Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.
CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.
König
R
R
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Ashwell
G
G
Hanover
J A
JA
D000818:Animals; D015139:Blotting, Southern; D015704:CD4 Antigens / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics*; D005260:Female; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D015870:Gene Expression; D005796:Genes; D006801:Humans; D010053:Ovary; D010957:Plasmids; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D014162:Transfection
D015704:CD4 Antigens; D003078:Colchicine