The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system. Overexpression of Myc-SERT using the baculovirus system led to substantial quantities of inactive transporter, together with small amounts of fully active and, therefore, correctly folded molecules. The high levels of inactive Myc-SERT probably arose because folding was rate-limiting due, perhaps, to insufficient molecular chaperones. Therefore, Myc-SERT was co-expressed with the endoplasmic reticulum (ER) molecular chaperones calnexin, calreticulin and immunoglobulin heavy chain binding protein (BiP), and the foldase, ERp57. The expression of functional Myc-SERT, as determined by an inhibitor binding assay, was enhanced nearly 3-fold by co-expressing calnexin, and to a lesser degree on co-expression of calreticulin and BiP. Co-expression of ERp57 did not increase the functional expression of Myc-SERT. A physical interaction between Myc-SERT-calnexin and Myc-SERT-calreticulin was demonstrated by co-immunoprecipitation. These associations were inhibited in vivo by deoxynojirimycin, an inhibitor of N-glycan precusor trimming that is known to prevent the calnexin/calreticulin-N-glycan interaction. Functional expression of the unglycosylated SERT mutant, SERT-QQ, was also increased on co-expression of calnexin, suggesting that the interaction between calnexin and SERT is not entirely dictated by the N-glycan. SERT is the first member of the neurotransmitter transporter family whose folding has been shown to be assisted by the molecular chaperones calnexin, calreticulin, and BiP.

Insect cells have been widely utilized as hosts for the production of numerous glycoproteins through the baculovirus expression system (1, 2). Native insect cell glycoproteins also serve as models for developmental processes in eucaryotes (3, 4). While the structure, synthesis, and function of oligosaccharides in mammalian glycoproteins are well characterized, information on the carbohydrate structures and processing pathways present in insect cells is limited and sometimes contradictory (5-7). The oligosaccharides in glycoproteins can play critical roles in cellular targeting, structural stability, resistance to proteolysis, immunogenicity, and circulatory halflife (8, 9). With insects representing more than half of the animal species classified (5), there is a need to obtain more information on the carbohydrate structures and processing of glycoproteins from insect cells.Many initial studies of N-glycans in insect cell-derived heterologous glycoproteins indicated the presence of only high mannose-type or short truncated, paucimannosidic oligosaccharides, 1 sometimes containing L-fucosyl 2 residues (6, 7). These observations confirmed the earlier studies of endogenous insect cell glycoproteins which were similarly found to lack complex carbohydrate structures (10 -12). It was presumed that insect cells did not possess the capacity to synthesize complex-type oligosaccharides since the levels of sialyl-, galactosyl-, and N-acetylglucosamine transferases were found to be insignificant (12).In contrast, studies with the recombinant human plasminogen indicated that insect cells could synthesize complex Nlinked oligosaccharides (13). Several more recent studies on homologous glycoproteins also have indicated that certain insect cell lines can synthesize hybrid and complex oligosaccharides. Honeybee venom phospholipase A 2 (PLA 2 ) 3 was found to

Human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) are being studied for cell replacement therapies, including the treatment of acute spinal cord injury. Current methods of differentiating OPCs from hESCs require complex, animal-derived biological extracellular matrices (ECMs). Defined, low-cost, robust, and scalable culture methods will need to be developed for the widespread deployment and commercialization of hESC-derived cell therapies. Here we describe a defined culture system that uses a vitronectin-derived synthetic peptide acrylate surface (VN-PAS; commercially available as Corning(®) Synthemax(®) surface) in combination with a defined culture medium for hESC growth and differentiation to OPCs. We show that synthetic VN-PAS supports OPC attachment and differentiation, and that hESCs grown on VN-PAS are able to differentiate into OPCs on VN-PAS. Compared to OPCs derived from hESCs grown on ECM of animal origin, higher levels of NG2, a chondroitin sulfate proteoglycan expressed by OPCs, were observed in OPCs differentiated from H1 hESCs grown on VN-PAS, while the expression levels of Nestin and PDGFRα were comparable. In summary, this study demonstrates that synthetic VN-PAS can replace complex, animal-origin ECM to support OPC differentiation from hESCs.

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