[CANCER RESEARCH 47, 2763-2766, June 1, 1987] Presence of Cytidine S'-Monophospho-TV-acetylneuraminic Acid:Gal/21- 3GalNAc-R a(2-3)-Sialyltransferase in Normal Human Leukocytes and Increased Activity of This Enzyme in Granulocytes from Chronic Myelogenous Leukemia Patients1 M. A. Baker,2 A. Kanani, I. Brockhausen, H. Schachter, A. Hindenburg, and R. N. Taub Department of Medicine, Toronto General Hospital, University of Toronto, Tarato, Ontario, M5G IL7 fM. A, B., A. K.J, the Department of Biochemistry, University of Toronto, and the Research Institute, Hospital for Sick Children, Toronto, Ontario, M5G 1X8 [I. B., H. SJ, and the Department of Medicine, Columbia University, New York, New York 10032 ABSTRACT We have examined granulocytes from patients with chronic myeloge- nous leukemia (CML) and from normal subjects to determine whether activity of a specific sialyltransferase might account for the aberrant sialylation of CMinked membrane oligosaccharides in CML cells. Total membrane preparations of morphologically mature CML and normal granulocytes were tested for sialyltransferase activity using the substrates galactosy!-/31-3-A'-acetyl-D-galactosamine-a-O-nitrophenyl and A'-ace- tyl-D-galactosamine-a-phenyl. W-Acetyl-D-galactosamine-a-phenyl was not an acceptor with either CML or normal cells. With galactosyl-j81-3- Af-acetyl-D-galactosamine-a-O-nitrophenyl, sialyltransferase activity was 2.8 times higher in CML cells compared to normal cells. Product iden tification by high performance liquid chromatography showed that en zyme from both normal and CML granulocytes linked sialic acid to galactosyl-fll-S-AT-acetyl-D-galactosamine-R by the o(2-3) and not the a(2-f>) linkage. The enzyme CMP-/V-acetylneuraminic acid: galactosyl- 01-3-,/V-acetyl-D-galactosamine-R a(2-3)-sialyltransferase has not pre viously been described in human granulocytes. The marked increase in activity of this enzyme in CML and the resulting increase in sialylation may contribute to the pathophysiological behavior of CML granulocytes. INTRODUCTION Chronic myelogenous leukemia is characterized by inappro priate early release of immature granulocyte precursors from the bone marrow and by a major delay of exit of mature granulocytes from the blood into the tissues (1). CML3 granu locytes show a marked decrease in binding to PNA when compared to normal granulocytes; they are less adhesive to nylon wool (2) than normal cells; their cell membranes show decreased labeling after treatment with galactose oxidase- NaB3H4 and increased labeling after sodium periodate-NaB3H4 treatment (3). All these effects can be partially or completely reversed by treatment with Vibrio cholera neuraminidase. These studies suggest that the cell surface oligosaccharides are more fully sialylated in CML cells and this may account in part for Received 8/25/86; revised 12/22/86; accepted 2/18/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by Medical Research Council of Canada Grant MT-3285 to H. S., by National Cancer Institute (United States) Grants CA 31761 and CA 37162, a grant from the William J. Matheson Foundation to R. N. T. and A. H., and by National Cancer Institute of Canada and Medical Research Council of Canada Grants to M. A. B. M. A. B. is a Research Associate of the Ontario Cancer Treatment and Research Foundation. A. H. is a Fellow of the Leukemia Society of America. I. B. did this work while holding a Canadian Cystic Fibrosis Foundation Studentship. 2To whom requests for reprints should be addressed, at Toronto General Hospital, Mulock Larkin Wing 1-005, Toronto, Ontario M5G 1L7, Toronto, Ontario, Canada. 3 The abbreviations used are: CML, chronic myelogenous leukemia; Gal, r>- galactose; GalNAc, W-acetyl-D-galactosamine; GalNAcOH, JV-acetylgalactosa- minitol; NANA, /V-acetylneuraminic acid; PNA, peanut agglutinin; HPLC, high performance liquid chromatography, Gal/31-3GalNAc, galactosyl-/31-3-Ar-acetyl- D-galactosamine; NeuAc, JV-acetylneuraminic acid; FMLP, formyl-methionine- leucine-phenylalanine. the pathophysiological behavior of the circulating cells. The aberrant sialylation involves the major granulocyte glycoprotein with a molecular weight of 150,000 which has been shown to be important for granulocyte adhesion (4,5). CML granulocytes show decreased binding of the synthetic chemotactic peptide FMLP (6). Sialylation of glycoproteins is effected by sialyltransferases specific to the structure of the substrate (Table 1; Ref. 7). Two enzymes are known to transfer sialic acid from CMP-NANA to Gal/31-3GalNAc-R: (a) CMP-NANA:Gal/31-3-GalNAc-R a(2-3)-sialyltransferase (sialic acid to galactose) (EC 2.4.99.4), where R can be polypeptide or a nitrophenyl group, and (h) CMP-NANA:R,/31-3GalNAc-R2 a(2-6)-sialyltransferase I (sialic acid to ./V-acetylgalactosamine (EC 2.4.99.3), where RI can be H-, Gal-, or sialyla2-3Gal-and R2 must be a polypeptide. The «3-sialyltransferase has been reported in rat, porcine and fetal calf liver, human placenta, and porcine and ovine submax- illary glands (8-12). The a6-sialyltransferase I has been found in the submaxillary glands of several species (9, 11, 13-16). The structures formed by these enzymes, sialyla-2-3-GaljSl- 3GalNAc- and Galj81-3(sialyla2-6)GalNAc-, respectively, have been detected on many mucins and on glycoproteins such as antifreeze glycoprotein, RBC glycophorins, etc. (7). Addition ally, a third sialyltransferase has been described which acts on O-glycosyl oligosaccharides. This enzyme, CMP-NANA:si- alyla2-3Galj31-3GalNAc-R a(2-6)-sialyltransferase II, also transfers a residue of sialic acid to W-acetylgalactosamine (EC 2.4.99.7); R can be a polypeptide or a nitrophenyl group (10, 14). This enzyme has been found in fetal calf liver, but is probably quite widely distributed (10, 14). It can act neither on Gal,81-3GalNAc-R nor on GalNAc-R. In a previous study (17) we compared the sialyltransferase activities of membranes prepared from CML granulocytes that appeared mature by microscopic criteria and from normal gran ulocytes of identical morphology. The CML preparations showed significantly higher sialyltransferase activities toward asialofetuin, asialo fucose-free blood group A-negative porcine submaxillary mucin and antifreeze glycoprotein, all of which are known to contain appreciable amounts of O-linked Gal/31- 3GalNAc oligosaccharides. Asialo-ovine submaxillary mucin, which contains mainly O-linked GalNAc residues and only a small amount of Galj81-3GalNAc, was a poor sialyltransferase substrate with either normal or CML cells. There was no significant difference in sialyltransferase activity between CML and normal cells when asialo-o, acid glycoprotein was used as an acceptor; this substrate contains only TV-linked oligosaccha rides with terminal Gal residues. These observations suggested that the increased sialyltransferase activity in CML cells was acting on O-linked Gal|81-3GalNAc groups. The present studies were carried out to prove this hypothesis and to establish the nature of the sialic acid linkage to Gal/31- 2763 Research. on January 22, 2022. © 1987 American Association for Cancer cancerres.aacrjournals.org Downloaded from