Reconstitution, Identification, and Purification of the Rat Liver Golgi Membrane GDP-fucose Transporter

Human membrane 17␤-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17␤-hydroxysteroids into their inactive keto forms in a variety of tissues. 17␤-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-␤-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-␤-D-maltoside-destabilized liposomes prepared from L-␣-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 mol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 mol/ min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 ؎ 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17␤-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K m of NAD being 110 ؎ 10 M and that of NADP 9600 ؎ 100 M using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20␣-dihydroprogesterone as substrates were also determined.

Section: Discussionsupporting

confidence: 74%

Purification, Reconstitution, and Steady-state Kinetics of the Trans-membrane 17β-Hydroxysteroid Dehydrogenase 2

Huang

Lin

2002

“…In fact, we tried several approaches to determine whether Slc35c2 has GDP-fucose transport activity in vitro. Both ER and Golgi membrane fractions isolated from LEC11B CHO cells stably expressing CHO Slc35c2 were assayed for GDP-fucose compared with UDP-Gal transport activity under varying conditions (21). However, although UDP-Gal transport was enriched ϳ10-fold in Golgi versus ER vesicles as expected, no consistent evidence that Slc35c2 affected GDP-fucose transport activity was obtained in several independent experiments.…”

Section: Discussionmentioning

confidence: 96%

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Hou

Shi

et al. 2010

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1-and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.

“…The membrane-bound localization of the protein O-fucosyltransferase identified so far and the presence of mostly high mannose type oligosaccharides on it suggest that this protein is probably localized in either the endoplasmic reticulum or the cis-Golgi region (43). Other than localization of GDP-L-fucose transport activity to the Golgi apparatus (46), no information is presently available on the distribution of the GDP-L-fucose transporter within the Golgi system. It is not known if transport occurs through only one protein species or other isoforms that can be expressed at different Golgi sites.…”

Section: Fig 3 Elution Profiles From Sephadex G-50 Column Obtained mentioning

confidence: 95%

Differential Terminal Fucosylation of N-Linked Glycans Versus Protein O-Fucosylation in Leukocyte Adhesion Deficiency Type II (CDG IIc)

Sturla

Rampal

Haltiwanger

et al. 2003

LAD II/CDG IIc is a rare autosomal recessive disease characterized by a decreased expression of fucosylated antigens on cell surfaces that results in leukocyte adhesion deficiency and severe neurological and developmental abnormalities. Its molecular basis has been identified as a defect in the transporter of GDP-L-fucose into the Golgi lumen, which reduces the availability of the substrate for fucosyltransferases. During metabolic radiolabeling experiments using [ 3 H]fucose, LAD II fibroblasts incorporated significantly less radiolabel compared with control cells. However, fractionation and analysis of the different classes of glycans indicated that the decrease in [ 3 H]fucose incorporation is not generalized and is mainly confined to terminal fucosylation of N-linked oligosaccharides. In contrast, the total levels of protein O-fucosylation, including that observed in Notch protein, were unaffected. This finding demonstrates that the decrease in GDP-L-fucose levels in the fibroblast Golgi caused by the LAD II defect does not impair bulk protein O-fucosylation, but severely affects the bulk addition of fucose as a terminal modification of N-linked glycans. These data suggest that the severe clinical abnormalities including neurological and developmental ones observed in at least some of the LAD II patients may be related to alteration in recognition systems involving terminal fucose modifications of N-glycans and not be due to a defective O-fucosylation of proteins such as Notch.