The presence of delta-crystallin in the plasma membrane of chick lens fiber cells

E.vperimental

THE PLASMA

PRESENCE

Cell Research 109 (1977) 63-69

OF DELTA-CRYSTALLIN

MEMBRANE J. ALCALA,

OF CHICK H. MAISEL

IN THE

LENS

FIBER

CELLS

and N. LIESKA

Department of Amztomy, Wayne State University, Detroit, MI 48201, USA

SUMMARY Analyses by SDS-polyacrylamide gel electrophoresis of plasma membranes isolated from a rebion of the chick lens (outer cortex) where little or no (< 1.O%) soluble &crystalline is present confirmed the presence of 6-crystallin in the membranes, ruling out the possibility that this component constitutes a contaminant in the membranes of soluble fraction origin. Delta-crystalline (polypeptide 11) comprised 38 % of the protein recovered in the membrane fraction of the chick lens outer cortex. Immunological reactions utilizing an antiserum prepared in rabbits against the isolated membrane component confirmed its identity as Ccrystallin. Several (2-3) immunologically identical &crystalline subunits, ranging in molecular weight from 43 000 to 48 000 D, were consistently observed to be present in membranes from the 17-day embryonic and adult cortical lens fiber cells; only the 43000 molecular weight subunit appeared to be present in the membranes of the adult lens nucleus. The exact role of membrane crystallins in lens fiber cell differentiation now remains open to further investigation.

The presence of lens crystallin (soluble) polypeptides in lens fiber cell plasma membranes on the basis of their mobility in SDS-polyacrylamide gel electrophoresis has been recently noted [ 1, 2, 31. Prior reports that lens fiber ghosts reversibly bind soluble lens proteins [4] raised the possibility that these membrane crystallins constituted methodological contaminants of otherwise purified lens fiber cell plasma membrane preparations [2, 31. The present investigation was undertaken to clarify this point and to enlarge on the characterization of any crystallin-membrane linkage. To test the hypothesis that lens crystallins are a significant constituent of the lens fiber cell plasma membranes, analyses were conducted of plasma membranes isolated from a region of the chick lens cortex (outer cortex) where no cytoplasmic 5-771803

(soluble) 6-

crystalline

is detected

[5]. The immunologi-

cal relatedness between soluble and membrane &crystallins was determined. MATERIALS Isolation

AND METHODS

techniques

Adult White Leghorn chicken eyes, obtained from a local poultry concern within 2 h of slaughter, were N It

Fig. I. Diagrammatic representation of concentric adult chick lens regions employed for plasma membrane analyses. The view corresponds to the crosssectional chick lens area. OC, outer cortex; IC, inner cortex; N, nucleus.

64

Alcala,

Maisel and Lieska

Fig. 2. Electron micrograph of adult chick whole-lens plasma membrane fraction. x 20 496.

WL

lentectomized and the isolated lenses dissected free of their capsule and anterior epithelium (including annular-pad cells). The decapsulated lenses were dissected into three concentric regions of advancing fiber cell age and maturity (fig. 1); the three regions corresponded approximately to 0.2 (OC, outer cortex), 0.3 (IC, inner cortex), and 0.5 (N, nucleus) of the crosssectional lens equatorial diameter. Corresponding lens regions were pooled and homogenized in 9 vol (w/v) of a standard salt solution, SEM buffer (0.1 M KCl, 0.001 M EDTA, 0.01 M 2mercaptoethanol, 0.006 M sodium phosphate buffer, pH 7.2) at 4°C in all-glass tissue grinders. The homogenate was centrifuged at 37000 g for 20 min at 4°C and the supernatant, designated as the soluble fraction (SF), was retained. The insoluble pellet, consisting of the plasma membrane and intracellular matrix complex, was washed repeatedly with SEM buffer with rehomogenization and centrifugation as above until soluble proteins were no longer detected in the washing fluid [3]. Plasma membranes were isolated from the insoluble pellet by solubilization of the intracellular matrix with repeated treatment by homogenization with 8 M urea in SEM buffer [3]. The plasma membrane fraction (MF) was washed repeatedly with SEM buffer to remove urea, characterized electron microscopically as previously described [3], and analysed by SDS-polyacrylamide gel electrophoresis. Plasma membranes were isolated in a similar manner from whole, undivided adult and 17-day embryonic lenses. Eq,

Cdl

Res 109 (1977)

SF

MF *I

P

(195 000) (190000)

Fig. 3. Electrophoresis of the adult chick whole-lens (WL) soluble (SF) and plasma membrane (MF) fractions in 5.13 % polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The identification of individual lens crystallins in the SF, and molecular weight (parentheses) determinations of the major (numbered) polypeptides of the MF were carried out as previously described [3]. The origin is at the top of the figure.

Delta-crystallin N

IC MF

65

in chick lens plasma membranes

SF

MF

MF

i

i -11

*

SDS-polyacrylamide

gel electrophoresis

Electrophoresis [3] was performed on 9 cm polyacrylamide gel columns (T=5.13 %; C=2.5%) in the presence of 1% sodium dodecyl sulfate (SDS). Pellets of the membrane fractions (MF) were solubilized by homogenization (100 mg wet weight/ml) in a solution of 0.01 M Tris-acetate, 1% SDS, 0.001% EDTA and 0.1% 2-mercaptoethanol (pH 9.0) and incubated at 37°C for 1.5 h; samples of the lens-soluble fractions (SF) were mixed with the solubilization solution in a ratio of 1 : 9. Applications consisted of mixtures of two-thirds samples and one-third 30% sucrose, 0.05 % bromophenol blue in the solubilizing solution. Electrophoresis was at 2 mA/gel column at 25°C and was terminated when the bromophenol blue front had migrated 80 mm into the gel (approx. 3.5 h). Polypeptide bands were visualized by fixation, staining of the gels with 1% Amido Black and destaining as previously described [3]; the gels were scanned in a Canalco model G densitometer with automatic integration of peak areas. Molecular weight estimation of the polypeptides was performed utilizing standard markers [3]. Protein was determined by the method of Lowry et al. [6].

Immunological

corresponding in relative mobility (R, 0.750) to polypeptide 11 was sliced from 12 such gels, the gel slices were pooled in 6 ml of a solution of 0.05 M TrisHCI, 0.005 M MgCl,, and 10 mM 2-mercaptoethanol, pH 7.4 (TM buffer), and dialysed against repeated changes of TM buffer to effect elution of protein from the gel and remove protein-bound SDS (minimal amounts of SDS remain protein-bound following this procedure). The amount of protein recovered by this procedure consisted of 250 pg (dry weight basis). Portions of the solution containing 100 pg of protein were mixed with an equal volume of Freund’s complete adjuvant and utilized for immunization. The antisera were characterized by analytical immunodiffusion and immunoelectrophoresis as previously described [3] and utilized in similar reactions to determine the immunological relatedness of polypeptide 11 to chick-soluble &crystallin.

Table 1. Relative

abundancea

of the poly-

peptides Soluble fraction

techniques

Antisera to chick lens-soluble crystallins were prepared as previously described [3]. The antiserum to polypeptide 11 of the chick lens fiber cell plasma membranes was prepared by immunization of rabbits with repeated injections of solutions of the polypeptide in Freund’s complete adjuvant. The polypeptide was isolated by elution from its sliced gel segment as follows: following electrophoresis, SDS gels of wholelens fiber cell plasma membranes were subjected to repeated changes of a solution of ammonium sulfate at 40% saturation to visualize the polypeptide bands by reversible precipitation and to remove non-proteinbound SDS. The ammonium sulfate-precipitated band

Fig. 4. SDS-polyacrylamide gel electrophoresis of the soluble (SF) and membrane (MF) fractions of the three concentric adult chick lens regions studied (see fig. 1). Note the absence of a &crystallin band in the gel of the SF from the outer cortex (OC) (see fig. 3). OC, outer cortex; IC, inner cortex; N, nucleus.

Membrane fraction

Deltacrystallin 17-day embryonic lens Whole lens, adult Outer cortex Inner cortex Nucleus

85.0 40.0
32.0 27.0 38.0 30.0 24.0

a Rel. peak areas (percentage of stain) obtained by densitometric scanning of the gels. Exp Cdl

RP\

109 (1977)

66

Alcala,

Maisel and Lieska

IC

N

MF

SF

MF

‘, ,-11

A,

,I1

“i,,

5. SDS-polyacrylamide gel electrophoresis of the membrane fraction 04F) of the inner cortex (ZC) and of the soluble (SF) and membrane (MF) fractions of the nucleus (A’) of the adult chick lens. The gels had been underloaded relative to the gels of the corresponding fractions in fig. 4.

Fig.

bility with crystallin polypeptides of the lens-soluble fraction (fig. 3). Polypeptide 11, of identical mobility to subunits of soluble &crystallin, comprised 27 % of the protein recovered in this fraction (table 1). Electrophoretic analysis of the soluble and membrane fractions isolated from fiber cells of the chick lens outer cortex (OC) revealed the presence of polypeptide 11 in the membrane fraction although little or no b-crystallin was detected in the soluble fraction of this region (fig. 4, OC). Polypeptide 11 of the membrane fraction presented 2-3 closely spaced bands, ranging in molecular weights from 48000 D for the slower and 43000 D for the faster moving These components were component. treated as a unit in all further analyses conducted for this study. A comparison of the soluble and mem-

17DW

RESULTS

BF

Urea treatment of the chick whole-lens in soluble residue solubilizes the filamentous intracellular matrix and releases morphologically intact plasma membrane vesicles of various sizes (fig. 2). No obvious damage to the structural integrity of the membranes could be detected as a result of this treatment.

L MF

.; ii.93

! ,. A-

SDS-polyacrylamide

gel electrophoresis

Electrophoresis of the whole-lens insoluble residue [3] yielded sixteen major polypeptide bands ranging in molecular weights from 22 500 to 200 000 D (data not shown). Electrophoresis of the membrane fraction (MF) resulting from the urea treatment of the whole-lens insoluble residue shows that this fraction retains eight of these bands, three of which share electrophoretic moExp

Cd

RPS 109 (1977)

B-

B _ 6 : 6. SDS-polyacrylamide gel electrophoresis of the soluble (SF) and membrane MFI fractions of the 17day embryonic whole lens (Z7DiL). Note the presence of Gcrystallin in the soluble fraction (SF) and the multiple closely spaced bands of polypeptide II of the membrane fraction (MF). The soluble fraction (SF) gel at the right had been underloaded relative to the SF gel at the left.

Fig.

Delta-crystallin

in chick lens plasma membranes

67

lens revealed the presence of S-crystallin in the soluble fraction and of polypeptide 11 in the membrane fraction (fig. 6). The multiple nature of the membrane polypeptide 11 band of the 17-day embryonic lens confirms this as a feature characteristic of membranes of younger fiber cells (fig. 6). The relative abundance of soluble S-crystallin and membrane polypeptide 11 within the various fractions studied expressed as percentage of stain (relative peak areas) obtained by densitometric scanning of the gels is summarized in table 1. The values for the two polypeptides obtained from the adult whole-lens fractions proved to be misleading when compared with the values obtained from the fractions derived from the three concentric lens regions. While little or no S-crystallin (
brane fractions from the three concentric lens regions (fig. 4) revealed that although little or no soluble S-crystallin was detected in the outer cortex (OC) its presence was evident in the soluble fractions derived from the lens inner cortex (IC) and nucleus (N), while polypeptide 11 was present in the membrane fractions of all three lens regions. Its multiple band appearance was evident from membranes derived from the younger fiber cells of the lens outer and inner cortex but not from the nucleus (figs 4 and 5). Analysis of the soluble and membrane fractions from the 17-day embryonic whole

Immunodiffusion reactions of comparison utilizing the antisera against membrane polypeptide 11 and soluble S-crystallin from whole-lens and the two antigens are shown in fig. 7a. The reactions revealed a single confluent precipitin line indicating complete immunological identity between these components; the two antisera were consistently indistinguishable from each other in these reactions. Identical findings were obtained by utilizing an antiserum prepared in rabbits against the whole-lens soluble fraction in reaction with the two antigens (fig. 76). Immunoelectrophoretic analysis utilizing the antisera in reaction with samples of the E.~I Cd

Re,s 109 (/Y77)

68

Alcala,

Maisel and Lieska

&s

.

t1Cl.9

c

Aas

iibs Aas

?lGls Aas

Fig. 8. Analytical immunoelectrophoretic reactions in agar. (a) Reactions between the whole-lens soluble fraction, SF-W (well) not exposed to SDS, and the antisera to soluble %crystallin, Aus (upper trough), and to polypeptide 11, Zlas (lower trough); (b) reactions between the two antisera (troughs) and the soluble fraction from the lens outer cortex, SF-OC (well), similarly not exposed to SDS; (c) alternating reactions of the two antisera (troughs) and soluble &crystallin (A), polypeptide 11 (II) and the whole-lens (MF-WL) and outer cortical (MF-OC) plasma membrane fractions (wells) as antigens. The membrane fractions had been subjected to dialysis to remove excess SDS. The anode is to the right of the figure.

whole-lens and outer cortical soluble fractions which had not been exposed to SDS showed that the antiserum to polypeptide 11 reacted with native, undissociated soluble &crystallin, as well as its homologous antiserum (fig. 8a, 6). Reactions utilizing the two antisera and the polypeptides isolated from the SDS-polyacrylamide gels showed that, although residual proteinbound SDS altered the electrophoretic mobility of the polypeptides, it did not interE.r/> Cdl

Rrs 109 (1977)

outer cortical membrane fractions subjetted to prior dialysis to remove excess SDS following their solubilization in the anionic detergent (see fig. 8c, MF-WL and MF-OC). The results of these analyses also showed that the two antisera were indistinguishable from each other, conlirming the antigenic similarity between polypeptide 11 and soluble 8-crystallin. Since the multiple components of polypeptide 11 of the outer cortical membranes were incorporated together in reaction with the two antisera, the absence of any spurring phenomena in these reactions indicates the lack of immunological differences between these components as judged by the immunological techniques employed here. DISCUSSION The detection of a relatively high amount (38 %) of 6-crystallin (polypeptide 11) in the fiber cell plasma membrane fraction from a region of the chick lens where little or no soluble &crystallin (< 1.O %) is present, rules out the possibility that this component constitutes a methodological contaminant of this fraction and confirms the original hypothesis. The results of immunological reactions utilizing the antiserum to membrane polypeptide 11 confirmed its identity as S-crystallin. The results of this study thus support previous reports that lens crystallins are significant constituents of the lens fiber cell plasma membranes [l, 2, 31. A major species difference would appear to exist between the chick and bovine lenses in as much as a-crystallin is reported to be the principal crystallin constituent in the bovine fiber cell plasma membrane [2].

Delta-crystallin

in chick lens plasma membranes

69

Evidence obtained in this study indicates tering essential for lens transparency [ 11, that heterogeneity of membrane-bound 6- 121. The presence of &crystallin in memcrystallin subunits may be a feature characteristic of membranes of developmentally branes of differentiating lens fiber cells younger chick lens fiber cells. Multiple might indicate a regulatory function for the (2-3) &crystallin subunits were consistently membrane in the process of fiber cell difobserved to be present only in plasma mem- ferentiation. The presence of &crystallin branes from the 17-day embryonic lens and mRNA has been detected in ectodermal from adult lens cortex. Heterogeneity in cells prior to their elongation and lens plaotherwise immunologically identical soluble code formation and the accumulation of 6-crystallin subunits has been reported in a cytoplasmic (soluble) &crystallin [ 131. The variety of studies [7, 81 and is believed to exact role of membrane &crystallin in chick be the result of post-translational modificalens fiber cell differentiation now remains tions [9]. The presence of these subunits open to further investigation. The lens cell in the younger fiber cell plasma membranes plasma membrane provides a useful model indicates that 6-crystallin exists in the mem- for studying the role of the plasma membrane in vivo as a larger macromolecular brane in cellular differentiation and growth, aggregate of its subunits. and the maintenance of cell specialization. Delta-crystallin is probably inserted in This work was supported by NIH Grant EY 01855. the lipid bilayer of the chick lens fiber cell plasma membranes, since it is released only following detergent solubilization of the REFERENCES membranes. Such a strong interaction was 1. Bloemendal, H, Zweers, A, Vermorken, F, Dunia, suggested by observations that crystallins I & Benedetti, E L, Cell differ 1 (1972) 91. remained in the lens membrane fraction 2. Alcala, J, Lieska, N & Maisel, H, Exp eye res 21 (1975) 581. even after extensive washing with buffers, 3. Maisel, H, Alcala, J & Lieska, N, Documen treatment with 8 M urea and other agents ophthalmol8 (1976) 121. 4. Maraini, G & Fasella, P, Exp eye res 10 (1970) 133. [l, 2, 3, lo]. This linkage probably involves 5. Zwaan, J & Ikeda, A, Exp eye res 7 (1968) 301. membrane interactions with the strongly 6. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. hydrophobic regions of the lens crystallin 7. Clayton, R M, Curr top dev bio15 (1970) 115. polypeptides. Such interaction would ex8. Brahma, S K & v d Starre, H, Exp cell res 97 (1976) 175. plain prior reports that lens fiber ghosts 9. Piatigorsky, J, Exp eye res 21 (1975) 245. reversibly bind soluble lens crystallins [4], 10. Dunia, I, Sen Ghosh, C, Benedetti, E, Zweers, A & Bloemendal, H, FEBS lett 45 (1974) 139. and suggest that the lens plasma membrane 11. Trokel, S, Invest ophthalmol 1 (1962) 493. may have a role in the structural organiza12. Bettelheim, F A, Exp eye res 2 1 (1975) 23 1. 13. Shinohara, T & Piatigorsky, J, Proc natl acad sci tion of the intracellular fiber cell matrix. US 73 (1976) 2808. Regularity in the organizational structure of the intracellular fiber cell matrix is a pre- Received October 26, 1976 Revised version received April 5, 1977 requisite to the minimization of light scat- Accepted May 6. 1971