Affinity, kinetics, and thermodynamics of E-selectin binding to E-selectin ligand-1.

E-selectin is an endothelial adhesion molecule, which mediates the tethering and rolling of leukocytes on vascular endothelium. It recognizes the glycoprotein E-selectin ligand-1 (ESL-1) as a major binding partner on mouse myeloid cells. Using surface plasmon resonance, we measured the kinetics and affinity of binding of monomeric E-selectin to ESL-1 isolated from mouse bone marrow cells. E-selectin bound to ESL-1 with a fast dissociation rate constant of 4.6 s(-1) and a calculated association rate constant of 7.4 x 10(4) m(-1) s(-1). We determined a dissociation constant (K(d)) of 62 microm, which resembles the affinity of L-selectin binding to glycosylation-dependent cell adhesion molecule-1. The affinity of the E-selectin-ESL-1 interaction did not change significantly when the temperature was varied from 5 degrees C to 37 degrees C, indicating that the enthalpic contribution to the binding is small at physiological temperatures, and that, in contrast to typical protein-carbohydrate interactions, binding is driven primarily by favorable entropic changes. Interestingly, surface plasmon resonance experiments with recombinant ESL-1 from alpha 1,3-fucosyltransferase IV-expressing Chinese hamster ovary cells showed a very similar K(d) of 66 microm, suggesting that this fucosyltransferase is sufficient to produce fully functional recombinant ESL-1. Following the recent description of the affinity and kinetics of the selectin-ligand pairs L-selectin-glycosylation-dependent cell adhesion molecule-1 and P-selectin-P-selectin glycoprotein ligand-1, this is the first determination of the parameters of E-selectin binding to one of its naturally occurring ligands.

The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter.

Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.

Tumor therapy with bispecific antibody: the targeting and triggering steps can be separated employing a CD2-based strategy.

For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 x tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab')2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.

Dependence of T cell antigen recognition on the dimensions of an accessory receptor-ligand complex.

The T cell antigen receptor (TCR) and its ligand peptide-major histocompatibility complex (MHC) are small (approximately 7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23-50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small "accessory" molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide-MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol. Today. 17:177-187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide-MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size.

CD2 and the nature of protein interactions mediating cell-cell recognition.

Rapid progress has recently been made in characterising the structures of leukocyte cell-surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co-receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell-cell recognition. As an example of biological recognition, the defining characteristic of cell-cell contact is that it involves the simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell-cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand-binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface-shape complementarity, is the dominant feature of specific, low-affinity protein recognition at the cell surface by CD2. The crystallographic analysis of the CD2-binding domain of CD58 strongly supports this concept.

The receptor function of CD2 in human CD2 transgenic mice is based on highly conserved associations with signal transduction molecules.

The activation of human T cells via CD2 in response to mitogenic monoclonal antibodies (mAbs) typically requires that one mAb is specific for an epitope within the N-terminal Ig domain of CD2 and the other for a partially hidden epitope. We have examined the proliferative response of human T cells and human CD2 (huCD2) transgenic murine T cells to two novel CD2 monoclonal antibodies, AICD2.M1 and AICD2.M2, and have partially mapped the epitopes of these and other mitogenic CD2-specific monoclonal antibodies by way of recognition of CD2:CD58 chimeric proteins possessing either the N-terminal or the membrane proximal immunoglobulin domains of CD2. To understand the molecular basis of proliferation in huCD2 transgenic murine T cells, the interactions of huCD2 with signaling proteins in murine T cells were analyzed. The transgenic huCD2 molecule was found to interact with the murine tyrosine kinases p56lck and p59fyn and the CD3-epsilon and zeta chains of the TCR/CD3 signaling complex and to coimmunoprecipitate tyrosine phosphatase activity. These molecular associations resemble the situation in human T cells and suggest that human CD2 couples to the same signal transduction pathways in humans and transgenic mice.