Both MP20 and galectin-3 have been shown to be expressed in lens fiber cells 15161720. The alleged involvement of both proteins in adhesion processes raises the possibility that they interact with each other in the lens fiber cell membranes. As a first step to examine this possibility, the spatial distributions of these two proteins were determined using immunocytochemistry (Figure 1). MP20 was expressed widely in the lens, both in elongating fiber cells near the lens periphery, as well as in mature cells deeper in the lens, which had already lost the cell nuclei (Figure 1A). In the peripheral elongating fiber cells, a significant portion of MP20 appeared to be concentrated in vesicles, possibly representing a precursor state to insertion into the plasma membrane (Figure 1B). Similarly, galectin-3 appeared to be present predominantly in vesicles, occasionally in the same ones as for MP20 (Figure 1B). A strikingly different pattern was observed in the mature fiber cells deeper in the lens: both MP20 and galectin-3 were entirely membrane associated (Figure 1C). MP20 was more uniformly distributed in the plasma membrane than galectin-3, which had a more punctate appearance. In many areas, the two proteins appeared to co-localize, supporting the notion that they might interact with each other. Some galectin-3 also localized in membrane regions where MP20 appeared to be missing, suggesting that galectin-3 might also have other binding partners.

Following on from the observation that MP20 and galectin-3 co-localized in many areas of the fiber cell plasma membrane, we now investigate their ability to bind to each other biochemically using two protocols. In the first protocol, we purified MP20 and galectin-3 individually and characterized their molecular mass. The purified proteins were then combined and the mixture was analyzed for complex formation. In the second protocol, conditions were optimized for the solubilization of native complexes of MP20 and galectin-3 directly from the fiber cell membranes.

MP20 was purified using a previous procedure 26 with some modifications. Outcomes of the individual steps in the procedure are documented in Figure 2. Ovine fiber cell membranes were "fully stripped" of peripheral proteins leaving predominantly the major integral membrane proteins including the 26 kDa major intrinsic polypeptide (MIP) and its 22 kDa cleaved form, MP20 (20 kDa), and the 38 kDa cleaved form of connexin46 and 50 (Figure 2A, lane 2). N-decyl-beta-D-maltopyranoside (DM) solubilized more than 50% of MP20 together with variable amounts of the other integral membrane proteins (Figure 2A, lane 4). All of these proteins including about half of MP20 bound to a MonoQ column. The other half of MP20 appeared in the flow-through fraction and could thus be easily separated from the other proteins (Figure 2B, arrow; Figure 2A, lane 5). Immunoblotting analysis confirmed the identity of MP20. Note that the antibody also recognizes a dimer of MP20 that is not normally detected in stained gels (Figure 2A, lane 8). Gel filtration also indicated that MP20 existed as an oligomer: the protein eluted from the column as a single peak at 15 ml (v_o = 8 ml) giving it an apparent molecular mass of 60 kDa (Figure 2A lane 7, C & D). This is consistent with a dimer of MP20 associated with a substantial amount of detergent, or less likely, with a trimer with no detergent present.

The purification of galectin-3 started from crude fiber cell membrane preparations, which included the membrane-integral proteins mentioned above and in addition a large number of membrane-adherent proteins (Figure 3A, lane 2). A single alkaline-extraction step removed a sufficient amount of galectin-3 for purification together with variable amounts of other membrane adherent proteins (Figure 3A, lane 4). The presence of galectin-3 in this fraction was confirmed by immuno blotting (Figure 3A lane 7). Galectin-3 from was purified by affinity chromatography on a α-lactose-agarose column 27 taking advantage of the lectin binding site of the protein. A single peak was eluted with 100 mM lactose (Figure 3B) and contained exclusively galectin-3 with an apparent molecular mass of 31 kDa by SDS-PAGE (Figure 3A, lane 5). Its identity was confirmed by immuno blotting analysis (Figure 3A lane 8). On a gel filtration column (Figure 3C) galectin-3 eluted at 12.5 ml (Vo = 8.3 ml) (Figure 3A lane 6) corresponding to an apparent molecular mass of 22 kDa (Figure 3D), which is smaller then the apparent molecular mass on SDS PAGE. This suggests that the purified galectin-3 is in a (hydrodynamically) compact conformation and is monomeric.

Having obtained pure preparations of MP20 and galectin-3, their ability to interact with each other was investigated by mixing them and monitoring for the formation of a complex composed of both proteins. This was achieved by gel filtration of the mixture, which should either show a peak for each protein individually with the predetermined molecular weights thus indicating a lack of interaction, or a major peak at a higher molecular weight containing both proteins thus indicating a positive interaction. When mixing pure MP20 (Figure 4A lane 2) and pure galectin-3 (Figure 4A lane 3) with a molar ratio of 1:6, the predominant peak eluted at 13 ml (v_o = 7.8 ml) corresponding to an apparent molecular mass of 104 kDa (Figure 4B and 4C). SDS-PAGE revealed the presence of MP20 and galectin-3 (Fig 4A, lane 4), the latter appearing as a doublet due to minor proteolysis. Such proteolysis had previously been observed and apparently did not abolish binding 28. The peak at the right edge of Figure 4B contained unbound galectin-3 but no free MP20 was observed. When a similar experiment was carried out using a molar ratio of 1:2, the elution profile was similar to the above except that an additional peak was obtained that contained exclusively MP20 (data not shown). Hence an excess of galectin-3 had to be used to saturate all MP20 indicating that not all galectin-3 had retained the ability to bind to MP20, or that purified MP20 and galectin-3 have a moderate affinity to each other. Nevertheless the data confirm that galectin-3 is a ligand to MP20 and that the two proteins can form a complex in vitro.

This ability of MP20 and galectin-3 to bind to each other was further supported by the second protocol aimed at purifying native MP20/galectin-3 complexes directly from fiber cell membranes. Starting from "partially stripped" membranes, which still contained some galectin-3 (Figure 5A, lane 2), MP20 consistently co-purified with galectin-3 on the MonoQ column (Figure 5B arrow; 5A, lane 3). Gel filtration of this fraction (indicated by the arrow on Figure 5B) produced a single peak eluting at 13.8 ml (vo = 8 ml) corresponding to an apparent molecular mass of 102 kDa (Figure 5C &5D) and containing both proteins (Figure 5A lanes 5–7). This strongly indicates that galectin-3 and MP20 interact with each other also in vivo.

Since galectin-3 had previously been shown to bind other protein ligands via its lectin domain or other segments of the molecule 21, the interaction between MP20 and galectin-3 was further analyzed to distinguish between these two possibilities. Native MP20/galectin-3 complexes were first solubilized from crude membranes, and then the involvement of the lectin site was investigated by immunoprecipitation with anti MP20 and protein A sepharose beads in the absence or presence of 100 mM lactose, which competes for the lectin site. In the absence of lactose, galectin-3 co-precipitated with MP20 as expected (Figure 5E lanes 1 and 3). However, in the presence of 100 mM lactose most galectin-3 was removed from the native complexes leaving only MP20 (Figure 5E lanes 2 and 4). Lactose did not affect MP20 binding to the protein A sepharose beads as equal amounts of MP20 bound to the beads irrespective of whether lactose was present or not (Figure 5E lanes 3 and 4). This result suggests that galectin-3 binds to MP20 via its lectin site.

Rat lenses were fixed in 0.75% paraformaldehyde in phosphate buffered saline (PBS) for 24 h at room temperature. They were washed 3 times for 10 min in PBS, then cryoprotected by incubation in 10% sucrose in PBS for 1 hr at room temperature, followed by 20% sucrose in PBS, and then 30% sucrose in PBS at 4°C overnight. Lenses were mounted in Tissue-Tek O.C.T. compound at 4°C on pre-chilled chucks. The chucks were immersed in liquid nitrogen for 25 s to freeze the lenses then stored on dry ice. 10 μm thick cryosections were cut at -18°C on a cryostat (Leica CM3050), and placed onto slides coated with poly-L-lysine. All following procedures were carried out either at room temperature for the times indicated, or at 4°C overnight, in a humid box. Slides were washed with PBS and treated for 1 hr with blocking solution (3% bovine serum albumin, 3% fetal calf serum in PBS), washed 3 times for 5 min in PBS, and treated for 2 hrs with a mouse monoclonal anti galectin-3 (Affinity Bioreagents Inc 24) and rabbit anti MP20 17 diluted 1:25 and 1:100, respectively, in blocking solution. Slides were washed in PBS to remove unbound antibodies and incubated for 1.5 hr in the dark with 1:200 diluted anti rabbit and anti mouse immunoglobulins conjugated with Alexa Fluor 488 and 568, respectively (Molecular Probes). After washing in PBS, cell nuclei were stained with propidium iodide (0.2% in PBS) for 5 min, and slides were washed and mounted in 10 l Citifluor AF1. Images of each chromophore-staining pattern were recorded digitally using a laser scanning confocal microscope (Leica TCS 4D), then pseudo-colored and combined using Adobe Photoshop software.

For binding studies of MP20 and galectin-3, proteins were purified from ovine lens membranes. Three different membrane preparations were made. All procedures were carried out at 4°C except where mentioned otherwise. Following removal of capsule and epithelium, batches of 100 lenses were homogenized in 100 ml 5 mM Tris-HCl pH 8.0, 5 mM EDTA and 5 mM EGTA. Membranes were pelleted at 18,000 g for 20 min, and were subsequently washed twice in the same buffer. This preparation was called "crude membranes". Membrane adherent crystallins and cytoskeletal proteins were removed from crude membranes with a urea/alkali stripping procedure 25. In one protocol, membrane adherent proteins were only partially removed; the crude membrane preparation was centrifuged and the pellet resuspended in 15 ml 4 M urea, 5 mM Tris-HCl pH 9.5, 5 mM EDTA, 5 mM EGTA. Following centrifugation at 110,000 g for 40 min the pellet was washed twice in 15 ml 20 mM NaOH, and finally resuspended in storage buffer containing 5 mM Tris-HCl pH 8.0, 2 mM EDTA, 2 mM EGTA. The resulting membranes were labeled "partially stripped membranes" as they contained membrane-integral proteins as well as some of the more strongly bound peripheral membrane proteins including galectin-3. They were stored at -20°C at a protein concentration of approximately 4 mg/ml. Protein concentrations were determined using the BCA protein assay method (Pierce).

"Fully stripped membranes" were also prepared from crude membrane preparations following a similar procedure to that outlined above but using larger quantities of the urea and alkaline solutions, and adding a lactose extraction step to remove any remaining galectin-3. Fully stripped membrane preparations contained only the membrane-integral proteins. The crude membrane preparation was centrifuged and the pellet resuspended in 100 ml 4 M urea, 5 mM Tris-HCl pH 9.5, 5 mM EDTA, 5 mM EGTA. Membranes were pelleted at 140,000 g for 1 hr and subsequently washed three times in 100 ml 20 mM NaOH. Next, membranes were incubated in 100 mM lactose in storage buffer for 10 min at room temperature, and pelleted as above. The lactose step was repeated once, and fully stripped membranes were resuspended in storage buffer and stored at -20°C at a protein concentration of approximately 1.5 mg/ml until further use.

MP20 was purified with a single ion exchange chromatography step. Aliquots of fully stripped membranes were solubilized in 1% n-decyl-beta-D-maltopyranoside (DM) (Sigma Chemical Co) in storage buffer at room temperature. Insoluble material was pelleted at 110,000 g for 1 hr. Soluble proteins were separated on a MonoQ column (Pharmacia Biotech) equilibrated with 0.3% DM in storage buffer on an AKTA FPLC (Pharmacia Biotech). All proteins except MP20 bound to the column. MP20 in the flow-through fraction was concentrated with Centricon-10 cartridges as required. MP20 was analysed by size exclusion chromatography using an S-200 10/30 column (Pharmacia Biotech) equilibrated with 0.3% DM, 0.5 M NaCl in storage buffer. The column was calibrated using ovalbumin, bovine serum albumin (BSA), aldolase, chymotrypsinogen A, and ribonuclease A as marker proteins, and blue dextran for determination of the void column volume. For the calculation of molecular weight a calibration plot of log Mw vs. Kav was constructed. Kav = (Vc-Vo/Vt-Vo) where Vc = protein elution volume, Vt = column volume (24 ml) and Vo = void column volume.

A single treatment step of crude membranes with 20 mM NaOH was used to solubilize sufficient galectin-3 for further purification. Insoluble material was pelleted at 140,000 g for 1 hr, and the supernatant dialyzed against 2 L 50 mM HEPES pH 6.8, 2 mM EDTA, 2 mM EGTA, at room temperature for 24 hours. The supernatant was then loaded onto an α-lactose-agarose column (Sigma) pre-equilibrated with 10 mM HEPES pH 6.8, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, and eluted with 100 mM lactose in the same buffer. Pure galectin-3 was obtained by this single purification step and was further analyzed by size exclusion chromatography using a S-75 10/30 column (Pharmacia Biotech) equilibrated with the same buffer. The column was calibrated using ribonuclease A, chymotrypsinogen A, ovalbumin, and BSA as marker proteins, and blue dextran to determine the void volume.

The ability of MP20 and galectin-3 to interact with each other was investigated in two ways: first, by analyzing the complex formed when combining the purified proteins, and second, by determining whether a similar complex could be isolated from lens fiber cell membranes. The protocol for the first approach was as follows. Lactose was removed from purified galectin-3 by dialysis against 2 L 10 mM Tris-HCl pH 8.0, 2 mM EDTA, 2 mM EGTA at room temperature for 24 hours. Galectin-3 was then incubated with purified MP20 for 24 hrs at 4°C at a molar ratio 6:1. The mixture was analyzed by size exclusion chromatography using an S-200 10/30 column (Pharmacia Biotech) equilibrated with 0.3% DM, 0.5 M NaCl in storage buffer. The column was calibrated using aldolase, BSA, ovalbumin, and chymotrypsinogen A as marker proteins, and blue dextran for the determination of the void column volume.

Alternatively, a native complex of MP20 and galectin-3 was isolated from lens fiber cell membranes. For this, the same protocol was employed as for the purification of MP20 except that "partially stripped" membranes were used as starting material. Isolated MP20/galectin-3 complexes were analyzed by gel filtration as above using carbonic anhydrase, BSA, alcohol dehydrogenase, and blue dextran for calibration.

Complexes of MP20 and galectin-3 were first solubilized from crude membranes, and the soluble complexes treated with lactose to determine whether this separated the two proteins. Crude membranes were treated with 1% DM in 10 mM HEPES pH 6.8, 2 mM EDTA, 2 mM EGTA for 1 hr at room temperature, and the soluble material was collected following centrifugation at 140,000 g for 1 hr. Soluble proteins were then incubated for 1 hr with anti MP20 at a dilution of 1:50 in 10 mM HEPES pH 6.8, 2 mM EDTA, 2 mM EGTA, 0.3% DM. Three volumes of a solution containing 50% protein A sepharose beads, 10 mM HEPES pH 6.8, 2 mM EDTA, 2 mM EGTA, 100 mM NaCl, 0.3% DM, and either none or 100 mM lactose, were added to the immunoprecipitated MP20/galectin-3 complexes and incubated overnight at room temperature. The beads were washed several times with the respective buffers (with or without lactose), collected by centrifugation, and analyzed by immunoblotting. For this purpose, 17.5% acrylamide gels were run in a Mini-PROTEAN II cell (BIO-RAD). Proteins were solubilized in sample buffer without boiling as heat tended to aggregate some of the membrane integral proteins. Proteins were visualized by silver staining. For immunoblotting, proteins were transferred electrophoretically in a Mini-PROTEAN Trans-Blot cell (BIO-RAD) onto Hybond-C pure nitrocellulose membranes (Amersham LIFE SCIENCES). Blots were stained with 0.1% Ponceau S/1% acetic acid to visualize and record the positions of lens proteins and molecular weight markers. Following washing in milliQ water, the blots were blocked overnight at 4°C in 5% non-fat milk powder in Tris-buffered saline (TBS). The blots were incubated with either anti galectin-3 (1:200 in TBS containing 1% BSA) or anti MP20 (1:1000), and bound antibodies detected with biotinylated secondary antibodies at 1:1000 and streptavidin-horseradish peroxidase at 1:1000 according to the manufacturer's instructions (ECL, Amersham LIFE SCIENCES).