CD15 Expression in Mature Granulocytes Is Determined by α1,3-Fucosyltransferase IX, but in Promyelocytes and Monocytes by α1,3-Fucosyltransferase IV

The CD15 carbohydrate epitope is expressed in mature human neutrophils, monocytes, and promyelocytes. We aimed to determine the α1,3-fucosyltransferase responsible for the expression of CD15 in each subpopulation of leukocytes. Three α1,3-fucosyltransferases, FUT4, FUT7, and FUT9, are expressed in human leukocytes. We demonstrated that FUT9 exhibits 20-fold stronger activity for CD15 synthesis than FUT4, whereas FUT4 exhibits 4.5-fold stronger activity for CDw65 synthesis than FUT9. By competitive reverse transcriptase-polymerase chain reaction, FUT9 was found to be strongly expressed in mature granulocytes and peripheral blood mononuclear cell, but not in monocytes. CD34+ and CD15+cells in cord blood and myeloid cell lines (HL-60 and U937) did not express FUT9 at all. FUT4 transcripts were ubiquitously expressed in all blood cells and all cultured cell lines, with HL-60 and U937 cells in particular expressing a number of FUT4 transcripts. Transfection of the FUT9 gene into Jurkat and U937 cells demonstrated that FUT9 has the potential to express CD15 in myeloid and lymphoid cells. These findings suggest that the expression of CD15 in mature granulocytes is directed by FUT9, whereas it is determined in promyelocytes and monocytes by FUT4. Measurement of CD15 synthesizing activity in cell homogenates of each cell population using the polylactosamine acceptor further supported these conclusions. The CD15 carbohydrate epitope is expressed in mature human neutrophils, monocytes, and promyelocytes. We aimed to determine the α1,3-fucosyltransferase responsible for the expression of CD15 in each subpopulation of leukocytes. Three α1,3-fucosyltransferases, FUT4, FUT7, and FUT9, are expressed in human leukocytes. We demonstrated that FUT9 exhibits 20-fold stronger activity for CD15 synthesis than FUT4, whereas FUT4 exhibits 4.5-fold stronger activity for CDw65 synthesis than FUT9. By competitive reverse transcriptase-polymerase chain reaction, FUT9 was found to be strongly expressed in mature granulocytes and peripheral blood mononuclear cell, but not in monocytes. CD34+ and CD15+cells in cord blood and myeloid cell lines (HL-60 and U937) did not express FUT9 at all. FUT4 transcripts were ubiquitously expressed in all blood cells and all cultured cell lines, with HL-60 and U937 cells in particular expressing a number of FUT4 transcripts. Transfection of the FUT9 gene into Jurkat and U937 cells demonstrated that FUT9 has the potential to express CD15 in myeloid and lymphoid cells. These findings suggest that the expression of CD15 in mature granulocytes is directed by FUT9, whereas it is determined in promyelocytes and monocytes by FUT4. Measurement of CD15 synthesizing activity in cell homogenates of each cell population using the polylactosamine acceptor further supported these conclusions. cluster of differentiation fucosyltransferase lactosamine galactose N-acetylglucosamine fucose N-acetylneuraminic acid Lewis x sialyl Lewis x 2-aminobenzamide all-trans-retinoic acid peripheral blood mononuclear cells high performance liquid chromatography monoclonal antibody reverse transcriptase-polymerase chain reaction There are three CD1markers of human leukocytes comprising fucosylated carbohydrate epitopes. As listed in Fig. 1 below, the distal lactosamine unit (LN; type 2 chain), Galβ1,4GlcNAc, of the polylactosamine chain is fucosylated through α1,3-fucosyltransferase (α1,3FUT) activity to form the CD15 (Lewis x; LeX) epitope (1Lowe J.B. Kukowska Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 2Niemela R. Natunen J. Majuri M.L. Maaheimo H. Helin J. Lowe J.B. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The CD15s (sialylated CD15; sialyl LeX (sLeX)) and CDw65 (VIM-2) epitopes are also fucosylated structures related to CD15,i.e. CD15s is formed by α2,3-sialylation prior to the fucosylation of the distal LN unit of polylactosamine by α1,3FUT, and CDw65 is formed by fucosylation of the inner LN unit of α2,3-sialylated polylactosamine by α1,3FUT (2Niemela R. Natunen J. Majuri M.L. Maaheimo H. Helin J. Lowe J.B. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 3Kono M. Ohyama Y. Lee Y.C. Hamamoto T. Kojima N. Tsuji S. Glycobiology. 1997; 7: 469-479Crossref PubMed Scopus (133) Google Scholar).The CD15 epitope is expressed in some tissues, such as epithelial cells of intestinal tissues (4Hakomori S. Nudelman E. Levery S.B. Kannagi R. 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Immunol. Immunopathol. 1982; 23: 172-188Crossref PubMed Scopus (169) Google Scholar, 11Civin C.I. Mirro J. Banquerigo M.L. Blood. 1981; 57: 842-845Crossref PubMed Google Scholar). CD15 is considered to be involved in neutrophil functions, that is, cell-cell interactions, phagocytosis, stimulation of degranulation, and respiratory burst, although the function of CD15 is not clear (12Melnick D.A. Nauseef W.M. Markowitz S.D. Gardner J.P. Malech H.L. J. Immunol. 1985; 134: 3346-3355PubMed Google Scholar, 13Melnick D.A. Meshulam T. Manto A. Malech H.L. Blood. 1986; 67: 1388-1394Crossref PubMed Google Scholar, 14Skubitz K.M. Snook R.W. J. Immunol. 1987; 139: 1631-1639PubMed Google Scholar, 15Forsyth K.D. Simpson A.C. Levinsky R.J. Eur. J. Immunol. 1989; 19: 1331-1334Crossref PubMed Scopus (20) Google Scholar, 16Warren H.S. Altin J.G. Waldron J.C. Kinnear B.F. Parish C.R. J. Immunol. 1996; 156: 2866-2873PubMed Google Scholar).Six human α1,3FUT genes have been cloned to date, which are FUT3 (Fuc-TIII), FUT4(Fuc-TIV), FUT5 (Fuc-TV),FUT6 (Fuc-TVI), FUT7(Fuc-TVII), and FUT9 (Fuc-TIX) (1Lowe J.B. Kukowska Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar,17Kukowska Latallo J.F. Larsen R.D. Nair R.P. Lowe J.B. Genes Dev. 1990; 4: 1288-1303Crossref PubMed Scopus (469) Google Scholar, 18Weston B.W. Nair R.P. Larsen R.D. Lowe J.B. J. Biol. Chem. 1992; 267: 4152-4160Abstract Full Text PDF PubMed Google Scholar, 19Weston B.W. Smith P.L. Kelly R.J. Lowe J.B. J. Biol. Chem. 1992; 267: 24575-24584Abstract Full Text PDF PubMed Google Scholar, 20Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 21Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 22Kudo T. Ikehara Y. Togayachi A. Kaneko M. Hiraga T. Sasaki K. Narimatsu H. J. Biol. Chem. 1998; 273: 26729-26738Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar). FUT9, a new member of the human α1,3FUT family, which we have recently cloned, is expressed in human leukocytes, glandular compartments of the stomach, and forebrain (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar). The FUT9gene was mapped on chromosome 6q16 (24Kaneko M. Kudo T. Iwasaki H. Shiina T. Inoko H. Kozaki T. Saitou N. Narimatsu H. Cytogenet. Cell Genet. 1999; 86: 329-330Crossref PubMed Google Scholar). Interestingly, only FUT9 has a highly conserved amino acid sequence between human and mouse, the level of conservation being equal to that of α-actin (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar). Five human α1,3FUTs (FUT3, 4, 5, 6, and 7) share highly homologous sequences, whereas FUT9 has a different sequence altogether (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar). This indicated that the substrate specificity of FUT9 is unique among the α1,3FUTs. In fact, we demonstrated in a previous study that FUT9 preferentially transfers a fucose to the GlcNAc residue at the distal LN unit of the polylactosamine chain, resulting in the LeX (CD15) structure, whereas the other α1,3FUTs preferentially transfer a fucose to the GlcNAc residue at the inner LN unit of the polylactosamine chain (25Nishihara S. Iwasaki H. Kaneko M. Tawada A. Ito M. Narimatsu H. FEBS Lett. 1999; 462: 289-294Crossref PubMed Scopus (77) Google Scholar). This implied that FUT9 exhibits stronger activity than the other α1,3FUTs for forming the CD15 epitope that is recognized by anti-LeX (anti-CD15) antibodies.It has been reported that FUT4 and FUT7 are expressed in human leukocytes, but FUT3, 5 and 6 are not (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar, 26Clarke J.L. Watkins W.M. J. Biol. Chem. 1996; 271: 10317-10328Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 27Marer N.L. Clarke J.L. Palcic M.M. Davies D. Skacel P.O. Glycobiology. 1997; 7: 357-365Crossref PubMed Scopus (20) Google Scholar). The carbohydrates modified by both FUT4 and FUT7 can function as ligands for E-selectin and P-selectin (28Weninger W. Ulfman L.H. Cheng G. Souchkova N. Quackenbush E.J. Lowe J.B. von Andrian U.H. Immunity. 2000; 12: 665-676Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 29Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). FUT4, named a myeloid-type α1,3FUT, is ubiquitously expressed in a variety of tissues and not only in leukocytes (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar). In a previous study (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar), we could not find any human tissues in which FUT4 is absent. However, a number of the tissues do not necessarily express the CD15 antigen. Overexpression of theFUT4 gene by its transfection resulted in the expression of CD15 on the cell surface (1Lowe J.B. Kukowska Latallo J.F. Nair R.P. Larsen R.D. Marks R.M. Macher B.A. Kelly R.J. Ernst L.K. J. Biol. Chem. 1991; 266: 17467-17477Abstract Full Text PDF PubMed Google Scholar, 22Kudo T. Ikehara Y. Togayachi A. Kaneko M. Hiraga T. Sasaki K. Narimatsu H. J. Biol. Chem. 1998; 273: 26729-26738Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar, 30Goelz S.E. Hession C. Goff D. Griffiths B. Tizard R. Newman B. Chi Rosso G. Lobb R. Cell. 1990; 63: 1349-1356Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 31Kimura H. Shinya N. Nishihara S. Kaneko M. Irimura T. Narimatsu H. Biochem. Biophys. Res. Commun. 1997; 237: 131-137Crossref PubMed Scopus (39) Google Scholar), whereas FUT7 transfection resulted in the expression of CD15s but not CD15 (20Sasaki K. Kurata K. Funayama K. Nagata M. Watanabe E. Ohta S. Hanai N. Nishi T. J. Biol. Chem. 1994; 269: 14730-14737Abstract Full Text PDF PubMed Google Scholar, 21Natsuka S. Gersten K.M. Zenita K. Kannagi R. Lowe J.B. J. Biol. Chem. 1994; 269: 16789-16794Abstract Full Text PDF PubMed Google Scholar, 31Kimura H. Shinya N. Nishihara S. Kaneko M. Irimura T. Narimatsu H. Biochem. Biophys. Res. Commun. 1997; 237: 131-137Crossref PubMed Scopus (39) Google Scholar). Therefore, FUT4 had been believed to be the enzyme solely responsible for the expression of CD15 in leukocytes before the finding of FUT9 in those cells. It has been investigated whether the expression of FUT4 and FUT7 correlated with the expression of CD15 and CD15s epitopes during hematopoiesis. Clarke et al. (26Clarke J.L. Watkins W.M. J. Biol. Chem. 1996; 271: 10317-10328Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) reported that FUT7 definitely determined the expression of the CD15s epitope, consistent with other studies (32Hiraiwa N. Dohi T. Kawakami-Kimura N. Yumen M. Ohmori K. Maeda M. Kannagi R. J. Biol. Chem. 1996; 271: 31556-31561Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 33Knibbs R.N. Craig R.A. Natsuka S. Chang A. Cameron M. Lowe J.B. Stoolman L.M. J. Cell Biol. 1996; 133: 911-920Crossref PubMed Scopus (155) Google Scholar, 34Maly P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. Cell. 1996; 86: 643-653Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar), however, the expression of CD15 does not correlate well with the expression of FUT4, which indicated that an unknown α1,3FUT is involved in the expression of CD15 in leukocytes. Marer et al. (27Marer N.L. Clarke J.L. Palcic M.M. Davies D. Skacel P.O. Glycobiology. 1997; 7: 357-365Crossref PubMed Scopus (20) Google Scholar) also reported a discrepancy between the expression of FUT4 and that of CD15, in which the level of FUT4 expression does not necessarily correlate with the level of CD15 expression during myeloid cell maturation.Previously (23Kaneko M. Kudo T. Iwasaki H. Ikehara Y. Nishihara S. Nakagawa S. Sasaki K. Shiina T. Inoko H. Saitou N. Narimatsu H. FEBS Lett. 1999; 452: 237-242Crossref PubMed Scopus (104) Google Scholar), we cloned a human cDNA encoding FUT9, and examined the tissue distribution of the six α1,3FUTs. We found that FUT9 is expressed in peripheral blood leukocytes. This indicated that FUT9 might be the enzyme responsible for determining the expression of CD15 in leukocytes. In this study, we examined which α1,3FUT is responsible for the CD15 expression, in conjunction with the expression of CDw65, in human leukocytes and demonstrated that the expression of CD15 is determined differentially either by FUT9 or FUT4, depending on the subpopulation of leukocytes.DISCUSSIONThe CD15 antigen is expressed on mature granulocytes and on myeloid cells from the promyelocyte stage onwards. Anti-CD15 antibody reacts with promyelocytes, and less strongly with myelocytes and metamyelocytes, but not bone marrow myeloblasts, as detected by flow cytometric analysis and an immunohistochemical method (10Hanjan S.N. Kearney J.F. Cooper M.D. Clin. Immunol. Immunopathol. 1982; 23: 172-188Crossref PubMed Scopus (169) Google Scholar, 11Civin C.I. Mirro J. Banquerigo M.L. Blood. 1981; 57: 842-845Crossref PubMed Google Scholar, 36Schienle H.W. Stein N. Muller-Ruchholtz W. J. Clin. Pathol. 1982; 35: 959-966Crossref PubMed Scopus (38) Google Scholar). CD15 epitopes are carried as terminal sequences on the oligosaccharide chains in both glycoproteins and glycolipids on mature granulocytes (37Spooncer E. Fukuda M. Klock J.C. Oates J.E. Dell A. J. Biol. Chem. 1984; 259: 4792-4801Abstract Full Text PDF PubMed Google Scholar). Some anti-CD15 antibodies also have a high immunoreactivity for normal peripheral blood monocytes (10Hanjan S.N. Kearney J.F. Cooper M.D. Clin. Immunol. Immunopathol. 1982; 23: 172-188Crossref PubMed Scopus (169) Google Scholar), but the majority of T and B lymphocytes do not express CD15 antigens (36Schienle H.W. Stein N. Muller-Ruchholtz W. J. Clin. Pathol. 1982; 35: 959-966Crossref PubMed Scopus (38) Google Scholar).The preferential activity of FUT9 to transfer fucose to the distal GlcNAc residue suggested that FUT9 synthesizes the CD15 epitope more efficiently in vivo than FUT4. The relative initial rate of transfer to the distal LN unit of neutral polylactosamine, 3LN-2AB, was ∼20 times higher for FUT9 than FUT4. On the other hand, FUT4 preferentially fucosylates the inner LN unit of polylactosamine on both the neutral and α2,3-sialylated polylactosamine chains, so that FUT4 can efficiently synthesize CDw65 epitope (2Niemela R. Natunen J. Majuri M.L. Maaheimo H. Helin J. Lowe J.B. Renkonen O. Renkonen R. J. Biol. Chem. 1998; 273: 4021-4026Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 25Nishihara S. Iwasaki H. Kaneko M. Tawada A. Ito M. Narimatsu H. FEBS Lett. 1999; 462: 289-294Crossref PubMed Scopus (77) Google Scholar). In the present study, we demonstrated that FUT9 can also transfer a fucose to the inner LN unit of the sialylated polylactosamine chain with a relative activity one fifth that of FUT4, resulting in the synthesis of CDw65 epitope (Fig. 2). In fact, the FUT9-transfected cells, Namalwa-FUT9 and U937-FUT9 cells, showed increased CDw65 expression. This confirmed the FUT9 activity to transfer fucose to the inner LN unit of the sialylated polylactosamine chain.Human mature granulocytes strongly express CD15 and CDw65 on their surface (10Hanjan S.N. Kearney J.F. Cooper M.D. Clin. Immunol. Immunopathol. 1982; 23: 172-188Crossref PubMed Scopus (169) Google Scholar, 11Civin C.I. Mirro J. Banquerigo M.L. Blood. 1981; 57: 842-845Crossref PubMed Google Scholar, 38Macher B.A. Buehler J. Scudder P. Knapp W. Feizi T. J. Biol. Chem. 1988; 263: 10186-10191Abstract Full Text PDF PubMed Google Scholar). Lactosaminoglycan on glycoprotein is a major carrier for the CD15 epitope in human granulocytes, because the CD15 epitope is three times more abundant in glycoproteins than in glycolipids (37Spooncer E. Fukuda M. Klock J.C. Oates J.E. Dell A. J. Biol. Chem. 1984; 259: 4792-4801Abstract Full Text PDF PubMed Google Scholar, 39Fukuda M.N. Dell A. Oates J.E. Wu P. Klock J.C. Fukuda M. J. Biol. Chem. 1985; 260: 1067-1082Abstract Full Text PDF PubMed Google Scholar). A typical feature of granulocyte lactosaminoglycan was a multiple fucosylated structure on LN units. Also, some of the side chains contain two or more fucose residues. However, the majority of fucose-containing neutral oligosaccharides possess a Galβ1–4(Fucα1–3)GlcNAc terminal structure (37Spooncer E. Fukuda M. Klock J.C. Oates J.E. Dell A. J. Biol. Chem. 1984; 259: 4792-4801Abstract Full Text PDF PubMed Google Scholar). This is consistent with the finding in the present study that mature granulocytes express substantial amounts of FUT9, which preferentially fucosylates the GlcNAc residue at the distal LN unit (25Nishihara S. Iwasaki H. Kaneko M. Tawada A. Ito M. Narimatsu H. FEBS Lett. 1999; 462: 289-294Crossref PubMed Scopus (77) Google Scholar). The repertoire of α1,3FUTs expressed in human mature granulocytes is distinct from that in promyelocytes, that is, a substantial amount of FUT9 transcript was detected in mature granulocytes, but not in promyelocytes. FUT4 can also be detected in mature granulocytes and promyelocytes, although its activity for the synthesis of CD15 is known to be minor in contrast to that for the synthesis of CDw65.The absence of FUT9 in the CD15+ and CD34+cells indicated that FUT4 is mainly responsible for the CD15 expression in the immature promyelocytes in cord blood cells. The human promyelocytic leukemic cell line, HL-60, which is composed of cells arrested at the promyelocytic stage, showed strong staining with both anti-CD15 and CDw65 antibodies, as described by others (26Clarke J.L. Watkins W.M. J. Biol. Chem. 1996; 271: 10317-10328Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The enormous amount of FUT4 expressed in HL-60 cells was able to give rise to bright CD15 and CDw65 staining of HL-60 even in the absence of FUT9. HL-60 cells are able to differentiate into myeloid mature cells and monocytes when cultured in the presence of dimethyl sulfoxide (Me2SO) and all-trans-retinoic acid (RA), respectively (40Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1421) Google Scholar, 41Nojiri H. Takaku F. Tetsuka T. Motoyoshi K. Miura Y. Saito M. Blood. 1984; 64: 534-541Crossref PubMed Google Scholar). However, we could not detect FUT9 transcripts in Me2SO-differentiated and RA-differentiated HL-60 cells (data not shown). FUT9 was not involved in the CD15 expression even in the differentiated HL-60 cells. It is difficult to exclude the presence of unidentified α1,3FUT in HL-60 cells and monocytes. However, each profile of the peaks (P1, P2, and P3) of HPLC in Fig. 2 is very characteristic of each α1,3FUT, enabling one to demonstrate that the α1,3FUT activity detected in the HL-60 cell homogenates corresponded to a typical pattern of FUT4 specificity. The peak areas were large enough for the CD15 and CDw65 expression, respectively, on the cell surface. These findings are consistent with the interpretation that FUT4 is responsible for the CD15 expression in the myeloid cells at the promyelocyte stage (26Clarke J.L. Watkins W.M. J. Biol. Chem. 1996; 271: 10317-10328Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).U937 and Jurkat cells expressed less FUT4 (9.5 and 7.0, respectively) than HL-60 cells and no detectable FUT9 leading to the weaker expression of CD15 and CDw65 on the cell surface. The transfection of the FUT9 gene to U937 cells demonstrated that FUT9 can potentially synthesize the CD15 epitope in myeloid cells. In U937-FUT9 cells, the intensity of CDw65 staining increased in conjunction with the increase of CD15 reactivity. The transcript levels forFUT4 and FUT9 genes expressed in Jurkat-FUT9 cells were found to be almost the same as those in mature granulocytes isolated from peripheral blood cells. The profile of α1,3FUT activity in Jurkat cells was typical of that of FUT4, and the profile in Jurkat-FUT9 cells converted to that of FUT4 plus FUT9. The P1 peak responsible for the CD15 synthesis was demonstrated to be directed by FUT9 in the Jurkat-FUT9 cells, not by FUT4. This fact strongly suggested that the CD15 expression in mature granulocytes is directed by FUT9, not by FUT4.The CDw65 expression in the Jurkat-FUT9 cells contrasted with that in the U937-FUT9 cells. The CDw65 intensity in the Jurkat-FUT9 cells decreased in comparison to that in the wild-type Jurkat cells, even though the FUT9 activity was increased. One explanation for this discrepancy may be that U937 cells possess enough of the precursor structure, i.e. the sialylated polylactosamine chains, to be fucosylated at the inner LN unit resulting in the CDw65 expression. Thus, FUT4 and FUT9 in the U937-FUT9 cells additively fucosylated the sialylated polylactosamine chains for the CDw65 expression. On the other hand, Jurkat cells may not possess enough of the precursor structure, because of weak activity of sialyltransferase(s), which is supplied for both FUT4 and FUT9 activity. In such a case, FUT9 may preferentially fucosylate the distal LN unit by overwhelming the sialyltransferase activity, resulting in the CD15 increase and CDw65 decrease.FUT9 was detected in each subpopulation of human PBMCs, although the expression level differed among the populations. Unexpectedly, monocytes do not express FUT9 despite expressing CD15 on their surface. The flow cytometric analysis indicated that the CD15 intensity in monocytes was dull and lower than that of CDw65, consistent with the substrate specificity of FUT4. FUT4 was found to synthesize CDw65 more than CD15, because P2s is 3.5 times larger than P1 (Fig. 2,B and C). Monocytes expressed FUT4 to almost the same extent as granulocytes, however, granulocytes showed bright CD15 staining with a much higher level of CD15 expression than monocytes due to the additional expression of FUT9. CD4+ T cells, CD8+ T cells, B cells, and CD56+ cells expressed FUT9, although flow cytometric analysis did not usually show CD15-positive staining in those cells. However, the transfection of theFUT9 gene to Jurkat cells demonstrated that FUT9 can potentially express the CD15 epitope on the cell surfaces of lymphoid cells (Fig. 5). Western blot analysis clearly demonstrated that all subpopulations of PBMCs can produce CD15-carrying glycoproteins intracellularly, which are not transported to be expressed on the cell surface. Considering the stronger activity of FUT9 for the synthesis of the CD15 epitope, the intracellular CD15 epitopes in PBMCs must be mainly synthesized by FUT9. The reasons why there were very few CD15 epitopes on the cell surface of PBMCs is unclear, but it is possible that either the intracellular CD15-positive glycoproteins are not translocated to the surface of resting PBMCs for some reason, or the expression level of FUT9 in each subpopulation of PBMCs is not sufficient to synthesize enough CD15-positive glycoproteins to be expressed on the cell surface. In fact, the level of FUT9 expression in PBMCs is lower than that in granulocytes and Jurkat-FUT9 cells. Among many Jurkat clones transfected with the FUT9gene, we have selected some, which expressed FUT9 at a range of 0.5 to 1.0. These clones with a relatively low expression level of FUT9 did not express CD15 on their cell surface (data not shown). These findings indicated that peripheral lymphoid cells do not express a high enough level of FUT9 to induce the cell surface expression of CD15.Although the function of CD15 in leukocytes is still not clear, CD15 may be involved in cell-cell interaction. The carbohydrate structure associated with CD15 on myeloid cells may be another ligand for human CD2 (16Warren H.S. Altin J.G. Waldron J.C. Kinnear B.F. Parish C.R. J. Immunol. 1996; 156: 2866-2873PubMed Google Scholar). As reported by others (14Skubitz K.M. Snook R.W. J. Immunol. 1987; 139: 1631-1639PubMed Google Scholar, 42Tetteroo P.A. Mulder A. Lansdorp P.M. Zola H. Baker D.A. Visser F.J. von dem Borne A.E. Eur. J. Immunol. 1984; 14: 1089-1095Crossref PubMed Scopus (37) Google Scholar, 43Albrechtsen M. Kerr M.A. Br. J. Haematol. 1989; 72: 312-320Crossref PubMed Scopus (24) Google Scholar), the CD15-positive glycoproteins in mature granulocytes showed characteristically broad bands spanning 140–180 kDa and 95–110 kDa on Western blot analysis. Surface-labeling studies revealed that only the 165-kDa and 105-kDa CD15-reactive glycoproteins are localized on the cell surface (43Albrechtsen M. Kerr M.A. Br. J. Haematol. 1989; 72: 312-320Crossref PubMed Scopus (24) Google Scholar, 44Stocks S.C. Albrechtsen M. Kerr M.A. Biochem. J. 1990; 268: 275-280Crossref PubMed Scopus (59) Google Scholar). They were identified to be a member of the LFA1/CR3/p150,95 (CD11/CD18) family (12Melnick D.A. Nauseef W.M. Markowitz S.D. Gardner J.P. Malech H.L. J. Immunol. 1985; 134: 3346-3355PubMed Google Scholar, 14Skubitz K.M. Snook R.W. J. Immunol. 1987; 139: 1631-1639PubMed Google Scholar) and NCA160 (44Stocks S.C. Albrechtsen M. Kerr M.A. Biochem. J. 1990; 268: 275-280Crossref PubMed Scopus (59) Google Scholar). We found that bands corresponding to the molecular sizes of CD11/CD18 members and NCA160 were present not only in mature granulocytes but also in all subpopulations of PBMCs. Monocytes showed the highest density of these bands, compatible with the strong cell surface expression of CD15.Recently, it has been reported that not only FUT7 but also FUT4 generate selectin ligands that support in vivo rolling of some leukocytes, whereas FUT7-dependent carbohydrates determine the rolling fraction for most leukocytes (28Weninger W. Ulfman L.H. Cheng G. Souchkova N. Quackenbush E.J. Lowe J.B. von Andrian U.H. Immunity. 2000; 12: 665-676Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 29Huang M.C. Zollner O. Moll T. Maly P. Thall A.D. Lowe J.B. Vestweber D. J. Biol. Chem. 2000; 275: 31353-31360Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). FUT9 can synthesize the CD15 epitope with very strong activity by transferring a fucose to the distal GlcNAc residue, although FUT7 can only fucosylate this precursor after sialylation by α2,3-sialyltransferases. This distinct substrate preference may lead to competition between the three α1,3FUTs if the availability of acceptor substrates is limited. This would result not only in the synthesis of CD15 but also in the interference of the generation of some carbohydrate epitope(s), such as CD15s and CDw65. The availability of FUT9 provides a tool for investigating the biological functions of the polylactosamine chain with or without fucosylation in human leukocytes under certain inflammatory conditions. There are three CD1markers of human leukocytes comprising fuco