Stable expression of acetylcholinesterase and associated collagenic subunits in transfected RBL cell lines: production of GPI-anchored dimers and collagen-tailed forms.
We obtained a stable expression of acetylcholinesterase (AChE, E.C. 3.1.1.7) in the rat basoleukemia cell line, RBL-2H3, which possesses a well developed secretory pathway, but expresses only very little endogenous AChE. Metabolic labeling showed that AChEH and AChET, differing by C-terminal peptides encoded by alternatively spliced exons, were synthesized at a similar rate. When transfected with AChEH, RBL cells efficiently produced GPI-anchored dimers, which were mostly exposed at the cell surface, as shown both by activity and immunofluorescence labeling. In contrast, when transfected with AChET, RBL cells produced about tenfold less activity, which was essentially retained in the cell, and the enzyme could not be detected at the cell surface by immunolabeling. The fate of the enzyme is therefore determined by its C-terminal alternative peptides. We were also able to coexpress the AChET subunit with the collagenic Q subunit. The cells produced small but significant amounts of collagen-tailed forms, essentially A4. The expression of these different catalytic and structural subunits in stably transfected RBL cells will be useful to explore the regulated posttranslational processes involved in protein maturation and transport.
Coussen
F
F
Laboratoire de Neurobiologie Moléculaire et Cellulaire, CNRS URA 1857, Ecole Normale Supériure, Paris/France.
Bonnerot
C
C
Massoulié
J
J
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002463:Cell Membrane Permeability; D002499:Centrifugation, Density Gradient; D003094:Collagen / Q000096:biosynthesis*; D005455:Fluorescent Antibody Technique; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis*; D015471:Leukemia, Basophilic, Acute; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D003094:Collagen; D000110:Acetylcholinesterase