Additional Selectin Functions

Apart from the function of leukocyte rolling, the selectins have also been implicated in a number of other biologically important functions. For a normal immune response the circulation of mature T and B lymphocytes throughout the secondaiy lymphoid organs is required as it ensures that a full complement of antigen receptors aie exposed to the full range of antigens present in the system. Entiy to these secondary lymphoid organs by lymphocytes occurs across specialised endothelial cells, known as high endothelial venules (HEV) (Picker and Butcher, 1992), and L-selectin is the principal if not sole HEV receptor for lymphocyte traffic to the lymph nodes (Kansas, 1996). Additionally L-selectin may play a role in interactions between leukocytes, and in paiticulai- neutrophils, which may be important in the amplification of the inflammatoiy response. It has been noted that neutrophils can roll on other neutrophils and that this is blocked by monoclonal antibodies to L-selectin (Bargatze et a l, 1994). Neutiophil aggregation can also be blocked by a monoclonal antibody to L-selectin or carbohydrates that inhibit L-selectin activity (reviewed by Kansas, 1996).

The fact that P-selectin is found on platelets suggests a role in platelet-leukocyte interactions during wound healing and hemostasis, which could amplify the recruitment to sites of vascular injuiy (Larsen et a l, 1989). P-selectin is known to mediate platelet binding to monocytes which in turn induces tissue factor, thereby activating the blood coaggulation cascade (Cell et a l, 1994). E-selectin has been implicated as a tissue specific homing receptor for leukocyte recruitment specifically to the skin, particularly in the case of memory T cells (Picker <2/., 1991).

In summary, although the selectins are involved in the recruitment of leukocytes to areas of inflamed tissue, it is apparent that their* role is not limited to this alone and each selectin may have a more specialised function within the immune response.

Chapterl: Introduction 18

L2.4 Carbohydrate Ligands

The identification of the physiological ligands for the selectins is complicated because, like most lectins, they bind to a wide variety of carbohydrate epitopes in vivo. The dependence of the selectins upon a caibohydrate ligand was illustrated in two patients with leukocyte adhesion deficiency type II disease, which is an inherited inability to recruit neutrophils to the sites of inflammation (Harlan, 1993). This inability to generate the ligands required for selectin mediated rolling is attributed to a defect in the fucose metabolism, and they are, therefore, unable to synthesise fucosylated caibohydiates. The observation that cell adhesion is sensitive to neuraminidase, indicates that fucose and sialic acid residues are essential for selectin binding, and a prototype caibohydrate ligand, sialyl Lewis^, Neu5Aca2-3Gaipi- 4[Fucal-3]GlcNAc, was proposed as the minimal required structure (figure 1.9).

OH OH HO OH OH OH _NHAc GlcNAc NeuSAc _OH OH OH Fuc

Figure 1.9 - Schematic representation of the sialyl Lewis'" antigen, the minimal ligand proposed for selectin binding.

All three selectins can bind to sialyl Lewis^, although with varying affinities, but they also bind with higher affinity to carbohydrate determinant on a limited number of glycoproteins or proteoglycans (reviewed by Rosen and Bertozzi, 1994). Therefore, a distinction must be made between structures which can interact with the selectins under certain conditions in vitro, and those stiuctures which do interact under physiological conditions in vivo. Hence, the simple tetrasaccharides originally proposed as ligands and which may contribute to the biologically active ligand, may be thought as maikers for construction of the biological relevant ligands.

Chapter!: Introduction 19

1.2,5 Glycoprotein Ligands

Purified forms of the selectins or IgG fusion proteins exhibit a higher level of specificity in compaiison to monoclonal antibodies against their putative ligands. This suggests that the selectins recognise not just a simple oligosaccharide, but a three dimensional surface composed of several carbohydrate moieties contributed by several molecular species attached to a specific glycoprotein.

Mouse L-selectin binds to at least three heavily glycosylated mucin-like proteins in vivo, namely GLYCAM-1, CD34, and MAdCAM-1. Each glycoprotein bears sulfated, sialylated, and fucosylated 0-lrnked oligosaccharide chains that appear essential for L-selectin binding (reviewed by Kansas, 1996). Although first reported to be the ligand for L-selectin, and thought to be constitutively expressed on peripheral lymph node HEV, GLYCAM-1 contains no apparent transmembrane region, and it appears that it is predominately released into circulation where it competitively inhibits, rather than promotes, L-selectin mediated attachment (Tedder e ta l, 1995b).

The second glycoprotein, a glycoform of CD34, is constitutively expressed on most endothelial and hematopoietic stem cells, and therefore has been proposed to regulate tissue specific selectin binding because of the tissue-specific glycosylation of CD34. In mice it is universally expressed on the endothelium both during mouse development and in the mature animal, so it is perfectly placed as a ligand for L-selectin. In CD34 knockout mice normal levels of rolling and lymphocyte homing is observed, and in puiified human tonsil, CD34 accounts for approximately half of the L-selectin activity. Therefore, the third ligand, MAdCAM-1, may constitute the principal adhesive ligand(s) for L-selectin, although it has yet to be identified at the moleculai* level. Interestingly, in knockout mice of both CD34 and GLYCAM-1, an up-regulation of this third ligand is seen, indicating the evolution of dynamic mechanisms to maintain adequate levels of lymphocyte traffic and recirculation. The O-linked carbohydrates of GLYCAM-1 have been characterised (Hemmerich et a l,

1995), and two of the more prominent structures obtained from P-elimination are novel sulfated derivatives of the sialyl Lewis^ motif; 6’-sulfo-sialyl Lewis’^, where the sulfate group is at the 6 position of the galactose residue; and 6-sulfo-sialyl Lewis^, where the 6 position of the GlcNAc residue is sulfated, and both of these moieties are contained within the core-2 O-

Chapter!: Introduction 20

linked glycans, le., Gaipi-3[GlcNAcpl-6]GalNAc. These ligands provide all the requirements for L-selectins ligands, but whether these are the actual recognition motifs, or whether CD34 or MAdCAM-1 contain these or different oligosaccharides structures is as yet unknown.

The P Selectin Glycoprotein Ligand -1 (PSGL-1) is a disulfide linked homo-dimer of two identical ~120kDa type I mucin-like proteins which have a unique stmcture (Moore et al,

1992). This glycoprotein is expressed by all blood neutrophils, monocytes, and lymphocytes but requires specific glycosylation for its function. Studies on the P-selectin - PSGL-1 binding have provided the first direct evidence that leukocyte rolling under physiological shear forces requires the specific interaction of a selectin with a single cell-surface glycoprotein. A specific anti-PSGL-1 monoclonal antibody completely inhibits P-selectin mediated rolling of leukocytes (Moore et a l, 1995). PSGL-1 is mainly O-linlced glycosylated, although it has two or three A-linked sugars which do not appear* to be necessary for binding, with extensive branched chain polylactosamine glycans, which contain fucose and sialic acid and many terminate in the sialyl Lewis* motif (Moore et al., 1994). The structure of these glycans has been characterised (Wilkins et a l, 1996) and contain N-

acetyllactosamine, sialyla2,3-A-acetylactosmine, Lewis*, and sialyl Lewis* motifs in differing quantities (figure 1.10).

Chapterl: Introduction 21 Gaipi-3GalNAc 1 4% Gaipi-4GlcNAcPK . oGalNAc 52% G aipK ^

{

Galpl^GlcNAcpK _ Neua2-3Gaipi-4GlcNAcpl\ ^ qGalNAc 6% gGalNAc 14% Gaipi"^^ Neua2-3Gaipr Fucal

I

3 Neua2-3Galpl-4GlcNAcpl\ gGaINAc 2% Neua2-3Gaipi"^

Fucal Fucal Fucal

1

I

I

3 3 3

Neua2-3Galpl-4GlcNAcpi-3Gaipi-4GlcNAcPl-Gaipi-4GlcNAcpl-

^GalNAc 12% G aip i^^

Figure 1.10 - Structures and percentages of the 0-glycans in PSGL-1 (from Wilkins et al., 1996)

In document Development of NMR methods for conformational analysis of ¹³C enriched oligosaccharides (Page 40-44)