Rango de hospedero

Sample preparation was carried out with help from Mr. Andrew Streets. The cells were washed three times with 10 ml of 5 mmol / 1 Tris-HCl and 0.15 mmol /1 phenylmethylsulfonylfluoride, pH 7.4. After washing, 10 ml o f fresh buffer was added to each flask and the cells removed with a cell scraper. The cells were lysed in a Parr cell distribution bomb apparatus (nitrogen

bomb) which was maintained at 700 psi for 15 min (apparatus loaned by the Department of Medicine, UCL). Conditions to achieve cell lysis had

previously been determined. To remove any gross cellular debris the cell lysate was centrifuged at 2,000 g for 20 min in a Centaur-2 MSE centrifuge. The supernatant was collected and filtered through a 0.2 p,m syringe top filter. Lipids were extracted from the cell lysates by phase-separation using one part o f chloroform to two parts methanol (Wessel and Flugge, 1984). Free sugars and salts were removed by passing the preparations through a Sephadex HRlO/10 'desalting' column fitted to a Pharmacia FPLC machine with in-line UV detector and X 254 nm filter. The HRlO/10 column retains material of less than 10,000 Da in size and larger proteins elute in the void volume. The buffer used was 20 mmol /1 Tris-HCl, 50 mmol /1 NaCl, pH 7.6, the flow rate was set to 3 ml / min and fractions of 1 ml volume were collected. The elution of the proteins from the cell lysate preparations was monitored by their absorbance in the UV. In practice, the proteins were collected in two 1 ml fractions. The fraction with the greatest amount of protein, as measured by optical density at X 254 nm, was used in the lectin-affmity

chromatography experiments.

4.2.2.2...HPA lectin affinity chromatography.

The experiments were conducted by Mr. Andrew Streets. First the HPA- agarose beads (Sigma) were ‘fined’ as follows: 6 ml of beads were suspended in 20 mmol /1 Tris-HCl, 50 mmol /1 NaCl, pH 7.6 and then transferred to a 10 ml measuring cylinder. After 30 min had elapsed the (cloudy) supernatant was removed. The procedure was repeated a further two times. After the beads had been ‘fined’, four HPA-agarose affinity

chromatography columns were prepared each containing 1 ml bed volume of beads. Each of the columns was washed with 20 column volumes o f 20 mmol /1 Tris-HCl, 50 mmol /1 NaCl pH 7.6 buffer. The proteins prepared from the cell lines, as detailed in 4.2.2.1, were loaded onto the HPA-agarose. In each case, the void volume, 0.8 ml, was allowed to flow, drip-wise, through the column. The column was stoppered for 40 min to allow interaction between the proteins and the beads, this time was determined in

previous experiments. After 40 min had elapsed, the column was

unstoppered and unbound / weakly bound proteins eluted from the column with five column volumes of buffer. 1ml fractions were collected. To elute the bound glycoproteins, 1 ml of freshly prepared 0.25 mol /1 GlcNAc in 20 mmol /1 Tris-HCl, 50 mmol /1 NaCl pH 7.6 buffer was added to the column. The column was stoppered and left at 4°C overnight. The following day the HPA lectin-binding glycoproteins were eluted from the column using 5 column volumes of freshly prepared 0.25 mol / 1 GlcNAc in 20 mmol /1 Tris- HCl, 50 mmol /1 NaCl pH 7.6 buffer. Again, 1 ml fractions were collected.

4.2.2. 3 Dot-blot to confirm the presence o f HPA lectin-binding proteins.

To confirm the presence of HPA lectin-binding glycoproteins, following the affinity chromatography experiments, a dot blot was performed. A 5 pi aliquot from each of the fractions was taken for this purpose, spotted onto a piece o f nitrocellulose membrane and allowed to air dry. The unreacted sites on the nitrocellulose membrane were blocked using a 1% ^ /y solution of bovine serum albumin (BSA, Sigma) in Tris buffered saline with 0.05% ^/y Tween 20, pH 7.6 (TBS-Tween). The BSA had previously been checked for non-reaction with HPA lectin. The blocking step was performed for 30 min with agitation, afterwhich the nitrocellulose was washed three times with the TBS-Tween solution to remove the BSA solution. The nitrocellulose

membrane was then incubated overnight in a solution o f 1 pg/ml HPA lectin.peroxidase label (Sigma) in TBS-Tween buffer. The next day, the nitrocellulose membrane was washed three times in TBS-Tween and six times with TBS to remove the lectin solution and then the blot was developed with diaminobenzidine and hydrogen peroxide for 10 min.

The dot blot confirmed the presence of HP A lectin-binding glycoproteins, as shown in figure 4.1.

4.2.2. 4 Dialysis prior to oligosaccharide release.

The fractions containing the HPA lectin-binding glycoproteins were collected and transferred to dialysis cassettes MWCO 10 kDa (Pierce). The samples

were dialysed at 4°C for 48 hours, with stirring and four changes of 500 ml HPLC grade water. After dialysis, the glycoproteins were transferred to 5 ml reactor vials (Chromocol) and freeze-dried for a minimum o f 48 hours as detailed in section 2.2.4.3.

4.2,3 Preparation ofpeptides from an HPA binding breast cancer specimen.

The experiments to release peptides from an HPA lectin-binding breast cancer specimen, reference number 1303, detailed in section 4.2.1.2, were conducted with help from Miss Stephanie Slinn. 50 sections, each 15 )im thick, were cut from the paraffin-wax embedded block. The sections were placed into a glass universal container and dewaxed using the method described in section 2.2.4.2 and lyophilised as detailed in section 2.2.4.3. Cyanogen bromide (CNBr) was used to cleave the lyophilised proteins into peptide fragments (Gross and Witkop, 1962) CNBr cleavage o f proteins has been reported not to damage the oligosaccharides attached (Brooks et al.,

1998).

48.1 mg of lyophilised protein was transferred to a round bottomed flask and a saturated solution of CNBr (Sigma), approximately 0.25 g in 25 ml of 70 % ^/v formic acid, was added. A nitrogen blanket was applied and the round bottomed flask corked with glass wool then incubated at 60°C for 2 hrs and then at 3 7°C for a further 60 hrs. At the end of the incubation period the reaction mixture was diluted to 7 % ^/y formic acid solution and filtered through nylon 140 fj,m mesh, the material was then centrifuged at 2,000 g for 20 min in a Centaur MSE centrifuge. The supematent was collected and freeze-dried for 48 hours to remove residual CNBr. The peptide preparation was then solubilised into water and transferred to a dialysis cassette, MWCO 10 kDa (Pierce), and dialysed at 4°C with stirring, for 48 hours to remove any remaining CNBr. Four changes of water were made, each 500 ml. After dialysis, the peptides were transferred to two 5 ml reactor vials (Chromocol) and freeze-dried for 48 hours. One of the reactor vials, containing 30 mg of freeze-dried peptides, was used for oligosaccharide release as shown below. The other was stored at -20°C for later use.

4.2.4 Oligosaccharide release and labelling.

The oligosaccharides were released, from the cell line preparations using 200

|Lil o f hydrazine and from the peptide preparation using 2.5 ml o f hydrazine,

then worked-up and labelled using the methods detailed in section 2.2.5.

4.2.5 A nalysis o f oligosaccharides released.