Biochemical Evidence for Adhesion-Promoting Role ... - Science Direct

Exp. Eye Res. (1995.), 61, 293-301

Biochemical Evidence for A d h e s i o n - P r o m o t i n g Role of M a j o r Intrinsic Protein Isolated from Both Normal and Cataractous H u m a n Lenses L U I S F. M I C H E A * , D A R I O A N D R I N O L O ,

HERN,AN C E P P I AND N I t S T O R L A G O S

Departamento de Fisiologfa y Biof/sica, Facultad de Medicina, Universidad de Chile. Casilla 70005, Correo- 7, Santiago, Chile (Received Rochester 13 October 1994 and accepted in revised form 30 March 1995) In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22 000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide. © 1995 Academic Press Limited Key words: Lens; lens junctions; cataract; liposome aggregation; adhesive role; proteins; lens major intrinsic protein; protein aggregates. 1.

Introduction

The lens fibers f o r m extensive areas of membrane appositions with a restricted extracellular space, known as lens fiber junctions. The most abundant intrinsic membrane protein of the lens fibers is the major intrinsic polypeptide (MIP, 28kDa), which represents about 5 0 - 6 0 % of the plasma membrane intrinsic protein (Benedetti et al., 1976). The physiological role of MIP is not well established. Immunocytochemistry studies show that MIP localizes in the junctional membranes (Bok, Donckstaer and Horwitz, 1982; Fitzgerald, Bok and Horwitz, 1983: Sas et al., 1985; Zampighi, Hall and Simon, 1989). These junctions measure 1 1 - 1 3 nm in width with an intercellular gap of about 0-5-0"7 nm (Zampighi, Simon and Hall, 1992). Freeze-fracture studies carried out both in h u m a n and bovine lenses show that MIP molecules in the junction abut and appose a particle-free membrane (Lo and Harding, 1984; Zampighi et al., 1989). Based on these observations it has been proposed that MIP promotes cell-to-cell adhesion in the lens membrane junctions (Zampighi et al., 1989). *

For correspondence.

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Gorin and colleagues (1984) proposed for MIP a structure of six transmembrane segments, with three extracellular domains. None of them present negatively charged amino acids. Two of these extracellular loops present sequences of 20 amino acids including arginine (112, 185, 196) and histidine (40, 122), which are positively charged at pH's below their pK~s. This prediction led Zampighi et al. (1989) to postulate that junctions could be stabilized by electrostatic attractions between positively charged amino acids, located in the external domain of the MIP molecule, and negatively charged lipids on the opposing membrane. Recently, Michea, De la Fuente and Lagos (1994) presented the first experimental evidence strengthening the hypothesis of an adhesive role for MIP. Using reconstituted bovine MIP in neutral phosphatidylcholine (PC) liposomes we studied the adhesion of the MIP reconstituted vesicles with negatively charged phosphatidylserine (PS) vesicles. These results prove that MIP promotes adhesion only with the negatively charged PS liposomes. Divalent cations reduced the rate of adhesion, suggesting that this interaction is primarily electrostatic in nature. Changes in the MIP molecule itself and its association with other proteins could influence the © 1995 Academic Press Limited

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stability of lens junctions. During aging (Horwitz et al., 1979; Roy, 1979; Garner, Roy and Spector, 1982) and cataratogenesis (Russell and Kinoshita, 1980; Alcal~i, Cendella and Katar, 1985; Kuck and Lo, 1987; Alcahi et al., 1990) MIP 28 kDa undergoes endogenous prote01ysis to give an MIP isoform of 22kDa. The de~'elopment of senile cataracts is associated with high molecular weight aggregates which act as scatter points of light (Spector et al., 1971; Jedziniak et al., 1973). The amount of water insoluble material and high-molecular-weight aggregates increase at the expense of the water soluble proteins (Garner, Garner and Spector 1979, 1981: Garner and Spector, 1980; Spector, 1984). Studies of the structure of human senile cataratic lenses have showed a disappearance of normal fiber structure in the opaque regions of tissue, with disruption of the fiber membrane (Philipson, 1973; Benedek et al., 1979 ; Clark, Mengel and Benedek, 1980; Clark et al., 1980; Garner et al., 1981; Bloemendal, 1991). Several biochemical studies in h u m a n senile cataracts indicate the formation of protein aggregates between oxidized cytoplasmic proteins and constituents of the plasma membrane of the fibers (Harding, 1973; Garner, W. H. et al., 1981; Takemoto and Hansen, 1982 ; Garadi, Giblin and Reddy, 1987: Bloemendal, 1991). The present study was undertaken to investigate the adhesive role of MIP from both normal h u m a n (cadaver) and senile cataratic lenses. MIP from both sources was reconstituted in neutral PC liposomes and the interaction of these liposomes with negatively charged ones was studied. The adhesive effect between both populations of vesicles were assayed by RET. We tested the effects of the primary modifications of MIP present in senile cataract, such as proteolytic alterations and its association to extrinsic cytoplasmic proteins. These studies are the first experimental evidence which show that MIP isolated from normal h u m a n lens junctional membranes, promotes adhesion when reconstituted into large unilamellar liposomes. In contrast, MIP isolated from h u m a n senile cataractous lens fails to promote adhesion.

2. Materials and Methods

Materials Phosphatidylserine (L-~-phosphatidylserine brain sodium salt) (PS) and phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) (PC) were purchased from Avanti Polar Lipids (Birmingham, AL, U.S.A.). Rhodamine-phosphatidylethanolamine (Rhodamine-DHPE) (Rh-PE) and NBD-phosphatidylethanolamine (N-(7-nitro-2-oxa-1,3-diazol-4-yl) 11,2dihexadecanoyl- sn-glycero- 3-phosphoethanolamine) (NBD-PE) were purchased from Molecular Probes (Eugene, OR, U.S.A.). Pronase from streptomyces griseus was obtained from Calbiochem (La Jolla, CA,

L.F. M I C H E A ET AL.

U,S.A.). Octyl-/~-glucopyranoside was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Lowmolecular-weight protein standards were purchased from Bio-Rad. All inorganic salts, reagents or buffers were analytical reagent grade or better. Lenses from adult bovines were obtained from the slaughterhouse, immediately cooled on ice and stored at - 2 0 ° C until use. Normal h u m a n lenses with no visible opacity were obtained from subjects ranging from 50 to 60 years old within 7 h of death, immediately cooled on ice and stored at - 2 0 ° C until use (Servicio MGdico Legal, Chile). Senile cataractous lens nuclei from nuclear cataracts were obtained from Hospital San Juan de Dios, by extracapsular extraction performed by local ophthalmologists. After removal, the cataractous nuclei were immediately cooled on ice and stored at - 2 0 ° C until use.

Isolation of Lens Fiber ]unctions The bovine lens junctional membranes were isolated as described previously (Zampighi et al., 1982: Ehring et al., 1992; Michea et al., 1994). The capsules of eight bovine lenses were removed. The lenses were cut with a razor blade and homogenized in 15 ml of solution A (2 mM NaHCO 3, 3 mM EDTA, 100/~M PMSF, pH 8"0). The suspension was filtered through eight layers of surgical gauze, diluted to 60 ml with solution A and centrifuged at 3640 g for 15 rain. The pellet was washed and centrifuged twice in solution A. Then the pellet was resuspended and homogenized in solution B (5 mM EDTA, 1 mM CaC12, 1'5 mM NaNa and 4 mM Tris pH 8-0) in 4 M urea. The suspension was centrifuged at 22 500 g for 25 rain. The white portion of the pellet was extracted by gentle agitation with 50 ml of solution B in 7 M urea. After homogenization it was centrifuged at 3 9 0 0 0 / / for 45 min. The pellet was washed three times with 50 ml of solution B and centrifuged at 1 0 0 0 0 0 g by 10 min. The isolated junctions were diluted in solution B plus 10% glycerol and stored at - 2 0 ° C . Decapsulated normal h u m a n lenses were separated into cortex and nucleus removing carefully with a surgical blade 2-3 mm of the external zone and 1 mm from both the anterior and posterior surface of the core. Typically, the nuclei obtained from four lenses were immediately used to isolate the junctional membranes as described by Michea et al. (1994). Human nuclei of senile cataract were used to isolate junctional membranes according to the same protocol. In order to extract the insoluble protein aggregates present in the junctional membranes isolated from cataractous lenses, the membranes were treated with 0"1 M NaOH for 15 rain at room temperature. The treated membranes were centrifuged at 1 4 0 0 0 g and the pellet was washed three times in solution B. The isolated junctional membranes were stored in solution B plus 10% glycerol and stored at - 2 0 ° C .

A D H E S I O N - P R O M O T I N G ROLE OF H U M A N MIP

Proteolysis of ]unctional membrane Proteins Intact bovine junctional membranes were centrifuged at 14 000 g for 5 min, resuspended in 1 mM CaC12, 25 mM Tris-HC1 pH 7"5 and incubated with trypsin at a mass ratio of 1 : 3 0 (protease/ junctional proteins) and 1:45 for chymotrypsin. After 15 min of incubation at room temperature, the membranes were centrifuged at 140OOg and suspended in solution B and washed three times. The treated membranes were immediately used in reconstitution experiments and analysed by SDS-PAGE (12.5% gels) and Western blot.

Solubilization of Lens Fiber ]unction Proteins Plasma membranes were solubilized by adding octyl-fl-glucopyranoside to the lens fibers junctions suspension [25 mg detergent (mg junctional protein) 1] and sonicated for 30 sec. The clear solution was centrifuged at 1 4 0 0 0 g for 5rain and the supernatant filtered through a 0-2/~m PVDF filter (Acro Disc, Gelman Sciences, Ann Arbor MI, U.S.A.).

Purification of MIP MIP was purified from the lens fibers junctions membranes isolated from h u m a n normal nuclei using the method described by Ehring et al. 1992. Briefly, 500/zl of solubilized membranes ( 3 0 0 - 4 0 0 #g) were injected into an analytical MA70 column (Bio-Rad) and eluted in buffer A with linear NaC1 gradient (0.025 to 0.5 M NaC1) in the presence of 34 mM octylfl-glucopyranoside. Protein was detected following the absorbance at 280 nm. MIP eluted with a retention time of 39 sec. This fraction exhibited a single band of an apparent molecular weight of 28 000 in SDS-PAGE (12.5%). The protein was stored at 4°C after concentrating to 0 - 5 - 0 " 7 m g m l 1 using microconcentrator tubes (Centricon 10, Amicon, Danvers, MA, U.S.A.

Preparation of Large Unilamellar Vesicles of Phosphatidylserine Large unilamellar vesicles (LUVs) were prepared by the extrusion procedure (MacDonald et al., 1991). A solution of phosphatidylserine (2 mg) and the fluorescent probe Rh-PE (2 mol%) in chloroform were dried under a nitrogen stream and thoroughly desiccated under vacuum. The dried lipids were hydrated with 200/zl of reconstitution buffer (NaC1 100 mM, MOPS 25 mM, pH 7"0) for 30 min at room temperature. The lipid was suspended by vortexing for 1 min and immediately extruded through a 0.1 # m polycarbonate filter (Costar Nuclepore, Pleasanton, CA, U.S.A.). The lipid content was determined in all

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preparations measuring phosphorus (Meek, 1986). The resulting vesicles were named Rh-PS.

Protein Reconstitution into Large Unilamellar Vesicles of Phosphatidylcholine The solubilized junctional proteins or purified MIP were reconstituted into phosphatidylcholine LUVs using the dialysis method previously described (Mimms et al., 1981 ; Ehring et al., 1990; Ehring et al., 1992; Michea et al., 1994). Phosphatidylcholine in chloroform (plus 2 mol % of NBD-PE), was dried under a nitrogen stream and suspended in 0-1 ml of reconstitution buffer plus octyl-fl-glucopyranoside (112 m g m l 1). The solution was mixed with solubilized junctional proteins or purified M[P and sonicated for 30 sec. This clear solution was dialysed against 3 1 of reconstitution buffer (three changes, 26 h at 4°C). The bovine lens junctions proteins reconstituted liposomes were obtained using a lipid/ protein ratio (expressed in micrograms) of 1 0 0 0 / 500. Protein (Bradford, 1976) and phospholipid (Meeck, 1986) contents were measured and the protein composition of the reconstituted vesicles was analysed by SDS-PAGE (12-5 % total acrylamide) and Western blot. Under our experimental conditions, 6 4 + 8 % (five different preparations) of the bovine junctional membrane protein, 5 2 + 6 % of h u m a n normal junctional membrane protein (four different preparations) and 45 4-5% of cataractous junctional membrane protein (four different preparations) were incorporated into liposomes. The presence of the bovine junctional proteins on PC vesicles was studied using SDS-PAGE. Like in lens junction membrane, the most abundant protein reconstituted was MIP, being about 60% of the total protein. The vesicles were named PC-Mbs when reconstituted with total junction protein, or PC-MIP when reconstituted with HPLC purified MIP. These names are preceded with NBD- or Rh- when containing fluorescent probes (as in NBD-PC-Mbs).

Adhesion Assay The method is based on the fact that if two vesicle populations come close together, the fluorescent emission of NBD-PE in a population of liposomes can be quenched due to resonance energy transfer (RET) to Rh-PE labelling the other population of liposomes (Stryer, 1978: Duzgunes et al., 1987; Michea et al., 1994). Experiments were carried out at 25°C in cuvette containing 3 ml of IOOmMNaCI, Bis-Tris 25 mM pH 6"0. Emission of NBD-PE containing liposomes (20/zM PC or PS) was followed at 520 nm (excitation at 450 nm) for 2 min, then Rh-PS liposomes were added (20/ZM).

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3. Results To test the adhesion-promoting role of h u m a n MIP we reconstituted lens junctions solubilized proteins into large unilamellar phosphatidylcholine (PC-Mbs) vesicles (LUVs). We studied the adhesion of these vesicles with phosphatidylserine LUVs using the resonance energy transfer assay previously described (Michea et al., 1994). In this assay the aggregation of both vesicle populations causes the quenching of the donor probe fluorescence NBD (Duzgunes et al., 1987). Figure I(A) shows the emission time course at 520 n m of NBD-PC vesicles reconstituted with normal h u m a n lens junction solubilized proteins. After the addition of Rh-PS vesicles (arrow) a rapid quenching on NBD emission occurs, due to vesicle aggregation. The quenching of NBD emission was only observed when PC LUVs reconstituted with solubilized junctional proteins were mixed with PS LUVs population. Dotted line shows the emission of NBD-PC vesicles mixed with Rh-PS vesicles. No quenching effect was observed when proteins were omitted in the LUVs reconstitution. To determine whether h u m a n MIP was responsible for the adhesive effect in the assay, we purified MIP from h u m a n lens sclubilized junctions using HPLC (Ehring et al., 1992; Michea et al., 1994). The pure MIP was reconstituted into NBD-PC LUVs. Figure I(B) (circles) shows that using these vesicles in the assay a rapid decrease in the fluorescence signal occurs after the addition of Rh-PS vesicles to the cuvette. Furthermore, the incubation of NBD-PC-MIP liposomes for 45 min with several dilutions of polyclonal antibody against the second extracellular domain of MIP produced the inhibition of more than 92% of the adhesive effect in a concentration dependent manner. No effect was observed using preimmune serum as a control (data not shown). Liposomes reconstituted with the remaining junctional m e m b r a n e proteins, that eluted as a broad peak during the HPLC purification of MIP (Ehring et al., 1992), did not aggregate with Rh-PS vesicles (data not shown). This experiment demonstrates that no other intrinsic protein present in the normal h u m a n junctional membranes is taking part in the adhesion, showing that only MIP exhibits this property. The pH dependence of the adhesion in our assay conditions was quantified measuring the initial quenching rate. As in our previous experiments with bovine MIP (Michea et al., 1994) the maximal initial quenching rate was observed at pH 6'0. There is no quenching effect below pH 5" 5 nor above pH 7"5 (data not shown). To study the capability of h u m a n senile cataract MIP to promote adhesion, we solubilized the m e m brane junctions obtained from h u m a n senile cataractous nuclei. These proteins were reconstituted in NBD-PC liposomes. Figure I(B) (squares) shows the emission time course of the fluorescent signal using

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FIG. l. Adhesion of PC liposomes reconstituted in presence of human MIP with PS vesicles detected by fluorescence assay. Adhesive capability of MIP of human senile cataract lens junctions: (A) NBD-PC-Mbs (solid line) or NBD-PC (dotted line) vesicles were preincubated in 1O0 mM NaC1 and 25 mM Bis-Tris (pH 6"0) at room temperature. Rh-PS liposomes were added to the cuvette after 2 min of preincubation (black arrow). The time course of NBD fluorescence emission was recorded (excitation, 450 nm; emission, 520 nm; slit width lOnm. (B) Emission time course of the NBD fluorescent signal at 520 nm of NBD-PC liposomes reconstituted with normal human HPLC purified MIP (lipid/MIP mass ratio of 500:1 circles) or membrane junctional proteins obtained from senile cataract nucleus (squares). The liposomes were preincubated in 1OOmM NaC1 and 25 mM Bis-Tris (pH 6'0) at room temperature for 2 min. The addition of Rh-PS vesicles is indicated by the arrow. LUVs reconstituted with cataractous lens proteins. Little quenching effect was recorded after mixing with Rh-PS vesicles (black arrow), showing that MIP isolated from senile cataractous lens fails to produce adhesion. Cataractous m e m b r a n e junctions were studied by SDS-PAGE. Figure 2 shows SDS-PAGE profiles of junctional membranes obtained from cataractous and normal h u m a n lens nuclei. In normal h u m a n lens junctions we found two major bands, both are MIP isoforms of apparent molecular weights of 28 000 and 22 000. Densitometric analysis demonstrates that the sum of both bands represents the 61% of the total intrinsic junctional protein, being distributed in almost equal amounts. In contrast, cataractous h u m a n lens junctions show a significative diminution of MIP 28 isoform, being MIP 22 the major band (lane 1). Both MIP isoforms were specifically recognized by Western blots using a polyclonal antibody raised against HPLC purified bovine MIP (data not shown). Also, both bands present cross-reactivity with a polyclonal antibody raised against a synthetic peptide belonging to the second loop of the extracellular domain of the MIP molecule (data not shown). The cataractous lens

A D H E S I O N - P R O M O T I N G ROLE OF H U M A N MIP

profile shows a continuous diffused background, revealing the presence of protein aggregates, similar to those described by other studies (Harding, 1973: Spector and Roy, 1978: Garner, W. H. et al., 1981; Spector, 1984: Garadi et al., 1987). The junctional proteins solubilized from cataractous lenses reconstituted with NBD-PC LUVs show a similar pattern to the one .observed with junctional membranes (Fig. 2, lane 1), revealing the presence of protein aggregates in the reconstituted liposomes. A possible explanation to the lack of adhesion in the experiments done with cataractous MIP, could be the endogenous proteolytic damage responsible for the increased amount of the 22 kDa isoform. To test this hypothesis we use as a model the bovine MIP 28 molecule. Several reports have showed proteolytic hydrolysis of MIP in vitro using proteases like trypsin and chymotrypsin (Wong, Mercola and Horwitz, 1980: Takemoto et al., 1983 : Horwitz, 1983: Peracchia and Girsch, 1985: Louis et al., 1985: ]ohnson et al. 1986: Lampe and ]ohnson, 1990). The incubation of the bovine lens junctions in the presence of these proteases produce the cleavage of MIP 28, mainly to 22 kDa isoform. This isoform cross-reacted with the polyclonal antibody raised against the synthetic peptide belonging to the second loop of the extracellular domain of the MIP molecule (data not shown). We also observed the appearance of small amounts of other proteolytic products. All of them were identified as MIP isoforms by Western blot using polyclonal serum against HPLC purified bovine MIP 28 (data not shown).

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FIG. 2. SDS-PAGE of human lens membrane junction proteins. Coomasie-blue-stained polyacrylamide gradient gel (6-20%). Lane 1 was loaded with lens membrane junctions isolated from senile cataractous nuclei (12 t,g of protein). Lane 2, with 8//g of molecular weight standard proteins (phosphorylase b 97400; bovine serum albumin 66200: ovalbumin 46600; carbonic anhydrase 31000: soybean trypsin inhibitor 21 500: lysozyme 14400). Lane 3, was loaded with lens membrane junction isolated from normal human nuclei (5/~g of protein),

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FIG. 3, Adhesion of Rh-PS and NBD-PCliposomes reconstituted with MIP 22 obtained by proteolytic treatment. (A) NBD-PC-MIP22 (solid line) or NBD-PC-MIP28 vesicles were preincubated in 100 mM NaCI and 25 mM Bis-Tris (pH 6"0) at room temperature. The time course of NBD fluorescence emission was recorded (excitation, 45Onto; emission, 520 nm: slit width 10 nm). Rh-PS liposomes were added to the cuvette after 2 rain of preincubation {black arrow). (B) The initial quenching velocity (V~) expressed as percentage of the control, obtained with vesicles reconstituted with untreated membranes. NBD-PC reconstituted with MIP 22 obtained after trypsin treatment (T, filled column) and NBDPC reconstituted with MIP 22 obtained after chymotrypsin treatment (C, empty column). Each column represents the mean_+s.D, of four independent determinations from tbur different preparations.

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Fro. 4. NaOH removes protein aggregates from cataractous lens junction membranes and restores MIP adhesive capability. (A) SDS-PAGE profiles of normal and cataractous junctional membranes after the treatment with 0"1 M of NaOH for 15 min. Coomasie-blue-stained polyacrylamide gradient gel (6-22To). Lane 1 was loaded with lens membrane junctions isolated from normal human nuclei (12 Fg of protein). Lanes 2 and 3 were loaded with lens membrane junction isolated from senile cataractous nuclei (5 and 7 tCg of protein respectively). Lane 4 was loaded with 8 tlg of molecular weight standard proteins (as in Fig. 2). (B) Adhesion of Rh-PS and NBD-PCliposomes reconstituted with cataractous lens proteins treated with NaOH 0"1 M. Time course of fluorescence emission of NBD-PC liposomes reconstituted with solubilized lens junctional proteins obtained from senile cataractous nuclei treated with NaOH 0.1 M (filled squares) or normal lens junctional proteins (solid line). After 2 min of preincubation in 100 mM NaCI and 25 mM Bis-Tris (pH 6'0) at room temperature Rh-PS liposomes were added to the cuvette (black arrow).

ET AL.

After proteolytic treatment, the junctions were isolated by centrifugation, solubilized and reconstituted into NBD-PC liposomes. The SDS-PAGE profile of the reconstituted vesicles showed that the only recognized protein incorporated into the liposomes was the band corresponding to MIP 22. The NBD-PC-MIP 22 vesicles were tested in the adhesion assay. Figure 3 (A) shows the NBD quenching time course of these vesicles at the time that they were mixed with Rh-PS LUVs (arrow). A rapid fall in NBD emission follows the addition of Rh-PS vesicles. The time course of quenching observed when using MIP 22 reconstituted vesicles was comparable to those obtained with MIP 28. The initial quenching velocity (V~) of three different junctional membrane preparations, treated with trypsin or chymotrypsin before reconstitution were recorded. Figure 3(B) shows the initial quenching velocity expressed as percentage of the control V, obtained with vesicles reconstituted with non treated membranes. In all cases we observed that the proteolytic treatment did not blunt the adhesive capacity of MIP. Moreover, a slight increase in the Vi was recorded. Membrane junctional proteins form insoluble complexes with oxidized cytoplasmatic proteins (Harding, 1973; Spector et al., 1978: Garner et al., 1981a; Spector, 1984; Garadi et al., 1987). Maybe, a protein complex formed between MIP and an extrinsic protein could be responsible for the adhesion deficiency when the liposomes reconstituted with cataractous lens junctional proteins were assayed. In order to remove the protein aggregates, the cataractous junctional membranes were treated with 0.1 M NaOH. Figure 4(A) shows the SDS-PAGE profile of normal and cataractous junctional membranes after the 0"1 M NaOH treatment. Contrasting with untreated cataractous lens junctional membranes the continuous diffuse background was not observed. The densitometric analysis of the cataractous lens junctions (lanes 2 and 3, 5 and 7 Fg respectively), shows that MIP 22 isoform is the major band, being 48% of the total junctional protein. The cataractous lens junction membranes treated with NaOH were reconstituted in NBD-PC liposomes and tested in the adhesion assay. Figure 4(B) shows the quenching time course of NBD-PC-Mbs liposomes reconstituted with normal (left profile) and NaOHtreated cataractous membranes (right profile). After the addition of Rh-PS liposomes a rapid quenching of NBD emission was observed. Both profiles were alike to those normally observed when MIP 28 reconstituted LUVs were tested (Michea et al., 1994 and Fig. 1 in this paper). The study of the initial rate of quenching of three different membrane preparations (treated and untreated) demonstrated that the NaOH treatment restores the adhesion capability. The supernatant obtained after the treatment of cataractous junctional membranes with NaOH, was collected and concentrated by microconcentrators.

ADHESION-PROMOTING

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Fro. 5. Effect of NaOH extracted proteins from cataractous lens junctions on NBD-PC-Mbs and Rh-PS vesicle adhesion. Time course of fluorescence signal from NBD-PC liposomes reconstituted with solubilized bovine junctional proteins (solid line) or reconstituted with a mix of bovine junctional solubilized proteins (0-75 mg) and proteins from cataractous lens, extracted with NaOH (1.5 mg, dotted line) were recorded. After 3 min of preincubation in 100 mM NaC1, 25 mM Bis-Tris, Rh-PS liposomes were added (black arrows). This fraction (1"5 mg of proteins) was mixed with solubilized bovine lens junction proteins (750 #g) and reconstituted in NBD-PC liposomes and assay for adhesion. Figure 5 shows the quenching time course recorded when normal bovine m e m b r a n e was assayed (solid line), typical adhesion was seen. In contrast, the NBDPC vesicles reconstituted with a mix of solubilized bovine junctional proteins and NaOH cataractous supernatant, failed to produce the quenching effect (dotted line). Furthermore, NBD-PC vesicles reconstituted with bovine solubilized proteins preincubated with NaOH cataractous supernatant, also failed to show NBD quenching. The soluble proteins, extracted from bovine lenses after the first homogenization, were used as a control. These proteins did not produce any inhibition of adhesion w h e n added in the reconstitution of liposomes. Also, the preincubation of NBD-PC vesicles reconstituted with bovine m e m b r a n e proteins did not inhibit the agregation with Rh-PS liposomes. 4. Discussion It is possible that during cataractogenesis changes in the MIP molecule itself and its association with other proteins could influence the stability of lens

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junctions, altering the adhesive promoting role of MIP. In order to test these hypothesis we assayed the adhesive capability of MIP from both normal h u m a n (cadaver) and senile cataractous lenses. Junctional m e m b r a n e solubilized proteins, obtained from both types of lenses, were reconstituted in neutral PC liposomes and the interaction of these liposomes with PS vesicles (negatively charged) was studied. The adhesion effect between both population of vesicles was assayed using the probe mixing method. Here, the NBD fluorescence decreases as consequence of the resonance energy transfer to Rh (Duzgunez et al., 1987; Michea et al., 1994). Our results show that normal h u m a n lens junction solubilized proteins and pure MIP isolated from them, promotes adhesion in a similar way as MIP 28 isolated from bovine lens. Similarly, maximal initial quenching rate was observed at pH 6.0, presenting a similar pH adhesion dependence. No quenching effect was observed when MIP was omitted in the vesicle reconstitution, no other intrinsical protein of h u m a n junctional m e m b r a n e produced the adhesive effect. When MIP reconstituted vesicles were preincubated with a polyclonal antibody raised against a synthetic polypeptide corresponding sequence of the second extracellular loop of the MIP molecule the agregation was inhibited. We conclude that MIP from normal h u m a n lens, when reconstituted into large unilamellar liposomes, promotes adhesion in a similar way to MIP isolated from bovine lens. In contrast, MIP isolated from h u m a n senile cataractous lens fails to induce adhesion. SDS-PAGE analysis of cataractous junctional membrane intrinsical proteins shows a significant diminution of MIP 28 kDa, MIP 22 kDa being the major band in this preparation. We found that the in vitro proteolytic cleavage that originates MIP 22 kDa did not blunt the adhesive capability of MIP. The MIP 22 reconstituted vesicles induced adhesion and its effect was comparable to those obtained with MIP 28. This suggests that the proteolytic modifications of MIP 28 are not related with the incompetence of h u m a n senile cataractous lens junctions to induce adhesion. These results are not a surprise, because in vitro proteolytic cleavage of MIP 28 involves largely the C-terminus with only limited N-terminal residues being cleaved and none of the extracellular domains (Takemoto et al., 198B; Peracchia and Girsch, 1985: Johnson et al., 1985, 1986; Lampe and Johnson, 1990). In a similar way, it has been reported that the age-dependent proteolitic processing of MIP 28 in the h u m a n s lens occurs from both ends of the molecule (Takemoto and Takehana, 1986). In fact in our case, polyclonal antibody raised against a synthetic peptide including the middle region of the molecule (aminoacids 106 to 119, SVTPPAVRGNLALNT), cross-reacted with the MIP 22 originated from bovine MIP 28 and with the MIP 22 present in the h u m a n normal and cataractous lens

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junctions. Gorin et al. (1984) proposed for MIP a six transmembrane structure with three extracellular domains. Two of these extracellular loops display sequences of 20 amino acids including arginine (113, 187, 197) and histidine (40, 122), which are positively charged at pHs below their PKaS. These domains would be responsible for the interaction between MIP and negatively charged phospholipids. Consequently, the domains required to induce adhesion, which has net positive charge, should be present in the MIP 22 sequence. Since the treatment with trypsin and chymotrypsin do not cleave these domains, NBD-PC-MIP 22 vesicles do aggregate with PS vesicles. The SDS-PAGE profile of cataractous junctional membrane intrinsical proteins also shows the appearance of protein aggregates. These complexes also were seen in the reconstituted liposomes which failed to aggregate with the PS vesicles. To dissociate possible proteins forming complexes with MIP, cataractous junctional membranes were treated with O' 1 M NaOH. First, a clean SDS-PAGE profile was obtained, being MIP 22 the major intrinsic polypeptide of the total junctional protein. Secondly, the junctional cataractous membrane treated with NaOH and reconstituted in NBD-PC vesicles, shows adhesion comparable to those normally observed with MIP 28. The treatment with NaOH restored the adhesive capability. Moreover, the supernatant obtained after the treatment of cataractous junctional membranes with NaOH, inhibits the quenching effect. Also, NBD-PC vesicles reconstituted with bovine solubilized proteins, preincubated with NaOH cataractous supernatant, fail to show NBD quenching. These experiments prove that protein aggregates, which can be removed with NaOH, are accountable for lacking adhesion when h u m a n senile cataractous lens junction membranes are assayed. Also, they demonstrate that the endogenous proteolysis that MIP 28 suffers during cataract development does not hinder its adhesive capacity. The ability of MIP 22 to induce adhesion also suggests that the extracellular domains of the molecule are responsible for the adhesion. We did not find any evidence of protein aggregates in normal h u m a n lens. These were only seen in junctional membranes isolated from cataractous lenses. From the data presented here, we propose that in the cataractous lens extrinsic proteins, including agemodified crystallins (Garner, W. H. et al., 1981), link to MIP forming protein complexes. This interaction does not allow K4IP to promote adhesion with negatively charged phospholipids, destabilizing the MIP junctions, loosing the membrane integrity, producing a disappearance of normal fiber architecture and the concomitant appearance of vesicular and multilaminar structures observed in cataract lenses.

L.F. M I C H E A ET AL.

Acknowledgements We gratefully acknowledge Drs ]eanette Larrondo and Mauricio Yahez from the slaughterhouse Agrfcola Industrial Lo Valledor AASA SA. for providing us with bovine lenses. We are grateful to Dr A. Morello and Dr Y. Repetto for the use of the spectrofiuorometer. We thank Servicio M4dico Legal for providing us with normal human lenses. This investigation was supported by FONDECYT 1096-91, DT13245-9015. L. M. was supported by a doctoral fellowship from CONICYT, FONDECYT 2930001-1993 and Financiamiento Parcial Tesis de Doctorado Universidad de Chile. References Alcahi, J., Cendella, R.J. and Katar, M. (1985). Limited proteolysis of MP26 in lens fiber plasma membranes of the U18666A-induced cataract in rat. Curt. Eye Res. 4, 1001-5. Alcala, ]., Unakar, N., Katar, M. and Tsui, ]. (1990). Reversal of the limited proteolysis of MP26 during the reversal and prevention of the galactose cataract in rat lenses. Curt. Eye Res. 9, 225-32. Benedek, G.B., Clark, J.I., Serrallach, E.N., Young, C.I., Mengel, L., Sauke, T., Bagg, A. and Benedek, K. (1979). Light scattering and reversible cataracts in the calf and human lens. Phil. Trans. Roy. Soc. (London) 293, 329-40. Benedetti, E. L., Dunia, I., Bentzel, C. J., Vermorken, A. J. M., Kibelaar, M. and Bloemendal, H. (1976). A portrait of plasma specializations in eye lens epithelium fibers. Biochim. Biophys. Acta 457, 353-84. Bloemendal, H. (1991). Disorganization of membranes and abnormal intermediate filament assembly lead to cataract. Invest. Ophthalmol. Vis. Sci. 32, 445-55. Bok, D., Donckstaer, J. and Horwitz, J. (1982). Immunocytochemical localization of the lens main intrinsic polypeptide (MIP) in communicating junctions. J. Cell Biol. 92, 213-20. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of protein utilizing the principle of protein dye binding. Anal. Biochem. 12, 248-54. Clark, J. I., Mengel, L. and Benedek, G. B. (1980). Scanning electron microscopy of opaque and transparent states in reversible calf lens cataracts. Ophthal. Res. 12, 16-33. Clark, J. I., Mengel, L., Bagg, A. and Benedek, G. B. (1980). Cortical opacity, calcium concentration and fiber membrane structure in the calf lens. Exp. Eye Res. 31, 399-410. Duzgunes, N., Allen, T., Fedor, J. and Papahadjopouls, D. (1987). Lipid mixing during membrane aggregation and fusion: why fusion assays disagree. Biochemistry 26, 8435-42. Ehring, G. l., Zampighi, G. A., Bok, D., Horwitz, ]. and Hall, ]. E. (1990). Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes. ]. Gen. Physiol. 96, 631-64. Ehring, G.J., Lagos, N., Zampighi, G.A. and Hall, J.E. (1992). Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of the lens fiber membranes. J. Membrane Biol. 126, 75-88. Fitzgerald, P. G., Bok, D, and Horwitz, J. (1983). Immunocytological localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens. J. Cell Biol. 97, 1491-9. Garadi, R., Giblin, F. J. and Reddy, V. N. (1987). Disulfide-

ADHESION-PROMOTING

ROLE OF H U M A N MIP

linked proteins in X-ray-induced cataract. Ophthalmic. Res. 19, 141-9. Garner, W.H., Garner, M.H. and Spector, A. (1979). Comparison of the 10000 and 4 3 0 0 0 dalton polypeptide populations isolated from the water soluble and insoluble fractions of human cataractous lenses. Exp. Eye Res. 29, 257-76. Garner, M. H. and Spector, A. (1980). Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc. Natl. Acad. Sci., U.S.A. 77, 1274-7. Garner, W.H., Garner, M.H. and Spector, A. (1981). Gamma-crystallin, a major cytoplasmatic polypeptide disulfide linked to membrane proteins in human cataract. Biochem. Biophys. Res. Comm. 98, 439-47. Garner, M.H., Roy, D., Rosenfeld, L., Garner, W.H. and Spector, A, ( 1981). Biochemical evidence for membrane disintegration in human cataracts. Proc. Natl. Acad. Sci., U.S.A. 78, 1892-5. Garner, M. H., Roy, D. and Spector, A. (1982). Immunochemical characterization of the main intrinsic proteins of the human lens membrane. Exp. Eye Res. 34, 781-8. Gorin, M. B., Yancey, S. B., Cline, J., Revel, J. P. and Horwitz, J. (1984). The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning. Cell 39, 49-59. Harlow, E. and Lane, D. (1988) in Antibodies. A Laboratory Manual. Pp. 92-120, Cold Spring Harbor Press. New York. U.S.A. Harding, J. J. (1973). Disulfide cross linked protein of high molecular weight in human cataractous lens. Exp. Eye Res. 17, 377-83. Horwitz, J., Robertson, N.P., Wong, M. M., Zigler, S. and Kinoshita, J. (1979). Some properties of lens plasma membranes from normal human lenses. Exp. Eye Res. 28, 359-65. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., Hocker, L. O. and Benedek, G.B. (1973). On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses. Exp. Eye Res. 15, 185-90. Johnson. K.R., Lampe, P.D.. Hur, K.C., Louis, C.F. and Johnson, R: G. (1986). A lens intercellular junction protein, MP26, is a phosphoprotein. J. Cell Biol. 102, 1334-43. Kuck, J. F. R. and Lo, W.K. (1987). Alterations of urea insoluble membrane fraction of Emory mouse lenses in aging and cataractogenesis. Invest. Ophthalmol. Vis. Sci. 28 (suppl.), 388. Lampe, P.D. and Johnson. R.G. (1990). Aminoacid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes. Eur. ]. Biochem. 194, 541-7. Lo, W. and Harding, C. (1984). Square arrays and their role in the ridge formation in human lens fibers. ]. Ultrastruc. Res. 86. 228-45. Louis, C.F.. Johnson, R., Johnson, K. and Turnquist, J. (1985). Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase. Eur. J. Biochem. 150, 279-86. MacDonald, R. C., MacDonald, R.I., Menco, B., Keizo, T.. Subbarao. N. and Hu, L. (1991). Small-volume extrusion apparatus for preparation of large unilamellar vesicles. Biochim. Biophys. Acta 1061, 297-303.

301

Meek, J.L. (1986). Inositol bis-, tris-, and tetrakis(phosphate)s: analysis tissues by HPLC. Proc. Natl. Acad. Sci., U.S.A. 83. 4162-6. Michea, L. F., De la Fuente, M. and Lagos, N. (1994). Lens major intrinsic protein (MIP) promotes adhesion when reconstituted into large unilamellar liposomes. Biochemistry 33, 7663-9. Mimms, L., Zampighi, G.A., Nozaki, Y., Tanford, C. and Reynolds, J. ( 1981 ). Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry 20, 833-40. Perachia, C. and Girsch, S. J. (1985). Is the C-terminal arm of lens gap junction channel protein the channel gate. Am. J. Physiol. 249, H765-82. Philipson, B. (1979). Changes in the lens related to the reduction of transparency. Exp. Eye Res. 16. 29-39. Roy, D. (1979). Age dependent changes in the abundance of the major intrinsic polypeptides of human lens membranes. Biochim. Biophys. Res. Comm. 88, 30-6. Russell. P. and Kinoshita, J.H. (1980). Changes in the mouse lens membranes during cataractogenesis. Invest. Ophthahnol. Vis. Sci. 19 (suppl.), 150-1. Sas, D., Sas, M. J., Johnson, K. R., Menko. A. S. and Johnson, R.G. (1985). Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26. J. Cell Biol. 100, 216-25. Spector, A., Freund, T., Li Lu-ku, and Augus~eyn, R. (1971). Age dependent changes in the structure of alphacrystallin. Invest. Opthahnol. 10, 677-86. Spector, A. and Roy, D. (1978). Disulfide-linked high molecular weight protein associated with human cataract. Proc. Natl. Acad. Sci., U.S.A. 75, 3244-8. Spector, A. (1984). The search for a solution to senile cataracts. Proctor Lecture. Invest. Ophthalmol. Vis. Sci. 25, 130-46. Stryer, L. (1978). Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47, 819~J~6. Takemoto, L.J. and Hansen, J.S. (1982). Intermolecular disulfide bonding of lens membrane proteins during human cataractogenesis. Invest. Ophthahnol. Vis. Sci. 22, 336-42. Takemoto, L. J., Hansen, J., Nicholson, B. J., Hunkapiller. M., Revel, J.P. and Horwitz, J. (1983). Major intrinsic polypeptide of lens membrane. Biochemical and immunological characterization of the major cyanogen bromide fragment. Biochim. Biophys. Acta 7 3 1 , 2 6 7 - 7 4 . Towbin, H., Staehelin, The. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose gels: procedure and some applications. Proc. Natl. Acad. Sci., U.S.A. 16, 4350-4. Wong. M. M., Mercola, M. and Horwitz. I. (1980). Peptide mapping and topography of the main intrinsic polypeptide from bovine and human lens plasma membrane. Invest Ophthahnol. 19 (suppl.), 87. Zampighi, G. A., Simon, S. A. Robertson, J. D., McIntosh, T. J. and Costello, M. J. (1982 ). On the structural organization of isolated bovine lens fiber junctions. ]. Cell Biol. 92, 175-89. Zampighi, G.A., Hall, J.E. and Simon, S.A. (1989). The structural organization and protein composition of lens fiber junctions. J. Cell Biol. 108, 2255-75. Zampighi, G.A., Simon. S.A. and Hall. J.E. (1992). The specialized junctions of the Lens. Int. Bey. Cytol. 136, 185-225.