Characterization of a soluble form of human CD4

Abstract

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class I1 major histocompatability (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells.This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P. J. et al. (1985) Cell 42, 93-1041. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345.The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-/3-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no 0-linked oligosaccharides.

Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells

“…In preliminary experiments (Figure 2; Harris et al, 1990), Asn-300 had been found to carry a mixture of endo H susceptible and endo H resistant oligosaccharides. To isolate oligosaccharides for structure elucidation, a sample (960 nmol) of the purified Asn-300-containing glycopeptides was first treated with endo H. At the conclusion of digestion, reversed-phase HPLC was used to recover the released oligosaccharides (which were detected by HPAE chromatography of aliquots of the reversed-phase HPLC column fractions) and the endo H resistant portion of the glycopeptide (not shown).…”

“…This work included assignment of the three intrachain disulfide bonds and demonstration that both potential N-glycosylation sites are glycosylated. Tryptic mapping of enzymatically deglycosylated rCD4 revealed that Asn-300 carried a mixture of endo H resistant and endo H susceptible structures whereas all of the structures attached to Asn-271 were endo H resistant (Harris et al, 1990). The goal of the present study has been the detailed elucidation of the carbohydrate structures of CHO-expressed rCD4, including the distribution of structures at each of the two glycosylation sites.…”

“…The extracellular domain has six cysteine residues that form three intrachain disulfide bonds (Carr et al, 1989;Harris et al, 1990) and two potential sites for N-glycosylation as recognized by the consensus sequence Am-Xaa-Ser/Thr (Kornfeld & Kornfeld, 1985). Konig et al (1 988) have examined the role of glycosylation in obtaining expression of CD4 on the cell membrane of human acute lymphoblastic leukemia cells.…”

Inhibitors of Protein-Disulfide Isomerase Prevent Cleavage of Disulfide Bonds in Receptor-bound Glycoprotein 120 and Prevent HIV-1 Entry

“…1C). Control experiments in which sCD4 was isolated with mAb Leu3a and probed with HRP-NeutrAvidin revealed no biotinylation, confirming that sCD4 does not contain unpaired cysteines (43) and indicating that disulfide bonds of sCD4 had not been reduced by PDI.…”

Failure of Human Immunoresponse to N-Glycolylneuraminic Acid Epitope Contained in Recombinant Human Erythropoietin

“…Various recombinant human glycoproteins synthe sized in CHO cells, such as y-interferon [9], FSH [10], E P O [ll, 12], plasminogen activator [13,14], HIV gp 120 [15], CD4 [16,17] and interleukin-5 [18] have been reported to have N-linked oligosaccharides with mono-, di-, tri-and tetra-antennary chains. They have a2-3 sialyl lactosamine structures at their carbohydrate chain terminals.…”