Winifred M. Watkins

1,3-L-Fucosyltransferase Expression in Developing Human Myeloid Cells ANTIGENIC, ENZYMATIC, AND mRNA ANALYSES

In an attempt to correlate the cell surface expression of Le and sialyl-Le structures in immature and mature myeloid cells with the genes expressing α1,3-fucosyltransferase(s) we have examined: 1) the properties of the cellular α1,3-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 α1,3-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 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 α1,3-fucosyltransferase expressed in COS-7 cells transfected with Fuc-TIV are consistent with this being the major gene responsible for the expression of Le 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 α1,3-fucosyltransferase controlling the expression of sialyl-Le on mature cells. However, Le 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 α1,3-fucosyltransferase gene remains to be identified.

Chapter 5 Biosynthesis 5. Molecular Basis of Antigenic Specificity in the ABO, H and Lewis Blood-Group Systems

Publisher Summary This chapter discusses the molecular basis of antigenic specificity in the ABO, H, and Lewis blood-group systems. The classical ABO blood group system provided the first examples of single human polymorphic characters that were not associated with inherited diseases, and the understanding of the serological relationships in this system laid the basis for the safe transfusion of blood from one individual to another. The antigenic structures classified within the ABO blood-group system are known to arise through the expression of genes encoding glycosyltransferase enzymes responsible for the terminal glycosylation of carbohydrate chains in glycoproteins, glycolipids and free oligosaccharides. The ABO classification is based on the presence or absence of two antigens, A and B, on the erythrocyte surface and two antibodies anti-A and anti-B which always occur in the plasma when the corresponding antigen is missing. It is the presence of these naturally occurring antibodies that makes knowledge of the ABO groups of crucial importance in blood transfusion.

Sda and Cad Antigens

In 1967 two independent groups (Renton et al., 1967; Macvie et al., 1967) recognized that a number of human red cell antibodies under investigation in different laboratories, belonged to a single system which has come to be known as the Sid blood group system. The antibodies in general gave only weak and rather nebulous reactions and were distinguished by the property of agglutinating only a proportion of a donor’s erythrocytes, even those of the more strongly positive reactors. The system has only one antigen Sda, and hence there are two phenotypes Sda(a+) and Sda(—): of the British population initially studied about 92% were Sd(a+) (Table I). Of these positive individuals only 1% gave relatively strong reactions, 80% gave distinct compact small agglutinates in a field of unagglutinated cells, and 10% gave very weak tiny agglutinates (Renton et al., 1967; Macvie et al., 1967; Pickles and Morton, 1977). Separation of the Sda-positive cells from the unagglutinated cells, followed by exposure of the remaining cells to more anti-Sda, gives a mixed field picture once more; indicating that the majority of cells carry the antigen but in variable amounts (Macvie et al., 1967).

The Genetic Regulation of Sialyl-Lewisx Expression in Haemopoietic Cells

Carbohydrate structures with terminal fucose and/or sialic acid residues are expressed on cell surfaces in a developmentally regulated and tissue specific manner. Such structures function as receptors for antibodies and lectins and have been implicated as tumour markers (1), as ligands for protein adhesion molecules (2) and in cell adhesion reactions based on carbohydrate-carbohydrate binding (3). This review is primarily concerned with the genetic regulation of the fucosyltransferases that are responsible for the expression on myeloid cells of the hybrid fucosyl/sialyl carbohydrate determinant sialyl-Lex (Fig 1.).

The Genetic Regulation of Fucosylated and Sialylated Antigens on Developing Myeloid Cells

The first part of this article reviews the stages of normal development of haemopoietic cells committed to the myeloid lineage, properties of leukaemic cell lines that are arrested at specific maturation stages along the granulocytic pathway, the structures of carbohydrate antigenic markers that appear on myeloid cell surfaces, with especial reference to sialyl-Le(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc), and the role of this antigen on mature granulocytes as a ligand for selectin molecules. The families of fucosyl- and sialyltransferase genes encoding enzymes responsible for the biosynthesis of sialyl-Le(x), and the pathways leading to the formation of this antigen, and more complex related structures, are described. The second part of the article outlines the work carried out in the authors laboratory with leukaemic cell lines in an attempt to ascertain the biochemical and genetic basis of the lowering of sialyl-Le(x) expression that occurs at intermediate stages of normal haemopoietic development. Analysis of enzyme levels and mRNA expression of the fucosyl- and sialyltransferase genes has led to the conclusion that depletion of substrate resulting from high levels of enzyme activity from co-expressed genes FUT4 and ST6Gal1 probably accounts for the dip in expression of sialyl-Le(x), rather than a change in the level of expression of FUT7, the gene in myeloid cells encoding the enzyme ultimately responsible for the synthesis of sialyl-Le(x). The possible significance of this change in relation to normal cell maturation is discussed.