Glycobiology

Materials

Plasmid pPIC9 and P. pastoris strain GS115 (his4) were obtained from InVitrogen (San Diego, CA). Type 1 and Gal[alpha]1-3-type 1-O-octyl were synthesized according to Augé et al. (1976) and to Lubineau et al., (1996), respectively. Dr. M. Palcic (Alberta Research Council, Edmonton, Canada) kindly provided the 8-methoxycarbonyloctyl type 2. H-Type 1-sp-biotin, H-type 2-sp-biotin, Le^a-sp-biotin, Le^x-sp-biotin, sialyl-type 1-O-sp-biotin, and sialyl-type 2-O-sp-biotin were purchased from Synthesome (Russia). GDP-Fractogel was a generous gift of Dr. H. Conradt (Protein Glycosylation, Braunschweig, Germany). Enzymes for DNA manipulations were obtained from Boehringer Mannheim Biochemicals (Mannheim, Germany) or New England Biolabs (Beverly, MA). Yeast extract, bactopeptone, and yeast nitrogen base were from Difco Laboratories (Detroit, MI). All other reagents were of molecular biology grade.

Transfection and expression of the FUT3 gene in COS7 cells

DNA sequence corresponding to the FUT3 gene (1125 bp) was obtained by amplification of the FUT3 cDNA (Mollicone et al., 1994). The amplified fragment containing the two engineered sites EcoRI/XbaI was digested with the respective enzymes and cloned between the EcoRI/XbaI restriction sites of the mammalian expression vector pcDNAI (Invitrogen) to give the pFUT3 vector. COS7 cells were transfected with DEAE-dextran (Davis et al., 1986) and harvested after a 48 h expression period (Weston et al., 1992).

Cloning of the truncated FUT3 gene into the pPIC9 expression vector

Routine cloning experiments were performed as described previously (Ausubel et al.,1989; Sambrook et al., 1989).

The pPIC9[FUT3] vector corresponded to the EcoRI/XbaI FUT3 fragment (1125 bp) excised from the pFUT3 vector and cloned into pPIC9. The pPIC9[FUT3t] vector resulted from cloning of the truncated FUT3 gene (AvrII/NotI digestion of pPIC9[FUT3]) in a pPIC9 plasmid (Figure 1).

The Pichia strain GS115 (his4) was transformed by electroporation (1500 V, 25 µF and 200½, Bio-Rad Gene Pulser) with 10 µg of SalI linearized pPIC9[FUT3t] vector and plated onto a histidine devoid RDB medium (1 M sorbitol, 1% (w/v) dextrose, 1.34% (w/v) YNB, 4·10^-5 % (w/v) biotin, 5·10^-3% (w/v) of each l-glutamic acid, l-methionine, l-lysine, l-leucine, and l-isoleucine). The integration at the expected locus was checked by replica plating on MM and MD agar (1.34% (w/v) YNB, 4·10^-5% (w/v) biotin and 0.5% (v/v) methanol or 1% (w/v) dextrose). Colonies were allowed to grow for 2 days at 30°C. Clones that grew rapidly on MM medium were picked for further investigation.

PCR analysis of Pichia integrants

Yeast genomic DNAwas extracted according to Ausubel et al. (1989), from selected clones grown overnight in 5 ml YPD medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% (w/v) dextrose) at 30°C.

PCR was performed in a mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 9), 1 mM MgCl₂, 100 µM of each dNTP, 1 µl of genomic DNA (about 100 ng), 10 pmol of PCR primers (5[prime]AOX1 : 5[prime]-GACTGGTTCCAATTGACAAGC-3[prime]; 3[prime]AOX1 : 5[prime]-GCAAATGGCATTCTGACATCC-3[prime]) and 1.25 U of Taq DNA polymerase (Promega) in a total volume of 25 µl. After heating at 94°C for 3 min, 25 cycles were performed (denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min). PCR products were analyzed on 1% (w/v) agarose gel electrophoresis.

Subcellular localization of Fuc-TIII activity

Subcellular fractionation of yeast cells was made as described by Gallet et al. (1995). Cells (48 h) were harvested by centrifugation (3000 × g for 5 min and 4°C) and washed in cold distilled water and then by mannitol/Tris buffer (0.6 M mannitol, 10 mM Tris-HCl pH 7.4). Cells were suspended in the same buffer and broken with acid-washed glass beads (6 × 30 s at 4°C). The homogenate was centrifuged at 1000 × g for 5 min and 4°C to pellet glass beads and unbroken cells. The resulting supernatant was centrifuged at 3600 × g for 5 min and 4°C to pellet cell walls. The nuclear fraction was obtained by centrifugation at 5000 × g for 5 min at 4°C while mitochondria were pelleted at 25,000 × g for 10 min and 4°C. The microsomal fraction was then sedimented at 120,000 × g for 1 h and 4°C. The resulting supernatant represented the cytosolic and periplasmic space fractions.

Pichia expression studies

Yeast preculture was made in 10 ml of MG medium (1.34% (w/v) yeast nitrogen base, 4·10^-5% (w/v) biotin, 1% (v/v) glycerol) for 12 h at 30°C to A_{60 0} = 4. Cells harvested by centrifugation (3000 × g for 5 min and 20°C) and washed with 10 ml of 1.34% (w/v) YNB, were then mixed to 10 ml of MM medium (A₆₀₀ = 1). Induction was carried out for 48 h with 1% (v/v) methanol under vigorous shaking at 30°C. Cells were harvested by centrifugation (3000 × g for 5 min and 4°C), washed with cold water, and suspended in 2.5 mM sodium cacodylate, pH 6.5. Cell breakage was made as described above. The protein content was determined by the Bradford assay (Bio-Rad) using bovine serum albumin as a standard (Bradford, 1976).

Fermentation

The inoculum for enzyme production, was obtained from 200 ml of overnight MG medium culture (A₆₀₀ = 10). Cells were harvested by centrifugation 3000 × g for 10 min and 20°C, washed by 50 ml of 0.67% (w/v) YNB and diluted to A₆₀₀ = 0.86 in the growth medium composed of 0.67% (w/v) YNB and 4·10^-5 % (w/v) biotin. During the 169 h induction, pH was maintained at 6.0 with 28% (v/v) ammonium hydroxide and temperature to 30°C. Methanol was continually added at a flow of 5 g/l·h. Agitation speed was 600 r.p.m., and air flow was 2.5 l per min. A supplement of YNB/biotine 5× was added to account for water evaporation. To prevent extensive foam formation, antifoam A (Sigma) was added. At the end (169 h), the culture was harvested by centrifugation 3000 × g for 10 min and 4°C. Supernatant and cells were recovered and stored at -20°C.

SDS-PAGE and Western blot analysis

Culture supernatant (20 µl) was analyzed by SDS-PAGE using a 12.5% acrylamide Tris-Tricine gel (SchSgger and Von Jagow, 1987). Proteins were visualized by silver staining (Blum et al., 1987). The amount of Fuc-TIII enzyme was determined by densitometry analysis (Vilbert-Lourmat, BioProfil software). Proteins were transferred onto nitrocellulose membrane (Schleicher & Schuell, Germany) which was blocked overnight at 4°C by phosphate-buffered saline containing 1% (v/v) Tween 20 and 5% (w/v) bovine serum albumin (PBS-T-BSA). After three washings in PBS-T, the membrane was incubated for 1 h at 20°C with a rabbit anti-Fuc-TIII antibody in PBS-T-BSA buffer at 1:500 dilution. Donkey anti-rabbit immunoglobulin coupled to horseradish peroxidase (Amersham, England), was used at 1:1000 dilution. The blots were developed according to the ECL detection Kit (Amersham, England).

Protein purification

Fifty milliliters of the culture broth were centrifuged for 20 min at 18,000 × g and 4°C and applied onto a 2 ml GDP-Fractogel column equilibrated with 20 mM Mes-KOH, pH 6.8, containing 0.02% (w/v) NaN₃ and 1 mM dithiothreitol at a flow rate of 0.8 ml·min^-1 at room temperature. The column was washed by 20 mM Mes-KOH buffer, pH 6.8 (19 ml), containing 50 mM NaCl, 0.02% (w/v) NaN₃, and 30% (v/v) glycerol at a flow rate of 0.8 ml·min^-1 and the same buffer (19 ml) containing 500 mM NaCl at the same flow rate. The enzyme was eluted from the column by the same buffer (14 ml) containing 1.5 M NaCl at a flow rate of 0.4 ml·min^-1. The eluted fraction was stored at -20°C or precipitated by 1 volume of acetone/[beta]-mercaptoethanol 0.7% (v/v) at -20°C for 24 h. After centrifugation for 1 h at 100,000 × g and 4°C, the protein pellet was suspended in 30 µl of elution buffer and analyzed by SDS-PAGE and immunoblotting.

Fucosyltransferase assay

Fucosyltransferase assays were performed according to Oulmouden et al. (1997). The enzyme activity with type 1, type 2, Le^a, or Le^x glycoside acceptors was tested at 37°C in 60 µl of the following reaction mixture: 25 mM sodium cacodylate, pH 6.5, 5 mM ATP, 20 mM MnCl₂, 10 mM [alpha]-l-fucose, 2.8 µM GDP-[¹⁴C]fucose (168 pmol/100,000 c.p.m.). Acceptors were used at 0.1 mM. The reaction was initiated by addition of 34 µl of culture broth or 10⁸ whole yeast cells or 50 µg of COS7 cell proteins. After 1 h of incubation, the reaction was stopped by addition of 3 ml of cold water, then centrifuged at 3000 x g for 5 min and 20°C. The supernatant was applied onto Sep-Pak C18 cartridges, which were washed with 10 ml of water. The products were eluted with 10 ml methanol. The incorporation of [¹⁴C]fucose was determined by liquid scintillation counting.

Enzyme kinetics was performed in reaction mixtures as described above except that the concentrations of the type 1, H-type 1, Gal[alpha]1-3-type 1, and sialyl-type 1 acceptors varied from 0 to 0.1 mM and GDP-[¹⁴C]fucose was 7 µM. The apparent kinetic parameters were determined from Michaelis-Menten curves fit to the experimental data using the least square method.

After GDP-Fractogel purification, the enzyme activity was measured in the presence of type 1 acceptor as described above, except that 0.2 mM (12.12 nmol/100,000 c.p.m.) GDP-Fucose was used. One unit of enzyme activity was defined as the amount of enzyme catalyzing the transfer of 1 µmol of fucose/min to the octyl-glycoside type 1 substrate.

To determine enzyme stability, 48 h methanol-induced Pichia cells (1 ml) were pelleted by centrifugation at 3000 × g for 5 min and 4°C, washed with cold distilled water, and suspended into 1 ml of 25 mM sodium cacodylate, pH 6.5. Aliquots of 2·10⁸ and 5·10⁷ cells were used to determine the Fuc-TIII activity. The reaction mixture was as described above, with 0.1 mM type 1 acceptor and 2.8 µM GDP-[¹⁴C]fucose. The incubation times were 1 and 4 h for 2·10⁸ and 5·10⁷ cells, respectively. The reaction was stopped with 3 ml of cold distilled water. The suspension was centrifuged at 3000 × g for 5 min and 4°C to pellet cells which were immediately stored at -20°C.