Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 microM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571-->Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.

No abstract

The antiviral activity of an aqueous extract of Podophyllum peltatum was investigated. The extract was fractionated by reversed-phase chromatography, and podophyllotoxin was found to be the most active component in inhibiting the replication of measles and herpes simplex type I viruses. beta-Peltatin and desoxypodophyllotoxin produced marginal antiviral effects, while alpha-peltatin and picropodophyllotoxin were inactive at the levels tested.

There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin b subunit (hCG-b), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-P fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the b subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.Keywords: disease states related to protein misfolding; hCG-P subunit as a model of secretory protein folding; molecular chaperones; protein misfolding; role of disulfide bonds and N-linked glycosylation in protein folding, assembly, and secretion Protein folding of secreted proteinsA growing number of human diseases are now known to be related to protein folding defects, and thus it is important to understand the rules that govern protein folding in vivo. The focus of this review is the mechanisms that regulate protein folding in intact cells, particularly those proteins synthesized in the endoplasmic reticulum (ER) of mammalian cells and targeted for translocation to the cell surface or for secretion.Many of the eukaryotic proteins whose folding and assembly have been studied in vivo are membrane or secreted proteins. They follow a similar route to the cell surface. (1) Synthesis occurs in the rough ER. (2) Nascent proteins are translocated into the cisternal space of the ER where the signal peptide is cleaved; initial co-translational folding involving secondary structure and some native tertiary structure occurs; addition of high-mannose N-linked oligosaccharides and initial processing of N-linked oligosaccharide chains (for glycoproteins) takes place; formation of disulfide bonds occurs, and for multimeric proteins, oligomerization or subunit assembly is attained along with achievement of native structure. (3) The proteins destined for the cell surface or secretion are translocated to the Golgi apparatus, further processed, and then either translocated to the cell surface or packaged into secretory vesicles for secretion.There are some similarities as well as differences between intracellular protein folding and protein folding in test tubes. For instance, for the tailspike protein of Salmonella typhimuriurn phage P22 (Fuchs et al., 1991;Mitraki et al., 1991) and the human chorionic gonadotropin b subunit (hCG-b) , the intermediates in the folding pathway for proteins appear to be the same in vivo and in vitro, but the rate and efficiency with which proteins achieve final native state in vivo is higher than in vitro. There are also many observations both in vivo and in vitro showing that correct folding is in competition...

The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)-beta subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-beta genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of p beta 1 into p beta 2, the rate-determining step in beta folding, was 7 min for wild-type beta but 33 min for beta lacking both N-linked glycans. The p beta 1-->p beta 2 half-time was 7.5 min in CHO cells expressing the beta subunit missing the Asn13-linked glycan and 10 min for the beta subunit missing the Asn30-linked glycan. The inefficient folding of hCG-beta lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG-beta-folding pathway. Unglycosylated hCG-beta was slowly secreted from CHO cells, and beta subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG-beta core fragment-like protein. However, coexpression of the hCG-alpha gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG-beta lacking N-linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG-beta and may be involved in the folding of this beta form. These data indicate that N-linked oligosaccharides assist hCG-beta subunit folding by facilitating disulfide bond formation.