Dependence of Conformation of D3/D4 Domains of Human CD4 on Glycosylation and Membrane Attachment

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L. F. Lideman^* and R. A. Gibadulin

Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, ul. Gamalei 16, 123098 Moscow, Russia; fax: (499) 190-2867; E-mail: info@virology.ru; llideman@mail.ru; gibadulin2@rambler.ru

^* To whom correspondence should be addressed.

Received January 29, 2008; Revision received June 30, 2008

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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.

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KEY WORDS: CD4, autoimmune epitope, glycosylation, protein folding, HIV-1

DOI: 10.1134/S0006297909020102

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