Characterization of galectins-carbohydrate interactions by affinity- gel

2.3 Characterization of galectins-carbohydrate interactions by affinity- gel electrophoresis

The fractions recovered from protein-carbohydrate affinity experiments were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), to verify (i) the successful preparation of affinity columns, (ii) the analyte affinity of the analyte for the immobilized ligand and (iii) the different binding and elution conditions employed in optimization tests. The experimental results were evaluated by visual or software-aided inspection of the gels.

Mass spectrometry is a rapid technique for analyzing affinity fractions, yet important information such as recovered vs. initial protein amounts can be obtained more accurately by gel electrophoresis. Compared to mass spectrometry, gel electrophoresis is also more tolerant to high concentrations of salts and carbohydrates commonly used to elute carbohydrate-binding proteins from immobilized carbohydrates.

The scheme of the affinity- gel electrophoresis procedure is shown in Figure 26. In the first gel lane the "molecular-weight marker" or "protein ladder", which designates a protein mixture of known composition and well-characterized migration pattern, is applied. The marker serves as reference to estimate the molecular weight (MW) of unknown samples run in the same gel. It was chosen according to the density and composition of the gel and to the estimated molecular weight of the sample, which should fall within the MW range of the marker. The second lane was used for a small volume of the protein stock solution (containing 1-5 μg protein), which serves as a reference for the protein sample subjected to an affinity experiment. Depending on the experimental conditions proteins may undergo (de-)oligomerization, proteolysis or autolysis, may form or lose disulfide bridges or suffer other changes in structure and conformation that affect their migration in gel. The reference band can deliver valuable information on such molecular changes and indicate the necessity of further optimization steps.

The sample loaded on the third lane consisted of the entire solution present in the affinity column after incubating the analyte with the immobilized ligand

(supernatant). A low intensity or absent protein band indicated strong binding of the analyte to the ligand, which lead to its decreased concentration or absence from solution. The very low protein concentration in this case was the reason for using the entire supernatant. The detection of an intense band in this lane, corresponding to the protein under study, indicated lack of analyte binding. This may occur due to faulty column preparation e.g. the ligand immobilization did not proceed with the expected yield or the structure of the affinity matrix was damaged. Another reason for a low intensity signal may be the lack of affinity due to analyte-ligand mismatch or suboptimal experimental conditions. Parameters such as pH and ionic strength of the buffer used for binding are especially important, because they have a strong influence on protein folding, charging of amino acid side chains and intermolecular bonds that form between analyte and ligand.

After washing the column with several column volumes, either continuously or stepwise, the last washing fraction was loaded on the fourth lane. The signal from this lane helped to establish the optimal number of washing steps required to remove all unbound analyte. In case several washing fractions are compared, the signals can also reveal if the analyte-ligand complex is easily dissociated. For the excision approach it was important that only carbohydrate-bound protein is retained on the column.

Insufficient washing leading to residual proteins in the supernatant when the protease is added might result in a mixture of proteolytic excision (from the protein bound on the immobilized carbohydrate) and extraction products (from the protein free in solution). The importance of carefully separating the excision and extraction approaches may be highlighted through the example of antigen-antibody complexes [158-163]. It is known that during excision cleavage sites within the epitope are shielded by the antibody from the action of the protease. The same cleavage sites are readily accessible to the protease when the antigen is digested in solution. Different peptide populations are generated in each approach, providing complementary information on the antibody-antigen contact sites.

The fifth lane was used for the elution fraction. A strong elution band in the conjunction with weak supernatant and washing signals indicated a high

ligand affinity, a good choice of eluent and generally optimal experimental parameters. In contrast, a weak or absent band indicated that the chosen eluent was not strong enough to dissociate the analyte-ligand complex or that there was no more bound analyte to be eluted. In case a protein mixture was used in the affinity experiment, protein bands visible in the stock lane but missing in the elution lane gave information on which components interact with the ligand.

An important step, even for known samples, before the experimental conditions were optimized, was to confirm the exact identity of the proteins in the gel bands. In the present work this was done using a proteomics approach by in-gel digestion followed by (LC-)MS analysis and evaluation of the MS results through database search. If multiple protein bands were observed, blotting of the gels onto PVDF membranes and subsequent N-terminal Edman sequencing of protein spots were also performed.

Concomitant with each protein-carbohydrate affinity-1DE experiment, a control was carried out using the unmodified matrix or an immobilized carbohydrate that did not interact with the protein. This was necessary in order to differentiate between the protein-carbohydrate affinity and unspecific protein-matrix binding. The conditions (buffers, binding and elution times, number of washing steps) were identical in both experiments. In some cases several runs were required to establish the optimal conditions under which the protein bound strongly to the carbohydrate and weakly or not at all to the matrix.

Wash

Figure 26. General scheme of affinity-1DE employed for evaluating protein-carbohydrate interactions.

The protein or protein mixture is added on an affinity column with immobilized carbohydrate. At the end of the incubation period the supernatant is collected and the column is washed. The protein-carbohydrate complex is then dissociated. A suitable MW marker (1) and the recovered fractions (2-5) are subsequently analyzed by gel electrophoresis. Valuable information on optimizing the experimental conditions is obtained by comparing the intensity of the protein bands.

To check the performance of an affinity column with lactose immobilized on divinyl-sulfone-activated Sepharose, a test was performed using human galectin-1 and the procedure described above. The results of the affinity test and of a control experiment with unmodified Sepharose are shown in Figure 27. The stock solution lane showed a strong band at ~15 kDa, corresponding to galectin-1 and a weaker band at ~30 kDa, corresponding to a dimer. The supernatant lane did not contain any protein bands, which indicated that all protein bound to lactose. The lane of the washing fraction was empty, indicating that the washing procedure was effective.

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Figure 27. Affinity-1DE of native hGal-1 interacting with lactose and control columns. The gels were stained with Coomassie blue. The first lane contained the molecular-weight marker (MW).

The stock solution lane (Stock) showed the hGal-1 monomer band at ~15 kDa and a weak dimer band at ~30 kDa. The supernatant lane (SN) did not show any protein bands, indicating complete binding of hGal-1 to lactose. The lack of protein spots in the wash lane (W) showed that the wash was effective. The first elution lane (E1) contained a strong band at ~15kDa, corresponding to hGal-1, while the second elution (E2) was empty, proving that the first elution was enough to dissociate the hGal-1/lactose complex. For the control experiment the strong signal in the supernatant lane and the absence of signals in the elution lanes indicated the lack of affinity between hGal-1 and the matrix.

Two different elution systems were employed. The first elution (E1) was performed with 60 % ACN, 0.1 % TFA in water, while the second elution (E2) was carried out with 0.3 M lactose in PBS. The lactose-based eluent is a well established competitive elution system that served as a reference to evaluate the success of the ACN elution. The reasons for establishing an elution system based on organic solvents as an alternative to carbohydrate-based, competitive eluents were described in Chapter 2.1.1. The strong signal from the first elution combined with the absence of a signal from the second elution demonstrated that the system based on organic solvents was effective at disrupting the protein-carbohydrate interactions. In the control experiment the supernatant lane showed, in contrast to the affinity experiment, an intense protein band similar to the band of the first elution lane from the carbohydrate column. This strongly indicated that all or most of the protein did not bind to the unmodified matrix.

The washing lane contained a weak band suggesting that some of the protein bound initially, but was removed afterwards easily since none of the elution lanes displayed

any signal. In conclusion, galectin-1 did not bind to the unmodified matrix under the conditions applied (Figure 27).

The same experimental setup described above for hGal-1 was used for intact hGal-3. The results obtained are shown in Figure 28. The supernatant and washing fractions were empty, while the elution provided an intense band. In contrast, the control experiment provided a strong signal in the supernatant lane and no band in the elution lane. This indicated that the column preparation was successful and that galectin-3 bound to lactose, but not to the unmodified matrix. In all lanes where the protein band was detected (stock, supernatant and elution) several bands corresponding to lower mass proteins were also present. Since these bands behaved similarly to full-length galectin-3, it is likely they were fragments of galectin-3 with residual affinity. However, since the bands were very weak, N-terminal Edman sequencing of the blotted bands did not provide any results. The corresponding proteins showed similar affinity as hGal-3 and, since the CRD is required for lactose-binding, they were most likely fragments generated by cleavage in the N-terminal domain. This conclusion is also supported by the proteolytic peptide mapping of galectin-3 (see Chapter 2.2).

An identical experiment was performed with the truncated form of human galectin-3 (hGal-3C). The strong band produced by the elution fraction and absence of signals from the supernatant fraction indicated that this form of galectin-3 strongly bound to lactose. The control experiment showed no binding of hGal-3C to the unmodified Sepharose matrix (Figure 29).

strong band at ~30 kDa, while the supernatant and washing fractions were clean, indicating a strong binding of hGal-3 to lactose. The fractions from the control column indicated a lack of affinity between hGal-3 and the Sepharose matrix.

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Figure 29. Affinity-1DE of hGal-3C interacting with lactose and control columns. The gel was stained with Coomassie blue. The elution fraction from the lactose column produced a strong band at ~16 kDa, while the supernatant and washing fractions were clean, indicating a strong binding of hGal-3C to lactose. The fractions from the control column indicated a lack of affinity between hGal-3C and the Sepharose matrix.

The two separate CRDs of human galectin-8, hGal-8N and hGal-8C, were also tested by affinity-1DE (Figure 30). Both lectins were added in identical amounts on identical affinity columns containing lactosyl-DVS-Sepharose from the same batch.

The experiments were performed under identical conditions, using the same buffers and number of washing steps. The supernatant band of the N-domain was weaker than that of the C-domain, while for the elution bands the situation was reversed.

Considering the identical experimental conditions, it was concluded that hGal-8N bound stronger to lactose than hGal-8C, in agreement with previously reported data [164].

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Figure 30. Affinity-1DE of human galectin-8N and -8C interacting with immobilized lactose. The gel was stained with Coomassie blue. The supernatant band of hGal-8N was weaker than the one corresponding to hGal-8C, while the band of the elution fraction was stronger for hGal-8N than for hGal-8C.

2.4 Identification of the carbohydrate binding site in galectins