fucosyltransferase with regard to sialylated acceptors, could be correlated with loss of expression of one fucosyltransferase gene and onset of transcription of one or more of the other cloned genes, we compared mRNA from differentiated and undifferentiated cells in Northern blot analysis with P-labeled probes for the five cloned alpha1,3-fucosyltransferase genes. On Me(2)SO-induced differentiation of the HL-60 cells a dramatic change occurs in the level of expression of mRNA corresponding to Fuc-TIV (Fig. 6) and this drop is concomitant with a fall in the level of alpha1,3-fucosyltransferase activity, and a loss in the preference of the enzyme(s) for non-sialylated acceptors ( Table 1and Fig. 3). At the same time there is a rise in the cell surface expression of sialyl-Le^x and a fall in the expression of the VIM-2 epitope. No significant expression of Fuc-TIII, Fuc-TV, or Fuc-TVI mRNA transcripts could be detected by Northern blot analyses of mRNA from undifferentiated HL-60 cells and none of the signals were intensified in mRNA from differentiated cells (Fig. 7). Transcripts corresponding to the Fuc-TVII gene were, however, well expressed in mRNA from the untreated HL-60 cells (Fig. 8) and the major transcript (2.0 kb) continued to be strongly expressed on Me(2)SO-induced differentiation of the cells (Fig. 8). As had been reported by others (34) transfection of COS-7 cells with a plasmid containing Fuc-TVII cDNA led to the expression of sialyl-Le^x and to a cellular alpha1,3-fucosyltransferase that reacted only with sialylated acceptors. The increase in expression of sialyl-Le^x did not therefore appear to correspond to a newly switched on gene but to a change in the balance of enzymes that allowed expression of the alpha1,3-fucosyltransferase encoded by the Fuc-TVII gene. This change could be associated with the sharp fall in the level of expression of Fuc-TIV which would decrease competition for the Type 2 acceptor substrate and therefore allow more alpha2,3-sialylated precursor structures to be synthesized as substrate for the product of the Fuc-TVII gene. A decrease in the expression of the strongly expressed alpha2,6-sialyltransferase which had been shown to occur on differentiation of HL-60 cells(14) , would also remove a competitor for the Type 2 acceptor substrate and thus act as a regulator for the appearance of sialyl-Le^x. At the same time the increase in polylactosamine branching structures that occurs as differentiation proceeds (64, 65) would provide further substrates for all the competing enzymes. The reactivity of the differentiated cells with antibody to the CD11b marker, but failure to express the CD16 antigen, indicated that the majority of the cells had matured to the myelocyte stage but not further along the granulocytic pathway; hence the decrease in reactivity with the antibody detecting the VIM2 epitope (Fig. 1) may indicate a specific down-regulation of cell surface expression of this antigen at the myelocyte stage concomitant with the drop in expression of the Fuc-TIV gene-encoded alpha1,3-fucosyltransferase.

Co-transfection of COS-7 cells with both Fuc-TIV and Fuc-TVII plasmids led to changes in the levels of cell surface expression of Le^x and sialyl-Le^x in comparison with the levels expressed when the cells were transfected with either of the plasmids singly (Fig. 2) and the activity of the cellular alpha1,3-fucosyltransferases with sialylated or non-sialylated acceptors was modified. In these experiments the enzyme encoded by the Fuc-TVII gene appeared to use up acceptor at the expense of the enzyme encoded by the Fuc-TIV gene since the levels of activity with the non-sialylated acceptors were the ones that decreased when both enzymes were present (Table 3). These experiments cannot be strictly compared with what is happening on differentiation of HL-60 cells because it is unknown how much of each cDNA is transfected, and also the nature and amount of the precursor acceptor molecules in the COS-7 cells probably differ from those in the myeloid cells and the number and activity of other competing enzymes, such as the sialyltransferases, may also be different. Moreover, the COS-7 cells are not undergoing the continuum of change in precursor structures and enzyme levels that are taking place in the maturing HL-60 cells. The results do, however, demonstrate the changes in cell surface structures that can occur when there is competition between two fucosyltransferases for the available substrate.

Examination of total RNA from mature granulocytes in Northern blot analyses with probes for Fuc-TIV and Fuc-TVII indicated very weak expression of Fuc-TIV transcripts and much more definite expression of Fuc-TVII transcripts (Fig. 9). The cell surface expression of sialyl-Le^x on the granulocytes was considerably stronger than on the differentiated HL-60 cells, which is in agreement the interpretation that the Fuc-TVII-encoded alpha1,3-fucosyltransferase is responsible for the appearance of sialyl-Le^x on the mature cells. Little is yet known about the rate of turnover of the cell surface carbohydrate antigens, the half-life of the alpha1,3-fucosyltransferase enzyme proteins, or their mRNA transcripts. However, the persistence of Le^x expression at about the same level as on the HL-60 cells and the continued activity of the cellular enzyme(s) with both sialylated and non-sialylated acceptors (Table 2) indicates that Fuc-TVII is not the only alpha1,3-fucosyltransferase gene that is being expressed in the granulocytes. The ratios of enyzme activity with sialylated and non-sialylated acceptors found for the alpha1,3-fucosyltransferases in granulocytes, in HL-60 cells, before and after differentiation, and in COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNAs, are summarized in Table 4. The ratios found with both the low molecular weight and glycoprotein acceptors in the granulocyte extracts resemble most closely the ratios found in the differentiated HL-60 cells. In these cells the ratios could be assumed to arise from a mixture of the enzymes encoded by the Fuc-TIV and Fuc-TVII genes, with a preponderance still at that stage of the Fuc-TIV-encoded enzyme. However, the alpha1,3-fucosyltransferase in the granulocytes acting on asialofetuin appears to be more resistant to heat than the enzyme in the COS-7 cells transfected with the Fuc-TIV plasmid and the enzyme acting on fetuin to be more resistant than the alpha1,3-fucosyltransferase utilizing this substrate in the extracts of the cells transfected with the Fuc-TVII plasmid (Fig. 5). The possibility that these differences might result from the protective effect of other proteins in the crude extracts cannot be excluded but the apparent anomalies raise the question as to whether the residual expression of the Fuc-TIV gene, together with the Fuc-TVII gene, can account for the specificity and properties of the alpha1,3-fucosyltransferases expressed in mature granulocytes or whether yet another myeloid alpha1,3-fucosyltransferase gene remains to be identified.




FOOTNOTES

*
This work was supported by a grant from the Wellcome Trust, U.K. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Haematology Dept., Royal Postgraduate Medical School, Hammersmith Hospital, London, W12 ONN, United Kingdom. Tel.: 44-181-743-2030 (ext. 2171); Fax: 44-181-742-9335.

(^1)
The abbreviations used are: alpha1,3-fucosyltransferase, GDP-L-fucose:N-acetyl-D-glucosamide 3-alpha-L-fucosyltransferase; Galbeta1-3GlcNAc, lacto-N-biose I; Le^x, Galbeta1-4[Fucalpha1-3]GlcNAc; sialyl-Le^x, NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc; sialyl-dimeric-Le^x, NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc1-3Galbeta1-4[Fucalpha1-3]GlcNAc; VIM-2, NeuAcalpha2- 3Galbeta1-4GlcNAcbeta1-3Galbeta1-4[Fucalpha1-3]GlcNAc; N-acetyllactosamine, Galbeta1-4GlcNAc; lactose, Galbeta1-4Glc; 2`-fucosyllactose, Fucalpha1-2Galbeta1-4GlcNAc; 3`-sialyllactose, NeuAcalpha2-3Galbeta1-4Glc; 6`-sialyllactose, NeuAcalpha2-6Galbeta1-4Glc; lacto-N-tetraose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc; lacto-N-neotetraose, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc; lacto-N-fucopentaose I, Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc; lacto-N-fucopentaose II, Galbeta1-3[Fucalpha1-4]GlcNAcbeta1-3Galbeta1-4Glc; lacto-N-fucopentaose III, Galbeta1-4[Fucalpha1-3]GlcNAcbeta1-3Galbeta1-4Glc; 3`-sialyl-N-acetyllactosamine, NeuAcalpha2-3Galbeta1-4GlcNAc; 6`-sialyl-N-acetyllactosamine, NeuAcalpha2-6Galbeta1-4GlcNAc; alpha1,2-fucosyltransferase, GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase; alpha1,4-fucosyltransferase, GDP-L-fucose:N-acetyl-D-glucosamide 4-alpha-L-fucosyltransferase; beta1,4-galactosyltransferase, UDP-D-galactose:beta-N-acetylglucosaminide 4-beta-D-galactosyltransferase; kb, kilobase; Me(2)SO, dimethyl sulfoxide; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter.


ACKNOWLEDGEMENTS

We are grateful to Dr. J. B. Lowe for supplying the fucosyltransferase cDNA probes, to Drs. S. Hakomori and P. Teraskai for the gifts of monoclonal antibodies, and Professor W. T. J. Morgan and the late Dr. A. S. R. Donald for providing substrates. We thank Drs. T. Vulliamy and C. Corcoran for advice on DNA and RNA isolation procedures, Dr. P. J. Mason for help with the transfection experiments, and Dr. Andrea Gordon for guidance with the FACS analyses.


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