14

Fuca

Sialyl-Le" Galßl -4GlcNAcß1 -3Galß1. I 2,3 | 1,3 NeuAca Fuca

Sialyl-Lea Galßl -3GlcNAcß1 -3Galß1. | 2,3 | 1,4 NeuAca Fuca

Sulfo-Lex Galßl -4GlcNAcß1 -3Galß1. I 3 | 1,3 HSOa Fuca

Sulfo-Lea Galßl -3GlcNAcß1 -3Galß1. I 3 | 1,4 HSOs Fuca

Figure 1 The inter-relationships between the carbohydrate antigens Le", Ley, sialyl- and sulfo-Le* (based on the type 2 backbone sequence Galßl 4GlcNAc), their isomers Lea, Leb, sialyl- and sulfo-Lea (based on type 1 backbone Galß1- 3GlcNAc) the ¡. I anc Fc10.2 antigens, and the major blood group antigens H, A and B. The i and I antigenic determinants are expressed on nonsubstituteo or accessible, repeating, type 2 backbone sequences, and Fc10.2 on a type 1 disaccharide backbone joined to a type 2 backbone The 'hybrid' backbone structure with a type 2 and a type 1 branch expresses one of the I antigenic determinants, I (Ma), which consists of the Galß1-4GlcNAcß1-6Gal sequence. The major blood group antigens are expressed on either type 1 or type 2 backbones anc on additional backbone families (not shown here). Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; NeuAc. N-acetylneura minic acid; HS03, sulfate.

structure termed Lex, is an isomer of blood group Lewis-1 (Lea) which consists of type 1 backbone (galactose 31-3 linked to N-acetylglucosamine) which is a1-4 fucosylated at N-acetylglucosamine. Further fucosylation of Le" and Lea with fucose ctl-2 linked to the terminal galactose constitute the Le> and blood group Le'1 antigens, respectively. The inter-relationships of Lex/y, Lei,/h with the major blood group antigens H, A and B are shown in Figure 1.

The backbone sequences are antigens in their own right when they are not further glycosylated and are accessible. Thus, the repeated type 2 backbones in linear sequence express the i antigen, and when branched, the large I antigen (Figure 1). Rather ahead of the applications of hybridoma antibodies, the human monoclonal autoantibodies, anti-i and anti-1, were used as sequence-specific reagents, and some striking changes were noted in the expression of these antigens during early murine cmbrogenesis, I being the predominant antigen in the early embryo, and i appearing coincident with the first differentiation event in the embryo, i.e. the formation of the primitive endoderm. From the observations with the monoclonal antibodies, together with earlier knowledge that marked changes occur in the expression ot the major blood group antigens in human fetal organs at various developmental stages, a picture emerged that some of the changes in antigenicities ot glycoproteins and glycolipids of cells during successive developmental stages may be brought about bv sequential addition or deletion of monosaccharide residues. This led to the proposal that Le\ I and i and other oligosaccharides of the blood group familv may have important biological roles as recognition structures. They seemed excellent candidates as 'area codes' which determine cell migration pathways, for example.

A functional antigen in early neural development, L5, is Lex

L.5 antibody, originally raised to glycoproteins of the murine nervous system, was shown to recognize neurons and astrocytes in the cerebellum. Later observations on the chick embryo have suggested that the 1.5 antigen plays a role during early neural development. The antigen has been detected on multiple brain glycoproteins including Thy-1, and on a chondroitin sulfate proteoglycan. The structure of this antigen has been elucidated recently by generating oligosaccharide probes (neoglycolipids) from N-glycans released from total antigen-positive glycoproteins, and from Thy-1 isolated from the brain. The L5 antigen was shown to consist of Le*. The various attributes for L5 antigen in the development of the nervous system can therefore be assigned to the Lex sequence. By other approaches, it has been shown that astrocytes in the developing brain and astrocyte subpopulations in adult cerebellum express the Lex antigen, and there is increased expression on the cell processes at sites of injury. It has been suggested that Le* and related sequences may be ligands for carbohydrate-binding proteins that mediate astrocyte-neuron and astrocyte-matrix interactions.

Lex and sialyl-Lex are markers of human granulocytes and monocytes, and have been designated CD 15 and CD 15s, respectively

The production of antibodies to distinguish different human blood cell types and lineages has been a major application of the hybridoma technology. Innumerable antibodies that clearly distinguish granulocytes from other blood cells were among the earliest to emerge. The antigenic determinants of one of these antibodies, VEP8, and the second, VEP9, which, in addition binds monocytes, were shown to be Le\ These findings highlighted a difference between the human and mouse cellular markers, for, in the adult mouse Le* is not readily detectable immunochemically on blood cells of any kind. Innumerable antibodies with similar specificities toward human granulocytes have been produced over the years, and are assigned as CD15. Desialyl-ation of human blood cells revealed a strong reactivity of human monocytes with anti-Lex as well as granulocytes, indicating that monocytes express pre dominantly the sialyl-Le", and that the sialic acid results in masking of the expression of Le" antigen. Antibodies that recognize the sialyl form of I.e" (Figure 1) on cells of the myeloid series are now designated CD 15s.

Lex and sialyl-Lex are tumor-associated antigens

Another intensive area of research that became stimulated by the advent of the hybridoma technology was the search for tumor-associated antigens that might be targets for immunotherapy. Among promising antibodies isolated, by comparing reactivities with tumor cells and their normal counterparts, were those which upon further investigation were found to be directed at carbohydrate sequences of glycoproteins and glycolipids. Disappointingly for those who were hoping to identify targets for a new generation of therapeutic monoclonal antibodies ('magic bullets'), the carbohydrates identified were not unique to the tumors, but were sequences that occur normally on other cells and tissues of the body. Notable examples were antibodies directed to Lcv and sialyl-Le". Thus, these oligosaccharides may have been inappropriately expressed on the cancerous cells being investigated, but, as discussed above, they were already established as 'distinctive markers' of myeloid cells among human peripheral blood cells. The conclusions of the present author were that either these findings were a reflection of limitations of the methods of selection of hybridoma antibodies, or they were pointers to roles for the carbohydrate structures not oniy as area codes but also as ligands for growth and differentiation.

Lex and Lea-related sequences as ligands for carbohydrate binding receptors, the selectins

The concept that oligosaccharides can serve as area codes has been corroborated by developments in leukocyte biology and inflammation research, notably from work with the leukocyte-endothelial adhesion molecules, the three selectins. The molecular cloning of these molecules, E- and P- selectins (CD62E, CD62P) of endothelia and L-selectin (CD62I.) of leukocytes, and the finding that each contains a lectin-like domain, stimulated intensive research into their oligosaccharide ligands. Knowledge that the E- and P-selectins bind to granulocytes and monocytes focused research on Le" and sialyl-Le" as obvious candidate ligands. Indeed, all three selectins can bind to sialyl-Le"-related sequences when these arc displayed in the clustered state on protein or lipid (E-

selectin can also bind the asialo-LeN sequence, although less avidly). The isomeric sialyl-Lea-related sequences (found at the surface of epithelial tumors) are rather more strongly bound than the Lex analogs. In addition, the sulfated Lea and Lex sequences (identified on an ovarian cyst adenoma glycoprotein) are also bound avidly. On endothelial venules (sites at which L-selectin-mediated leukocyte adhesion occurs), variously sulfated forms of the Le* sequence have been identified. Details of the macromolecular carriers of these oligosaccharide ligands are out of the scope of this entry: suffice it to say that oligosaccharides of this family are key players in the selectin recognition systems. Unlike the usual situation of receptor-ligand pairs, however, a picture is emerging of recognition systems that operate as triads: receptors, ligands and carriers. Here the receptors are lectins (the selectins), the ligands are oligosaccharides, but only when they are optimally expressed and presented on carriers (proteins or lipids) are functional counter-receptors formed. Also a possible involvement of these carbohydrate structures in the process of metastases has been suggested, the proposed mechanism being that sialyl-Le*- or sialyl-Lea-positive cancer cells hijack the leukocyte adhesion pathway for extravasation.

See also: Cold agglutinins; Selectins (CD62-E/L/P).

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