The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/)

Role of the human serotonin transporter external gate in substrate and antagonist recognition

The serotonin transporter (SERT) is a twelve transmembrane (TMH) alpha helical protein that is responsible for the clearance of serotonin (5-HT) from the synaptic cleft. The regulation of serotonergic levels has been implicated in a number of disorders including depression and addiction. As such, SERT is the target of most antidepressants including the tricyclic antidepressants as well as several drugs of abuse including the amphetamines. However, the sites of interaction with SERT for the antidepressants and amphetamines have remained elusive. The purpose of these studies was to better understand the role of the internal and external gating residues in the mechanism of SERT action. A recently constructed homology model, coupled with mutagenesis data between human SERT (hSERT) and Drosophila SERT (dSERT) was used to determine a potential binding site for the tricyclic antidepressants and propose a mechanism for amphetamine-induced efflux at the transporter. Specifically, a region near extracellular loop (ECL) 2 that appears important for tricyclic antidepressant recognition was identified. The importance of a salt-bridge at the external gate in the ability of the transporter to efflux 5-HT in response to amphetamines was also determined