STRUCTURAL-ANALYSIS OF THE CD5 ANTIGEN - EXPRESSION, DISULFIDE BOND ANALYSIS AND PHYSICAL CHARACTERIZATION OF CD5 SCAVENGER RECEPTOR SUPERFAMILY DOMAIN-1

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cell s and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) super family, for which no three-dimensional structure has been obtained. Re combinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, ha s been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leu1. Circular dichroism and NMR analyses indicat e that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding p attern was determined and is consistent with that found for the group- A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the m acrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5, P. pastoris expression provi des large quantities of correctly folded recombinant CD5d1 for multidi mensional NMR and for X-ray crystallographic studies. The whole extrac ellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbo hydrate analysis to gain an overview of the structure of the extracell ular portion of intact CD5, Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbo hydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linke r region.