Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.
CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has 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 Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular 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 carbohydrate 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 linker region.
McAlister
M S
MS
Department of Biochemistry and Molecular Biology, University College London, UK.
Brown
M H
MH
Willis
A C
AC
Rudd
P M
PM
Harvey
D J
DJ
Aplin
R
R
Shotton
D M
DM
Dwek
R A
RA
Barclay
A N
AN
Driscoll
P C
PC
D000595:Amino Acid Sequence; D000818:Animals; D018956:CD5 Antigens / Q000737:chemistry* / Q000235:genetics; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002942:Circular Dichroism; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008854:Microscopy, Electron; D010843:Pichia / Q000235:genetics; D051381:Rats; D011971:Receptors, Immunologic / Q000737:chemistry*; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B
D018956:CD5 Antigens; D002241:Carbohydrates; D004220:Disulfides; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B