CD23/Fc epsilon RII and its soluble fragments can form oligomers on the cell surface and in solution.

Human CD23 (also known as Fc epsilon RII) is a 45,000 MW glycoprotein with homology to C-type animal lectins. It is involved in B-cell differentiation and IgE regulation, and is naturally cleaved to give soluble products of 37,000, 33,000, 29,000, 25,000 and 16,000 MW. Previous work has suggested that the region between the transmembrane sequence and the extracellular lectin head is capable of forming an alpha-helical coiled coil, one of the main consequences of which would be formation of dimers or trimers. Here we present protein-protein cross-linking data showing that CD23 forms trimers on the cell surface and hexamers in solution, and we use several different fragments to determine the regions of the protein involved in this self-association. The region of the putative coiled coil is indeed responsible for trimerization, with additional interactions between the lectin heads resulting in the formation of hexamers observed in solution.

Partial characterization of natural and recombinant human soluble CD23.

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.

Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.