Dependence of conformation of D3/D4 domains of human CD4 on glycosylation and membrane attachment.
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.
Lideman
L F
LF
Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, Moscow, Russia. info@virology.ru
Gibadulin
R A
RA
D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000939:Epitopes; D006031:Glycosylation; D015658:HIV Infections / Q000097:blood; D006801:Humans; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012995:Solubility; D014162:Transfection
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D015704:CD4 Antigens; D000939:Epitopes; D011134:Polysaccharides