title: Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant.
abstract: The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.
authors.affiliation: Agency for Science, Technology and Research, Bioprocessing Technology Institute, Singapore.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Zhang
authors.forename: Ryan
authors.initials: R
authors.lastname: Haryadi
authors.forename: Kah Fai
authors.initials: KF
authors.lastname: Chan
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.forename: John
authors.initials: J
authors.lastname: Goh
authors.forename: Natasha Ann
authors.initials: NA
authors.lastname: Pereira
authors.forename: Huatao
authors.initials: H
authors.lastname: Feng
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D017124:Conserved Sequence; D006224:Cricetinae; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D054643:INDEL Mutation; D037102:Lectins / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D016366:Open Reading Frames; D019887:Peanut Agglutinin / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D016335:Zinc Fingers
chemical_list: D037102:Lectins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D019887:Peanut Agglutinin; C426650:SLC35C1 protein, human; C064549:lectin, Aleuria aurantia