Production and secretion in CHO cells of the extracellular domain of AMOG/beta 2, a type-II membrane protein.
A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/beta 2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/beta 2 (reAMOG/beta 2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/beta 2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/beta 2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immunoaffinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/beta 2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.
Gloor
S
S
Department of Neurobiology, Swiss Federal Institute of Technology, Zürich.
Nasse
K
K
Essen
L O
LO
Appel
F
F
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D027682:Cation Transport Proteins; D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002461:Cell Line, Transformed; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
C504190:ATP1B2 protein, human; C504191:Atp1b2 protein, mouse; D027682:Cation Transport Proteins; D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D021382:Protein Sorting Signals; D000251:Adenosine Triphosphatases