MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3

Abstract

Background: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.

Results: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction.

Conclusions: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Figure 1

Spatial distribution of MP20 and galectin-3 in the lens. (A) Overview of the distribution of MP20 (red) in relation to cell nuclei (blue) as a marker for fiber cell differentiation in equatorial sections. (B) Co-localisation of MP20 (red) and galectin-3 (green) in peripheral cortex as indicated by box b in panel A. (C) Co-localisation of MP20 and galectin-3 in an area approximately 500 m into the cortex as indicated by box c in panel A. Yellow indicates regions of overlap of both proteins.

Figure 2

Purification and characterization of lens MP20. (A) SDS-PAGE and Western blot documentation of the purification process. Lane 1: Molecular weight markers. Lane 2: Fully stripped membranes showing significant enrichment in MP20 (20 kDa), and other integral membrane proteins including MIP (26 kDa) and its 22 kDa cleavage product, and MP38 which is the cleavage form of connexins 46 and 50. Lane 3 and 4: Insoluble and soluble fractions, respectively, following treatment of membranes with DM. Lane 5 and 6: Soluble proteins that did not bind (mostly MP20) and proteins that did bind to the MonoQ, respectively. Lane 7: Pure MP20 eluted from size exclusion chromatography column S-200. Lane 8: Immunoblot of lane 5 identifying MP20 and its dimer that is not detected in the stained gel. (B) Single step purification of MP20 on a MonoQ column. MP20 is the only major integral membrane protein that does not bind to the column at pH 8 (arrow). (C) Size exclusion chromatography of pure MP20. The protein was eluted at 15 ml. AU and mAU are absorbance units at 280 nm. (D) Calibration of the S-200 column. Vo = 8 ml. MP20 has an apparent molecular mass of 60 kDa (arrow).

Figure 3

Purification and characterization of lens galectin-3. (A) SDS-PAGE and Western blot documentation of the purification process. Lane 1: Molecular weight markers. Lane 2: Crude lens fiber cell membranes. Lane3 and 4: Insoluble and soluble fractions, respectively, after treatment with 20 mM NaOH. Lane 5: Pure galectin-3 eluted from the α-lactose-agarose column with 100 mM lactose. Lane 6: Galectin-3 eluted from a size exclusion chromatography column S-75. Lanes 7 and 8: Immunoblot of the preparations in lanes 4 and 5 identifying galectin-3. (B) Galectin-3 bound to an α-lactose-agarose column, and was eluted with 100 mM lactose. (C) Size exclusion chromatography of the pure galectin-3. The protein was eluted at 12.5 ml. mAU refers to absorbance units at 280 nm (D) Calibration of the S-75 column. Vo = 8.3 ml. Galectin-3 has an apparent molecular mass of 22 kDa (arrow).

Figure 4

MP20/galectin-3 complex formation in vitro. (A) SDS-PAGE analysis of pure MP20 and pure galectin-3 and of the complex formed. Lane 1. Molecular weight markers. Lanes 2 and 3. Pure MP20 and galectin-3 used for the binding experiment, respectively. Lane 4. The complex eluted from the gel filtration column. Galectin-3 appears as a doublet due to minor proteolysis [28]. (B) Size exclusion chromatography of MP20/galectin-3 complexes formed following the mixing of purified lens MP20 with purified galectin-3. The complex eluted at 13 ml. mAU refers to absorbance units at 280 nm. (C) Calibration of the S-200 column. V_o = 7.8 ml. MP20/galectin-3 complex has an apparent molecular mass of 104 kDa (arrow).

Figure 5

Purification of a native MP20/galectin-3 complex from lens membranes and role of lectin site. (A) SDS-PAGE and Western blot documentation of the purification process. Lane 1: Molecular weight markers. Lane 2: Partially stripped lens fiber cell membranes showing both galectin-3 and enriched MP20. Lanes 3 and 4: Proteins that did not bind (including MP20 and galectin-3) and proteins that did bind to the MonoQ column, respectively. Lane 5: MP20 and galectin-3 eluted from the S-200 column. Lanes 6 and 7: Immunoblot of lane 5 identifying MP20 and galectin-3, respectively. Note that anti MP20 also recognizes dimeric MP20 that is not normally detected on stained gels. (B) Galectin-3 and MP20 did not bind to the column at pH8 (arrow). (C) Size exclusion chromatography of the complex. A single peak eluted from the S-200 column at 13.8 ml. AU refers to absorbance units at 280 nm. (D) Calibration of the S-200 m column. Vo = 8 ml. MP20/galectin-3 complex has an apparent molecular mass of 102 kDa (arrow). (E) Immunoprecipitation of MP20 and galectin-3 and protein detection by Western blotting. Lanes 1 and 2: galectin-3 detected without and with lactose present, respectively. Note the removal of galectin-3 from the complex in the presence of lactose. Lanes 3 and 4: Equal amounts of MP20 are detected without and with lactose present, respectively.