Interrelations between assembly and secretion of recombinant human acetylcholinesterase.
Transport and secretion of recombinant human acetylcholinesterase (rHuAChE) were studied in transfected human 293 cells expressing either the oligomerized soluble enzyme or a monomeric mutant derivative in which Cys-580 was substituted by alanine (C580A). In cells expressing the wild-type enzyme, the gradual assembly of newly synthesized intracellular rHuAChE monomers into oligomers occurs within the endoplasmic reticulum. Secretion of mature wild-type enzyme into the medium is efficient and appears to be exclusive to multimeric forms. Consistently, intracellular oligomers, but not monomers, are endoglycosidase H-resistant, indicating that only oligomers undergo terminal glycosylation in the wild-type enzyme. In contrast, in cells expressing the dimerization-defective C580A mutant, newly synthesized rHuAChE monomers undergo terminal glycosylation and are secreted into the medium as efficiently as wild-type multimers. No significant difference between the intracellular transport rates of wild-type rHuAChE oligomers and mutant C580A monomers was revealed by probing with specific lectins. In both systems, transport and processing prior to the trans-Golgi galactosylation compartment appear to be rate-limiting, whereas the following passage to the cell surface is rapid. In conclusion, we suggest that in the presence of a free cysteine at the COOH terminus of the rHuAChE polypeptide, secretion of monomers is not effectuated, whereas in its absence, monomers are exported from the endoplasmic reticulum and are capable of traversing the entire secretory pathway.
Kerem
A
A
Department of Biochemistry, Israel Institute of Biological Research, Ness-Ziona.
Kronman
C
C
Bar-Nun
S
S
Shafferman
A
A
Velan
B
B
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000110:Acetylcholinesterase