Structural and Binding Analysis of a Two Domain Extracellular Cd 2 Molecule by Peter H . Sayre , Rebecca E . Hussey , Hsiu - Ching

The human CD2 (T11) molecule is a 50-kD surface glycoprotein expressed on >95To of thymocytes and virtually all peripheral T lymphocytes that mediates both adhesion between these cells and their cognate partners, as well as subsequent activation events . Specific combinations of antibodies against the surface-bound molecule can activate IL-2-dependent T cell proliferation, helper T cell function, and cytotoxicity by NK cells and CTL (1-3) in the absence ofcellular adhesion . In addition, thymocyte activation can be mediated via CD2 (4, 5) . The role of CD2 in approximation ofTcells to various cell types, including human thymic epithelial cells, B cells, target cells, and sheep erythrocytes has been demonstrated to depend on direct interaction between CD2 and the broadly distributed human LFA-3 surface glycoprotein or its sheep homologue, T11TS (5-10) . Biochemical analyses using specific mAbs show that CD2 is Tlineage specific and exists on the cell surface in several differentially glycosylated forms (11-13) . CD2 cDNA clones predict a cleaved signal peptide of 24 amino acid residues, an extracellular segment of 185 residues, atransmembrane domain of 25 residues, and a cytoplasmic region of 117 residues (13-16). The corresponding genomic organization reveals a single exon encoding the signal peptide (less four residues), two exons encoding the extracellular segment, one exon encoding the transmembrane domain andcharged membrane anchor segment, and one exon encoding the cytoplasmic region (17) . A prerequisite for in-depth structure-function analysis of cell surface receptors is the capacity to produce and analyze sufficient quantities of material that bear a high degree of fidelity to the native structure. Preliminary observations suggested that a baculovirus expression system could provide such a capability. However, due to the nature of the cDNA construction, monomeric CD2 was not obtained . Here we report the production and characterization of a recombinant soluble CD2 molecule, termed T11,X2, that corresponds to the two extracellular segment exons and exists in solution entirely in monomeric form . The folding of the recombinant CD2 protein appears to be native since it reacts with mAbs that were produced against transmembrane CD2 on the surface ofhumanTlymphocytes . Surprisingly, the affinity of the CD2 extracellular segment monomer is only micromolar, implying that the avidity of CD2-mediated Tcell adhesion is dependent on cooperative binding resulting from the multiple copies of CD2 on the T lymphocyte surface. To our knowledge, this represents the first time that a lymphoid adhesion receptor has been produced on a milligram scale in a functional and pure form .