Dependence of conformation of D3/D4 domains of human CD4 on glycosylation and membrane attachment

• Published in: Biochemistry (Moscow) Vol. 74; no. 2; pp. 194 - 200
• Main Authors: Lideman, L. F, Gibadulin, R. A
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : SP MAIK Nauka/Interperiodica 01-02-2009; SP MAIK Nauka/Interperiodica; Springer Nature B.V
• Subjects: Amino acids; Antibodies, Monoclonal - immunology; Autoantibodies - immunology; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; CD4 Antigens - genetics; CD4 Antigens - immunology; CD4 Antigens - metabolism; Cell Line; Cell Membrane - metabolism; Epitopes; Glycoproteins; Glycosylation; HIV Infections - blood; Humans; Life Sciences; Microbiology; Polysaccharides - metabolism; Protein Conformation; Protein Folding; Protein Structure, Tertiary; Solubility; T cell receptors; Transfection; HIV-1; glycosylation; CD4; protein folding; autoimmune epitope
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297909020102

Conformational dynamics of human T-helper cell receptor protein CD4 has been studied with the help of monoclonal antibody (mAb) T6. The mAb T6 discriminates between s- and m-forms of CD4 and recognizes a specific conformation of the soluble (s) form of CD4 including the first nine amino acids of CD4 transmembrane sequence. However, change of tryptophan for serine in position 2 in this sequence destabilizes the T6-type conformation. By enzymatic deglycosylation and deletions of glycosylation sites, we show that T6-type conformation depends on glycosylation in both sites (Asn271 and Asn300). We show also that the sugars are not involved in direct binding to the antibody but stabilize the D3/D4 local conformation. Deglycosylated forms of sCD4 in vivo acquire a specific conformation similar to the wild type sCD4, which however cannot be restored after denaturation/renaturation under conditions of non-reducing Western blot. This observation indicates that the correct protein folding needs chaperone assistance and cannot be achieved in vitro. Completely non-glycosylated sCD4 is synthesized and secreted into the growth medium. In the medium, this mutant appears to be unstable and aggregates during time. In a contrast to soluble CD4, mutations in glycosylation sites abrogate expression of membrane CD4, thus demonstrating a different secretion pathways for soluble and membrane proteins.