The distribution of the fiber cell intrinsic membrane proteins MP20 and connexin46 in the bovine lens

Abstract

MP20 is an intrinsic membrane protein previously identified in mammalian lens fiber cells. To identify a possible role for this protein in the lens, the distribution of MP20 and connexin46 has now been examined. Western immunoblotting with an anti-peptide antibody generated to the C-terminal 8 amino acids of MP20 confirmed the presence of this protein in the lens of several different mammalian species. A monoclonal antibody 5H1 was prepared that, in Western blots of bovine lesn membranes, recognized the same component as an antibody to rat connexin46 (Cx46). The apparent molecular mass of this component decreased from 59 kDa to 55 kDa following treatment of lens membranes with alkaline phosphatase. A monoclonal antibody to connexin-related MP70 recognized a 70 kDa component in bovine lens membranes confirming the presence of these two different connexin proteins in bovine lens membranes. To localize MP20 and Cx46 in the bovine lens membrane, lens fiber cell bundles were immunofluorescently labeled with both the MP20 antibody, and the monoclonal antibody to Cx46. Cx46 was identified in large plaques on the broad faces of the lens fiber cells throughout the outer 1 mm of the lens cortex. MP20 colocalized with Cx46 only in a restricted area 0.5 mm to 1.0 mm into the lens. In other regions of the lens, MP20 appeared more diffusely distributed over the fiber cell surface, although apparently concentrated in the ball-and-socket regions at the corners of the narrow side of the inner cortical lens fiber cells. These inner cortical regions were devoid of Cx46. A difference in distribution of these two proteins was confirmed in studies of immunofluorescently labeled lens cryosections. Furthermore, immunogold electron microscopy of purified lens membranes identified MP20 in both junctional regions (with Cx46) and in single membranes. These results provide evidence for a role for MP20 in mammalian lens fiber cell junctional formation or organization.

Association of Alpha-Crystallin with Fiber Cell Plasma Membrane of the Eye Lens Accompanied by Light Scattering and Cataract Formation

“…Gap junctions, also known as membrane channels, are made of three kinds of connexins, Cx43, Cx46, and Cx50 [9,185]. Cx46 is found mainly in the cortex and the outer nuclear layers, and Cx50 is found mainly in the nuclear core [186][187][188]. Six connexins form a connexon, and two connexons from neighboring cells form a gap junction.…”

Levels and Modifications of Both Lens Fiber Cell Connexins Are Affected in Connexin Mutant Mice

“…In addition, these experiments indicate that the change in the electrophoretic mobility primarily represents the phosphorylation of the proteins. Both Cx46 and Cx50 are known to be phosphoproteins [ 6 , 19 , 20 , 21 , 22 , 23 , 24 ]. Previous immunoblotting experiments using ovine, bovine, and rat samples have shown that alkaline phosphatase treatment modifies the electrophoretic mobility of immunoreactive Cx46 and Cx50 [ 6 , 19 , 20 , 21 , 22 ].…”

“…Both Cx46 and Cx50 are known to be phosphoproteins [ 6 , 19 , 20 , 21 , 22 , 23 , 24 ]. Previous immunoblotting experiments using ovine, bovine, and rat samples have shown that alkaline phosphatase treatment modifies the electrophoretic mobility of immunoreactive Cx46 and Cx50 [ 6 , 19 , 20 , 21 , 22 ]. In rat lenses, Cx46 is mainly detected as two bands of 46 and 54 kDa; both forms are present at embryonic ages, but the slower (phosphorylated) form predominates in young adults [ 21 ].…”

Loss of fiber cell communication may contribute to the development of cataracts of many different etiologies

“…All of the lens connexins are phosphoproteins ( Musil et al, 1990 ; TenBroek et al, 1992 ; Jiang et al, 1993 ; Berthoud et al, 1994 ; Gupta et al, 1994 ; Jiang et al, 1994 ; TenBroek et al, 1994 ; Berthoud et al, 1997 ; Shearer et al, 2008 ; Wang and Schey, 2009 ; Wang et al, 2013 ; Myers et al, 2018 ). Changes in phosphorylation of connexins (at different sites) can affect many aspects of the connexin life-cycle and channel function ( Sáez et al, 2003 ; Solan and Lampe, 2005 ; Solan and Lampe, 2014 ; Leybaert et al, 2017 ).…”