The folding and cell surface expression of CD4 requires glycosylation.

C.J Tifft,R.L Proia,R.D Camerini-Otero

doi:10.1016/s0021-9258(19)50726-8

Abstract

Human CD4, a monomeric T cell surface glycoprotein, is required for T helper cell activation and is also the receptor for the human immunodeficiency virus. There have been conflicting reports as to whether glycosylation of CD4 is required for its cell surface expression. To clarify the effect of glycosylation on surface expression, folding, and intracellular sorting of CD4, we generated a series of mutant cDNAs in which one, the other, or both glycosylation recognition sites were eliminated. Using in vitro transcription and translation we confirmed that both potential glycosylation sites of CD4 were utilized. Transient expression of the mutants in HeLa cells demonstrated that glycosylation at either site was necessary and sufficient for cell surface expression. Finally, we showed that unglycosylated CD4 produced in HeLa cells was incorrectly folded and retained intracellularly, probably in the endoplasmic reticulum.

Highlights

Human CD4, a monomeric T cell surface glycopro- and to tcheell surface
Materials-Dulbecco’s modified minimal essential medium with 25 mM HEPES (DMEM) [22], fetal bovine serum (FBS), penicillin/ streptomycin, and trypsin/versene solutions were purchased from Biofluids
Following addition of proteinase K (0.1 mg/ml final concentration) fected HeLa cells were washed in PBS, trypsinized, and resuspended to removeCD4 protein outside the microsomal membranes and in 1 mlof cold PBS with 2.5% FBS (PBS/FBS).The cells were

Summary

THEJOURNOAF LBIOLOGICACLHEMISTRY

Vol 267, Nofo. 5, Issue February 15. pp. 3268-3273.1992 Printed in U.S.A. From the Genetics and BiochemistryBranch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. At the cell surface CD4 facilitates tein, is required for T helpecrell activation and is MHC class 11-restricted antigen recognition and T cell actithe receptor for the human immunodeficiency virus. Conflicting reports have appeared regardof the mutants inHeLa cells demonstrated that glyco- ing a glycosylation requirement for cell surface expression of sylation ateither site was necessaryand sufficient for CD4. Human CD4 is a monomeric integral membrane protein primarily present on a subsetof T lymphocytes [1] and other hematopoietically derived cells [2,3,4,5]. CD4, like other membrane glycoproteins, is synthesized in the rough endoplasmic reticulum (ER) where the signal pepthe receptor for HIV infection, indicating that it functioned at thecell surface. CD4 contains two such potential Asn-linked glycosylation sites at Asn2I3and Asn300between the third andfourth immunoglobulin-like domains.

EXPERIMENTAL PROCEDURES

Findings

RESULTS