Structural analysis of the CD5 antigen

Abstract

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 Leu1. Circular dichroism and NMR analyses indicate that CD5d1 has a high β-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 (cCD5d1Ϫ3-CD4d3ϩ4), 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.Keywords : CD5; scavenger receptor cysteine-rich ; glycosylation; disulphide bonds ; NMR spectroscopy.The cell surface glycoprotein CD5 is expressed on thymocytes, T lymphocytes and some B cells [1]. Functional data including studies of knock-out mice point to a role for CD5 in the control of lymphocyte responses (e.g. [2,3] 118 , CD5d1 residues 1Ϫ118 expressed in CHO cells ; cCD5d1Ϫ3-CD4d3ϩ4, a chimaera consisting of the entire extracellular region of human or rat CD5 fused to domains 3 and 4 of rat CD4; cCD5d1-CD4d3ϩ4, a chimaera of human CD5 residues 1Ϫ118 fused to rat CD4 domains 3 and 4; pCD5110, CD5d1 residues 1Ϫ110 expressed in Pichia pastoris ; CHO, chinese hamster ovary cells ; endo, endoglycosidase; ESI-MS, electrospray ionisation mass spectroscopy; gu, glucose units; MALDI-MS, matrix-assisted laser-desorption ionisation mass spectroscopy ; SEC, size-exclusion chromatography; SRCR, scavenger receptor cysteine-rich superfamily; WAX, weak anion-exchange chromatography. three scavenger receptor cysteine-rich (SRCR) domains [4]. In structure and tissue distribution CD5 resembles CD6 which has three SRCR domains [5,6] and SPA which also has three SRCR domains but is a soluble protein that binds to macrophages [7]. SRCR domains are found in various cell surface antigens of leukocytes ...

Frontline: Optimal T cell activation requires the engagement of CD6 and CD166

“…The schematic drawing in Fig. 1A is supported by electron micrographs of a CD5CD4d3+4 in which IgSF and SRCR domains have similar dimensions and the three domains of CD5 formed a linear array [39]. Recruitment of CD6 to the immunological synapse is a prerequisite for optimal T cell activation and blocking the CD6/CD166 interaction prevents this recruitment from occurring.…”

Three-dimensional Solution Structure and Conformational Plasticity of the N-terminal Scavenger Receptor Cysteine-rich Domain of Human CD5

“…23 The chimaera design allowed purification of the construct on a BIAcore sensor chip using a CD4 mAb, avoiding any bias towards a particular CD5 ext conformation resulting from the immobilization chemistry. V88D and V97K single-mutant chimaeras were not expressed as soluble proteins in human embryonic kidney (HEK) 293T cells, but the expression level of the double mutant V88D/V97K CD5 ext /CD4d3+4 was similar to that of the wildtype chimaera.…”

“…In SRCR domains with eight cysteines, the disulphide bond connectivity is (in order of occurrence of the cysteines in the polypeptide sequence) C1-C4, C2-C7, C3-C8 and C5-C6. [22][23][24] Every known SRCR domain has at least two putative intradomain disulphide bonds, one of which is always equivalent to C3-C8. Traditionally, the SRCR superfamily had been divided in two groups, SRCR A and SRCR B, according to the number and position of the cysteines present.…”

“…26,27 Earlier, we reported attempts to obtain human CD5d1 in recombinant form for analysis of its structure and function. 23,28 Recombinant N-glycosylated CD5d1 can be obtained in significant yield from Chinese hamster ovary (CHO) cells and Pichia pastoris yeast cultures, but these materials have proved refractory to structure determination. Here we report the production of CD5d1 expressed in Escherichia coli (eCD5d1).…”

Role of CD5-negative CD8⁺ T Cells in Adaptation to Antigenic Variation of Human Immunodeficiency Virus Type 1

“…The modulation of antigen-specific signalling on virtually all T cells in a ligand-independent manner is performed by group B cysteine-rich scavenger receptor CD5 [9][10][11][12][13]. The conjugation of T-cells with antigen-presenting cells is accompanied by co-localization of CD5 with Tcell receptors (TCRs).…”