Biosynthesis and oligosaccharide structure of human CD8 glycoprotein expressed in a rat epithelial cell line.
The biosynthesis, post-translational modifications, and oligosaccharide structure of human CD8 glycoprotein have been studied in transfected rat epithelial cells. These cells synthesized and expressed on the plasma membrane high amounts of CD8 in a homodimeric form stabilized by a disulfide bridge. Three different CD8 forms were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after metabolic labeling and immunoprecipitation: a newly synthesized, unglycosylated 27-kDa (CD8u), a palmitylated and initially O-glycosylated 29-kDa (CD8i), and the mature, terminally glycosylated 32-34-kDa doublet (CD8m). CD8i is a transient intermediate form between CD8u and CD8m: characterization of carbohydrate moiety of [3H]glucosamine-labeled CD8i showed that it comprises for the vast majority non-elongated O-linked GalNAc closely spaced on the peptide backbone. Structural analysis of oligosaccharides released by mild alkaline borohydride treatment from the [3H]glucosamine-labeled CD8 34-kDa form showed that the neutral tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAcOH, and an homologous monosialylated pentasaccharide, predominate; the disialylated NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6) GalNAcOH tetrasaccharide appeared to be poorly present. In the CD8 32-kDa form the neutral tetrasaccharide was by far the prominent O-linked chain, and no disialyloligosaccharides were identified. These results indicate that the maturation of CD8 glycoprotein in transfected rat epithelial cells results in the formation of branched O-linked oligosaccharides and that a higher degree of sialylation is responsible for the production of the heavier 34-kDa form.
Pascale
M C
MC
Department of Biochemistry and Medical Biotechnology, University of Naples, Italy.
Malagolini
N
N
Serafini-Cessi
F
F
Migliaccio
G
G
Leone
A
A
Bonatti
S
S
D000818:Animals; D016827:CD8 Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004848:Epithelium / Q000276:immunology; D005944:Glucosamine / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D014162:Transfection; D014316:Tritium
D016827:CD8 Antigens; D006020:Glycopeptides; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D014316:Tritium; D005944:Glucosamine