Interaction with CD2

Initially, LFA-3 was defined by the ability of a monoclonal antibody (mAb) to block human cytolytic T lymphocyte (CTL)-mediated lysis by binding to the target cell surface. However, even before this discovery, a method widely used by immunologists to purify human T cells by forming rosettes with sheep red blood cells utilized the molecular interaction between LFA-3 and its ligand. The CTL-mediated lysis of targets as well as T cell rosetting was also inhibited by mAbs which bound to CD2 on the T lymphocytes. These antibody-blocking studies suggested that CD2 and LFA-3 were both involved with cell-cell adhesion.

Subsequent studies demonstrated that purified LFA-3 incorporated into the lipid bilayer of large lipid vesicles could bind to cells expressing CD2. Protein micelles, another form of multivalent LFA-3 prepared by detergent dialysis of purified phosphoinosi-tol (Pl)-linked LFA-3, bound to CD2-expressing cells with a Kd in the range of 0.5 nM. Bivalent LFA-3-Fc, prepared by splicing residues 1-184 of LFA-3 to the hinge and CH2-CH3 region of human immunoglobulin G1 (IgGl), would also bind to cells expressing CD2 with a Kd in the range of 150 nM. Soluble monovalent LFA-3 prepared by expression of amino acid residues 1-180 in CHO cells demonstrated weak binding to CD2. In a recent study using surface plasmon resonance technology, the association of soluble LFA-3 to immobilized CD2 was found to occur quickly; however, the dissociation was also very rapid, feoffs4s"', resulting in a low affinity, Kd — 15 |xm. Since all interactions would not reverse at the same instant, weak interaction between individual LFA-3/CD2 molecules would be stabilized if present as multivalent interactions. Provided the density was sufficient, the fast rate of association would ensure new interactions that would maintain adhesion. The experimental data with bivalent LFA-3, LFA-3 micelles and LFA-3-containing lipid membranes support this idea.

Using fluorescence photobleaching recovery of a planar phospholipid membrane containing purified fluorescently tagged phosphatidylinositol-linked LFA-3, the interaction between CD2 on the T lym phocyte membrane and LFA-3 in a lipid planer membrane has been quantitated. This study reported the two-dimensional dissociation rate constant to be about 20 LFA-3 molecules p,m~2 of membrane. (This measurement was based upon the density of LFA-3 in the contact area (bound) and the density outside the contact area (free) to calculate the number of molecules per unit area of membrane to give equilibrium binding.) Most APCs express LFA-3 at levels several fold above this density suggesting that laterally mobile LFA-3 should readily promote adhesive interactions with cells expressing CD2. However, the transmembrane form of LFA-3 has a much slower membrane mobility than Pi-linked LFA-3 and would probably require a greater membrane density to achieve the same level of adhesion found for the lipid-linked LFA-3 in model membranes. This and a previous study indicated that the multivalent interaction between LFA-3 and CD2 in different membranes was favorable but dependent upon the membrane density and mobility of the receptor-ligand pair.

Human LFA-3 (CD58) and its receptor, CD2, are both encoded on chromosome 1 and consist structurally of two extracellular immunoglobulin-like domains. There is 21% homology between these two glycoproteins, suggesting that they arose from a single ancestral gene that encoded for a self-binding or homophilic type protein. Interestingly, a homolog of LFA-3 has not been found in mice and rats. However, a protein structurally similar to LFA-3, CD48, which is also encoded on chromosome 1 in humans, appears to be utilized in both rodent species comparably to LFA-3 in humans. In studies using surface plasmon resonance, binding between human soluble CD48 and CD2 was not found; however, binding between rat CD48 and CD2 was demonstrated and determined to have a of about 80 pM. Although somewhat less than the interaction of LFA-3 with CD2, this level of binding in multivalent form should be sufficient to mediate adhesion in the rodent immune system.

The regions of LFA-3 required for binding to CD2 have been mapped by limited mutagenesis of the extracellular domain. Two regions containing charged amino acids in the first Ig-like domain were required. Specifically, amino acids 30, 34 and 84 of LFA-3 were shown to be essential for binding to CD2. Modeling by comparison with the structure of CD2 places these sites in two loop-out regions on one face of the LFA-3 molecule. From the position and complementarity of these charged groups, it appears that the LFA-3-CD2 interaction is mediated in part by interactions between amino acids with opposite charges.

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