Post-translational modifications of aquaporin 0 (AQP0) in the normal human lens: spatial and temporal occurrence

Because of the lack of protein turnover in fiber cells of the ocular lens, Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, undergoes extensive post-translational modification with fiber cell age. To map the distribution of modified forms of AQP0 within the lens, normal human lenses ranging in age from 34 to 38 were concentrically dissected into several cortical and nuclear sections. Membrane proteins still embedded in the membranes were digested with trypsin, and the resulting C-terminal peptides of AQP0 were analyzed by HPLC tandem mass spectrometry, permitting the identification of modifications and estimation of their abundance. Consistent with earlier reports, the major phosphorylation site was Ser 235, and the major sites of backbone cleavage occurred at residues 246 and 259. New findings suggest that cleavage at these sites may be a result of nonenzymatic truncation at asparagine residues. In addition, this approach revealed previously undetected sites of truncation at residues 249, 260, 261, and 262; phosphorylation at Ser 231 and to a lower extent at Ser 229; and racemization/isomerization of l-Asp 243 to d-Asp and d-iso-Asp. The spatial distribution of C-terminally modified AQP0 within the lens indicated an increase in truncation and racemization/isomerization with fiber cell age, whereas the level of Ser 235 phosphorylation increased from the outer to inner cortex but decreased in the nucleus. Furthermore, the remarkably similar pattern and distribution of truncation products from lenses from three donors suggest specific temporal mechanisms for the modification of AQP0.     

Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.     

Degradation of human aquaporin 0 by m-calpain

Opacities (cataracts) in the lens of the eye are a leading cause of preventable blindness. Aquaporins function as water channels, and the C-terminus is postulated as a regulatory domain. The C-terminal domain of aquaporin 0 (AQP0) develops numerous truncation sites during lens aging. The purpose of the present experiment was to determine if the calcium-activated protease m-calpain (EC 3.4.22.17) was responsible for truncation of human AQP0. AQP0 was isolated from young human donors, incubated with recombinant m-calpain, and the cleavage sites on the released peptides were determined by on-line electrospray ionization mass spectrometry. We found that four cleavage sites on human AQP0 could be tentatively assigned to m-calpain. This is the first evidence for possible calpain activity in human lens. Because the cause(s) of 17 other cleavage sites was unknown, the data also suggested that other, as yet unknown, proteases or non-enzymatic mechanisms are more active than calpain in human lens.     

Developmental regulation of the direct interaction between the intracellular loop of connexin 45.6 and the C terminus of major intrinsic protein (aquaporin-0)

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.     

The Arg233Lys AQP0 mutation disturbs aquaporin0-calmodulin interaction causing polymorphic congenital cataract

Calmodulin (CaM) directly interacts with the aquaporin 0 (AQP0) C-terminus in a calcium dependent manner to regulate the water permeability of AQP0. We previously identified a missense mutation (p.R233K) in the putative CaM binding domain of AQP0 C-terminus in a congenital cataract family. This study was aimed at exploring the potential pathogenesis of this mutation causative of cataract and mainly identifying how it influenced the binding of AQP0 to CaM. Wild type and R233K mutant AQP0 with EGFP-tag were transfected separately into Hela cells to determine the expression and subcellular localizations. The co-immunoprecipitation (CoIP) assay was used to detect the interaction between AQP0 and CaM. AQP0 C-terminus peptides were synthesized with and without R233K, and the binding abilities of these peptides to CaM were assessed using a fluorescence binding assay. Localizations of wild type and R233K mutant AQP0 were determined from EGFP fluorescence, and the chimeric proteins were both localized abundantly in the plasma membrane. Protein expression levels of the culture cells showed no significant difference between them. The results from CoIP assay implied that R233K mutant presented more weakly in association with CaM than wild type AQP0. The AQP0 C-terminal mutant peptide was found to have 2.5-fold lower binding affinity to CaM than wild type peptide. These results suggested that R233K mutation did not affect the expression, location and trafficking of the protein but did influence the interaction between AQP0 and CaM. The binding affinity of AQP0 C-terminus to CaM was significantly reduced. Due to lack of the modulation of the Ca2+-calmodulin complex, the water permeability of AQP0 was subsequently augmented, which might lead to the development of this cataract.     

Novel fatty acid acylation of lens integral membrane protein aquaporin-0

Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.     

Aquaporin 6 binds calmodulin in a calcium-dependent manner

Aquaporin 6 (AQP6) is an anion channel that is expressed primarily in acid secreting α-intercalated cells of the kidney collecting duct. In addition, AQP6 anion channel permeability is gated by low pH. Inspection of the N-terminus of AQP6 revealed a putative calmodulin binding site. AQP6-expressing CHO-K1 cell lysates were mixed with calmodulin beads and AQP6 was pulled down in the presence of calcium. Mutagenesis of the N-terminal calmodulin binding site in full length mouse AQP6 resulted in a loss of calmodulin binding activity. Mouse and human AQP6 calmodulin binding site peptides bound dansyl-calmodulin with a dissociation constant of approximately 1 μM. The binding of AQP6 to calmodulin may be an important key to determining the physiological role of AQP6 in the kidney.     

Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin.

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin.     

Spatial differences in an integral membrane proteome detected in laser capture microdissected samples

The combination of laser capture microdissection and mass spectrometry represents a powerful technology for studying spatially resolved proteomes. Moreover, the compositions of integral membrane proteomes have rarely been studied in a spatially resolved manner. In this study, ocular lens tissue was carefully dissected by laser capture microdissection and conditions for membrane protein enrichment, trypsin digestion, and mass spectrometry analysis were optimized. Proteomic analysis allowed the identification of 170 proteins, 136 of which were identified with more than one peptide match. Spatial differences in protein expression were observed between cortical and nuclear samples. In addition, the spatial distribution of post-translational modifications to lens membrane proteins, such as the lens major intrinsic protein AQP0, were investigated and regional differences were measured for AQP0 C-terminal phosphorylation and truncation.     

Differentiation-dependent modification and subcellular distribution of aquaporin-0 suggests multiple functional roles in the rat lens

Using immunohistochemistry and mass spectrometry, differentiation-dependent changes in the subcellular distribution and processing of aquaporin-0 (AQP0) have been mapped in the rat lens. Sections labelled with C-terminal tail AQP0 antibodies yielded two concentric rings of labelling with minimal signal in the lens core. The rings were separated by a transient zone of decreased labelling located prior to the transition of differentiating fiber (DF) cells into mature denucleated fiber (MF) cells. Mass spectrometry showed that the loss of core labelling was due to AQP0 cleavage, while the transient loss of labelling was more likely caused by masking of the antibody epitope. AQP0 subcellular distribution changed with radial distance into the lens. In peripheral DF cells, AQP0 was found throughout both broad and narrow side membranes. In deeper-lying DF cells, AQP0 aggregated into plaque-like structures located on the broad sides. This shift occurred prior to the transient loss of AQP0 signal, and coincided with formation of broad-side membrane invaginations between adjacent fiber cells to which filensin, a known binding partner of AQP0, was also localized. After nuclei loss, AQP0 was once again distributed throughout MF cell membranes. In the absence of protein synthesis, the observed subcellular redistribution of AQP0 in DF and subsequent cleavage of AQP0 in MF are suggestive of a switch in the function of AQP0 from a water channel to a junctional protein.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

Phosphorylation determines the calmodulin-mediated Ca2+ response and water permeability of AQP0

In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.     

Simultaneous binding of the N- and C-terminal cytoplasmic domains of aquaporin 4 to calmodulin

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca²⁺-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional ¹H, ¹⁵N-HSQC NMR with ¹⁵N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a K_d of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; K_d1: 3.6 μM, K_d2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4^CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a K_d of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.     

Some Properties of Caldesmon and Calponin and the Participation of These Proteins in Regulation of Smooth Muscle Contraction and Cytoskeleton Formation

The interaction of caldesmon with different Ca²⁺-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca²⁺-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B" of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actinbinding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and a-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.     

Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0^+/−) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0^+/−/AQP1^+/−) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P_f) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA, possibly due to alteration in RING. To our knowledge, this is the first report identifying the role of AQP0 in RING development to ward off lens SA during focusing.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

Structural features underlying the unusual mode of calmodulin phosphorylation by protein kinase CK2: A study with synthetic calmodulin fragments

To shed light on the paradoxical behaviour of calmodulin, whose phosphorylation is inhibited by the regulatory beta-subunit of protein kinase CK2, a series of peptides encompassing the phosphoacceptor sites of calmodulin have been synthesized and assayed as substrates of CK2 alpha-subunit either alone or combined with the beta-subunit. The shortest peptide whose phosphorylation is reduced instead of being enhanced by the beta-subunit encompasses the sequence 68-106, including the central helix and the Ca2+-binding loop-III. In contrast, the phosphorylation of a peptide encompassing loop II and the central helix (54-92) is stimulated, like that of several shorter peptides, by the beta-subunit. Our data localize to the C-terminal domain of calmodulin the structural elements that are responsible for inverted susceptibility to beta-subunit regulation.     

Aquaporin-0 membrane junctions form upon proteolytic cleavage

Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.