Eur. J . Biochem. 52, 143-155 (1975)

Lens Glycoproteins : Biosynthesis in Cultured Epithelial Cells of Bovine Lens R. Colin HUGHES, Maryvonne LAURENT, Marie-Odile LONCHAMPT, and Yves COURTOIS National Institute for Medical Research, Mill Hill, London, Groupe de Recherches sur les Maladies de la Rttine, HBpital Cochin, and Unite de Recherches Gerontologiques, Institut National de la Sante et de la Recherche Medicale, Paris (Rcccived July 26/November 5, 1974)

1. Radioactivity from [3H]glucosamineis rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecylsulphate- polyacrylamide gel electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000are present. The relationships of these glycoproteins to collagen and the non-collagenous glycoproteins of lens basement membranes are discussed. 2. A plasma membrane fraction obtained from non-trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non-glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al. (1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes. 3. Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present. The columnar epithelial cells of the lens form a monolayer on the anterior surface of the lens capsule [l]. The cells rest on the lens capsule, a basement membrane largely comprised of collagen [2- 51 that completely .surrounds the internal structures of the lens cortex. The cells of the lens epithelium perform two important functions. First, these cells synthesise and lay down the components of the extracellular basement membrane, including collagen [4,5]. Additional glycoproteins of lower molecular weight have also been found in lens capsule but their relationship to collagen and mode of biosynthesis remain unclear [4,6]. Secondly, formation of the main cellular component of the lens, the fibre cells, results from differentiation of the epithelial cells [I, 7,8]. Differentiation involves strictly only the cells at the equatorial region of the lens. These cells elongate and protrude into the centre of the lens in strict alignment one with another and the nuclei decrease in size. The lens fibre cells contain few cell organelles and the cytoplasm accumulates large amounts of the lens-specific proteins, CI B and Enzyme. Collagenase (EC 3.4.24.3).

Eur. J. Biochem. 52 (1975)

y crystallins [7,8]. The lens epithelium therefore is

particularly interesting, both for study of basement membrane biosynthesis and also as a differentiating system. In the present paper we describe experiments using cultured lens epithelial cells. These cultures incorporate radioactive glucosamine rapidly into cellular glycoproteins. Most of the radioactive glycoproteins are exposed on the surface of the cells and are released by treatment with trypsin. A smaller proportion are removed from intact cells with EDTA in the absence of trypsin. A smooth membrane fraction isolated from cells treated with EDTA contains one major glycoprotein of high molecular weight. Comparison is made of the smooth membrane fraction, largely plasma membranes, obtained in this paper and similar fractions prepared from differentiated lens fibre cells by others [9]. Lens epithelial cells also secrete into culture fluids several radioactive glycoproteins of lower molecular weight than the major membrane glycoprotein. The relation of products secreted by cultured epithelial cells to glycoprotein components of the extracellular basement membrane is discussed.

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MATERIALS AND METHODS

Solubilization of Basement Membranes

Cell Cultures

Samples of lens capsule basement membranes were treated (10 mg/ml) with 5 sodium dodecylsulphate/l % 2-mercaptoethanol at 90 "C for 10 min. Insoluble material was removed by centrifugation at 2000 rev./min and the soluble fraction containing 60% of the capsular protein was examined by acrylamide-gel electrophoresis. In the absence of reducing agent, 5 % sodium dodecylsulphate removed only 10-16% of the total capsular protein. The same spectrum of proteins and glycoproteins was present in this fraction, however, compared to that obtained under reducing conditions.

Sterile lens were excised from cow eyes freshly brought from the slaughter house, and washed in Hank's buffer (Flow Laboratories Irvine, Scotland). The capsules, after removing the lens fibril material, were placed in a 10-cm diameter Falcon petri dish, with 10 ml of minimal essential medium (MEM medium, Flow Laboratories, Irvine, Scotland) with 6 %, calf serum, penicillin (100 units/ml), 100 pg/ml, streptomycin, in a 95% air 5% COz mixture. Four capsules were kept in place on the bottom of each petri dish by sterile glass coverslips. The medium was changed every third day. Cells began to grow out of the capsules very quickly and to attach to both the coverslips and the petri dish. About 15 days later, when the cells were confluent they were removed from the dish with 0.25 % trypsin. The detached cells remained in clumps that were very difficult to dissociate even by vigorous mechanical stirring [lo]. Cells were counted with an hemocytometer and plated at a density of 4.5 x lo5cells per 10-cm-diameterpetri dish. When cells had reached confluency, they were subcultured again by the same technique, and plated with a split ratio of 1 :2 to 1 : 4. 15 days after the first subculture cells were seeded into roller bottles (surface area 690 cm') at a density of 4 x lo6 cells per bottle and grown further until confluent. The density at cell saturation was about 3-5 x 104 cells/cm2. The cells were usually very resistant to trypsin but could be easily removed from the glass within a few minutes in 10 ml of 20 mM EDTA in Ca, Mg-free Hank's buffer. These cells have been maintained for up to 4 months without any noticeable sign of degeneracy but their doubling time increased from less than 3 days for a young culture to 7 days after continuous culture for 5 months. Similar results were obtained previously [15] for rabbit lens cells. Cells were fixed and stained for light microscopy with May-Grungwald fixative. Preparation of Lens Capsule Basement Membranes

Cow eyes were excised directly after death and kept on ice until dissection. The outer capsule of the isolated lens was cut, removed and washed with Tyrode's buffer made up as described in [IOa]. It was then cut into pieces and dried with acetone. In order to reduce as much as possible contamination of the capsule by cells, in some experiments they were washed with Tyrode's buffer containing 0.02 % (w/v) EDTA at 4 "C for 30 min before acetone treatment.

Collagenase Treatment of Extracts

Aliquots of the lens capsule extracts were dialysed extensively in the cold against sodium phosphate buffer, pH 7, and the non-diffusible materials were treated (about 3 mg of protein/ml) with Clostridium histolyticum collagenase (0.5 mg/ml, 200 U/mg, Sigma Chem. Corp., London, type 111) at 37 "C for 1 h then at 2 "C overnight. Suitable portions (100-500 pg of protein) of the treated extracts were then examined by polyacrylamide gel electrophoresis. Control gels were run containing respectively untreated extracts and collagenase alone. Membrane Preparation of' Cultured Epithelial Cells

Cells (approximately 10') grown in roller bottles for 2 days as above with [3H]glucosamine(100 pCi, specific activity 12 Ci/mmol) and almost confluent were washed once with cold Hank's medium. The cell monolayer was rinsed rapidly with cold 0.02 % EDTA in calcium and magnesium free Hank's solutions and both washes were combined with the culture fluids. The combined solutions were dialysed against water at 2 "C and the non-diffusible fraction was lyophilised. The cells were detached from the surface of the growth bottles by addition of 0.02 % EDTA in calcium and magnesium-free Hank's solution warmed to 37 "C. The cells within a few minutes rounded-up, detached rapidly and began to form large clumps of cells. The cells were isolated and washed twice with complete Hank's solution by centrifugation at 600 x g for 3 min at room temperature. The supernatants from these centrifugations were combined, dialysed against cold water and lyophilized. This fraction is referred to as the "EDTA extract". The cells were then suspended in 4ml of hypotonic reticulocyte standard buffer (10 mM Tris-HC1 pH 7.2, 10 mM NaC1, 1.5 mM MgC1,) and allowed to swell in the cold for approximately 30 min. The cells were broken Eur. J. Biochem. 52 (1975)

R. C. Hughes, M. Laurent, M. 0.Lonchampt, and Y. Courtois

with 10 strokes in a Dounce homogenizer and the homogenate was centrifuged at slow speed for 2 min at room temperature. The pellet consisted largely of intact nuclei (4 x lo7 total count) and unidentified fibrous material. A few unbroken cells were also present. The turbid supernatant was mixed with 65 % (wiw) sucrose (4ml), adjusted to 10ml with reticulocyte standard buffer and added [final concentration 30 % (w/w) of sucrose] to a sucrose gradient consisting of 65 % (w/w) sucrose (3 ml), 45 % (w/w) sucrose (7 ml) and 40% (w/w) sucrose (7 ml). The gradient was completed with 25 % (w/w) sucrose (5 ml) and reticulocyte standard buffer (1.5 ml). Sucrose solutions were all in reticulocyte standard buffer. The tube was centrifuged at 64000 x g (average) in the SW 25.1 rotor for 17 h at 2 "C. After centrifugation several bands had formed within the gradient. A prominent band at the interface of the 25% and 30% sucrose layers represents the smooth membrane fractions. A main band of membrane elements was present at the 30% and 40% sucrose layers. A minor band at the 40% and 45 % sucrose interface, was not examined further. The two main membrane bands were recovered from the gradient, diluted with reticulocyte standard buffer several-fold and isolated by centrifugation at 35 000 x g and 2 "C for 1 h. Further purification was carried out by centrifugation through continuous gradients from 20 % (w/w) sucrose to 40 % (w/w) sucrose. Centrifugation in the SW 50.1 rotor was overnight at 2 "C and 114000 x g (average). Single membrane bands were visible in the gradients at the expected relative densities. Fractions (0.5 ml) were collected and aliquots (0.05 ml) counted for radioactivity in 10 ml of Instagel. The peaks of radioactivity coincided exactly with the visible membrane bands. The membranes were harvested, diluted with reticulocyte standard buffer and pelleted as before. The washed membrane pellets were suspended finally in reticulocyte standard buffer (1 ml) for analysis. Gel Electrophoresis

Polyacrylamide gel electrophoresis in 0.01 M sodium phosphate buffer containing 0. l % sodium dodecylsulphate was performed as described previously [ll]. The gels contain 7.5 % acrylamide. Samples were disaggregated before electrophoresis by heating at 90 "C for 5 min in 1 % sodium dodecylsulphate 0.1 % 2-mercaptoethanol 0.01 M phosphate buffer, pH 7. Gels were stained for proteins with amido black or Coomassie blue. Glycoproteins were detected with a periodate-Schiff technique [12]. Other gels were sliced and counted for radioactivity. Calibration Eur. J. Biochem. 52 (1975)

145

of the gels was done by electrophoresis of adenovirus and identification of the major proteins of the virion [13]. Enzyme Assays

5'-Nucleotidase activity, L-leucyl-B-naphthylamidase, phosphodiesterase and succinate dehydrogenase activities were assayed by standard techniques [9,14]. Incorporation Experiments

Lens epithelial cells were grown on 50 mM Falcon plates in Eagle's minimal essential medium plus 10 % fetal calf serum (5 ml per plate). Cells were pulsed for different periods of time with [3H]glucosamine(2 pCi per plate, specific activity 12 Ci/mmol). At appropriate times the culture fluid was removed from a plate and the cell sheet rinsed with cold Hank's solution. Cells were detached from the plastic surface by incubation at 37 "C for 10 min with 0.25% trypsin phosphate-buffered saline (2 ml). The cells were centrifuged and washed with phosphate-buffered saline (2 ml). The supernatants were combined (4 ml) and are referred to as "trypsinates". In some experiments, trypsinates were passed through a Millipore filter (0.45 pm) and dialysed overnight in the cold against water. The non-diffusible fractions were lyophilized and dissolved in water (1 ml) for analysis. The cell pellets were extracted with cold 5 % trichloroacetic acid (2 ml) overnight and insoluble fractions were collected and washed by centrifugation. Supernatant fractions were combined (4 ml) and counted for radioactivity. Insoluble fractions were dissolved at 60 "C for 1 h in Hyamine (1 ml) and also counted for radioactivity as described previously [111. Chromatography

Materials solubilized with trypsin were chromatographed at room temperature on columns (2.5 x 60cm) of Sephadex G50 equilibrated with 1 % sodium dodecylsulphate/lO mM Tris-HC1 buffer pH 7. The fractions (1.6) ml) were eluted with the same buffer and aliquots (0.5 ml) were counted for radioactivity. Chromatography on columns (2.2 x 150cm)of Sepharose 6B used 1 % sodium dodecylsulphate in 10mM Tris-HC1 pH 7 and fractions (2.8 ml) were collected. Sodium dodecylsulphate was removed from pooled fractions by exhaustive dialysis, first at room temperature against 0.1 M sodium chloride followed by dialysis in the cold against water. The non-diffusible fractions were freeze-dried and re-dissolved either in water or in 0.1 % sodium dodecylsulphate/lO mM Tris-HC1 pH 7.0.

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A

B

Fig. 1. Lens epithelial cells in culture for two months after explant. Cells were fixed and examined when growing (A) exponentially and (B) one week after confluency was reached. Note the cells showing mitosis in the exponential culture and the fibrous material in the confluent culture. Magnification : x 700. ( C ) x 3 1200. Aspect of an epithelial cell after 2 months

in culture. N : nucleus; C: cytoplasm; Mb: plasma membrane; M : mitochondrion; F : filaments; V: autolysis vacuole. Bar represents: 0.5 pm. (D) x 44800. Electron micrograph of intracellularand extracellular material. Note the intracellular microtubules (Mt), filaments (F), and pinocytotic vesicles (P) of the plasma membrane (Mb). Bar represents: 1 pm

Electron Microscopy

confluent cells (Fig. 1A). A striking phenomenon was noticed in confluent cultures, that consistently displayed a mass of fibrillar material in and around the cells as shown by light microscopy (Fig. B) or electron microscopy (Fig. l C , D). This material appeared to be of two distinct kinds. The intracellular material was mostly fibrillar. Microtubules having a diameter of about 18 nm and filaments 7 nm might represent cytoplasmic contractile elements (Fig. 1 D). Microtubules and filaments which run in different directions are perhaps related to actin-like molecules although we have not tested the ablity to form “arrowhead” structures in conjunction with meromyosin. Micro-filaments also formed “tufts” of parallel fibres which extended from the region near the nucleus to the surface membrane (Fig. 1 C). Fibrous material was also detected surrounding the cell and obscuring almost completely the plasma membrane. The structure was less organized than the cytoplasmic fibrillar

culture was fixed in glutaraldehYde fixative, osmium-treated, dehydrated and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate for examination in an electron microscope (Siemens Elmiskop 101).

RESULTS Epithelial Cell Cultures

In culture, the cells kept their epithelial cuboidal character even after 20 subcultures (Fig. 1A). Sometimes spindle-like cells could be found. The cells were sensitive to contact inhibition of mitosis but nevertheless could form multilayers as shown in Fig. 1B. At confluency virtually no mitotic cells were found (Fig.1B) in contrast to nonconfluent cells or just

Eur. J. Biochem. 52 (1975)

R. C. Hughes, M. Laurent, M. 0. Lonchampt,andY. Courtois

147

Fig. 1 C

material and probably was chemically distinct (Fig. 1D). The chemical nature of this material is unknown but it might be surmised that it is related to components of the basement membrane (collagen and possibly other glycoproteins). Sensitivity to collagenase has not yet however been established. Conversely in exponential cells there was very little of this material in agreement with similar findings reported previously [15]. The possible relation of these phenomena to differentiation and basement membrane biosynthesis in vitro [8,10,16,17] will be described elsewhere. Eur. J. Biochem. 52 (1975)

Incorporation Experiments

The incorporation of [3H]glucosamine by cultured lens epithelial cells is shown in Fig.2. Incubation of cells in culture at a low cell density with labelled precursor led to a relatively rapid incorporation of 3H into glycoprotein. A major proportion of the labelled glycoproteins was removed from the surface of intact cells with trypsin. Dialysis of trypsinates prepared from cells labelled for various times removed approximately 30 % of the total radioactivity showing that the largest part (70%) of the radioactivity was

Lens-Membrane Glycoproteins

148

Fig. 1D

Eur. J. Biochem. 52 (1975)

R. C. Hughes, M. Laurent, M. 0. Lonchampt, and Y. Courtois

149

I

I

1

I

I

5

0

0

5

10

15 Time ( h i

33

25

Fig. 2. Incorporation of [3H]glucosamine into cultured lens epithelial cells. Radioactivity present in materials removed from intact cells with trypsin, before (0)and after (0)dialysis, and in the trichloroacetic-acid-insoluble (0)and soluble (A) fractions of trypsinised cells was measured as described in the text, and is expressed per lo5 cells

present in glycoproteins of relatively large molecular weight. A small amount of radioactively labelled glycoproteins precipitable with cold trichloroacetic acid remained with the cells after treatment with trypsin. The origin of these glycoproteins is not clear but could be intracellular membranes of cells. Alternatively the radioactivity may be present in glycoprotein components of the cell surface membrane that are resistant to trypsin. Incorporation of labelled precursor into the acid-soluble fractions rapidly reached a constant level within 2 to 3 h of incubation. Since at least 90% of the radioactivity in these fractions was lost on dialysis, the acid-soluble materials probably represent in large part the amino-sugar pools of the cells. Essentially the same results as those shown in Fig. 2 were obtained with cultures of confluent epithelial cells. Glycoproteins Solubilized from Cultured Epithelial Cells

The molecular size of the glycoprotein fragments removed from intact cells with trypsin and representing at least 75 per cent of the radioactivity incorporated into glycoprotein material by lens epithelial cells in culture was examined by gel filtration. Two Eur. J. Biochem. 52 (1975)

distinct peaks of radioactive glycoproteins were obtained during chromatography on Sephadex G 50 (Fig. 3). Approximately one half of the radioactivity was recovered in glycoproteins eluted in the void volume of the column. In addition glycoprotein material was eluted in later, included fractions from the column. A third peak of radioactivity eluted in the salts region represented the small amount, about 30 %, of radioactivity removed from trypsinates by dialysis. Glycoproteins of cultured lens epithelial cells were also isolated by procedures not involving proteolysis. Cells labelled for an extended period with [3H]glucosamine were detached from the surface of growth vessels by brief treatment with EDTA. The EDTA detachment medium contained 2.9 x 10' counts per min of 3H after dialysis to remove low-molecularweight precursors. The non-diffusible radioactive materials were fractionated on Sepharose 6B (Fig. 4). Radioactivity was distributed in a series of broad, poorly separated peaks. Reference to the point of elution of immunoglobulin on the same column suggested that the epithelial glycoproteins solubilized by EDTA had molecular weights ranging downwards from approximately 150000. Sodium dodecylsulphate disc-gel electrophoresis of pooled column fractions disaggregated in the presence of 2-mercaptoethanol is shown in Fig. 5 E and F. Although electrophoresis was carried out in the absence of reducing agent and reaggregation may have taken place to some extent the results were

Lens-Membrane Glycoproteins

150 4

4

4 000

3000 2000 1000

-

0

c .-

E

>-

1000

c

Fraction number

0 3 1

Fig. 4. Sepharose 6 B chromatogruphy of radioactive glycoproteins secreted by lens epithelial cells in culture ( 0 ) .The boxes indicate fractions combined for analysis. The chromatography was performed in buffer containing 1% sodium dodecylsulphate. The profile of radioactive glycoproteins removed from labelled cells by EDTA is also shown (0). 2.8-ml fractions were collectcd. The arrows indicate the points of elution respectively of blue dextran (leading peak only), immunoglobulin (mol. wt lSOOOO), '251-labelled adenovirus fibre (mol. wt 62000) and [3H]glucosamine

>

: - 1500

.-

0 m

$1000 cc

500 0 2000

I000

consistent in that chromatography on Sepharose 6 B gave fractions clearly differing in mobilities on polyacrylamide-gel electrophoresis. A major part of the radioactivity eluted early from Sepharose 6B gave a broad peak on electrophoresis with an apparent average molecular weight of 75000- 85000. Later fractions from Sepharose 6 B contained a broad spectrum of glycoproteins, largely of lower apparent molecular weights. The net charge of the glycoproteins eluted from cultured epithelial cells with EDTA was examined by isoelectric focussing in sucrose gradients and pH gradient 3- 10. The radioactive material was concentrated in the acid range and no radioactivity was present in glycoproteins of p l values greater than about 3.8. We therefore conclude that the glycoproteins present in the EDTA extract are acidic glycoproteins. Epithelial Cell Membranes

Two main membrane bands could be prepared from EDTA-detached cells : S 1banded at the 25 - 30 interface and S 2 at the 30-40% interface on centrifugation through a discontinuous sucrose gradient. The sedimentation characteristics of these bands are similar to results obtained with HeLa cells and chick fibroblasts using the same fractionation scheme [18, 191. Attempts to detect enzymic activities normally described as specific for plasma membranes, for example 5'-nucleotidase, leucyl aminopeptidase, and phosphodiesterase have so far been unsuccessful.

0

20

40 rnm

60

Fig. 5. Sodium dodecylsulphate -polyacrylumide gel electrophoresis patterns. Solubilization and electrophoresis were carried out as described in Materials and Methods. All fractions were obtained from lens epithelial cells cultured with [3H]glucosamine. (A) Membrane band S 1 ; (B) membrane band S 2; (C) secreted glycoproteins, Sepharose 6 B fractions 75 - 95; (D) secreted glycoproteins, Sepharose 6 B fractions 96 - 108; (E) EDTA-soluble glycoproteins, Sepharose 6 B fractions 76- 90; (F) EDTA-soluble glycoproteins, Sepharose 6B fractions 95- 120. (I) Glycoprotein of mol. wt 120000; (11) adenovirus fibre protein, mol. wt 62000; (111) position of dye marker. The peaks 1 - 5 in box C are discussed in the text

Activities were below the levels of detection as described by others for differentiated lens fibre cells [9]. Succinate dehydrogenase activity was undetectable in the purified membrane fractions S 1 and S 2 from the lens epithelial cells indicating the absence of mitochondria1 contamination. Bands S 1 and S 2 purified further by centrifugation through continuous 20 - 40 % sucrose gradients were radioactively labelled to different extents during the growth in the presence of [3H]glucosamine.About 80 % of the 3H recovered from the gradient was present in band S 1. The specific activity of this fraction was 2.9 x lo5 counts/min per mg protein while S 2 contained 0.68 x lo5 counts/ min per mg protein. Since a large part of the radioactivity incorporated by cultured lens epithelial cells is removed with trypsin from the surface of intact Eur. J. Biochem. 52 (1975)

R. C. Hughes, M. Laurent, M. 0.Lonchampt, and Y . Courtois

cells, membrane band S1 is identified with some confidence as a plasma membrane fraction. The origin of membrane band S2 may derive from intracellular smooth or rough membrane elements although the presence of some surface membrane cannot be excluded. The patterns of radioactive glycoproteins obtained by polyacrylamide-gelelectrophoresisin sodium dodecylsulphate of the membrane fractions S 1 and and S 2 are shown in Fig. 5A, B. The very low radioactivity obtained on electrophoresis of band S 2 was distributed throughout the gel. Electrophoresis under identical conditions of band S 1 showed a prominent radioactive peak with an apparent molecular weight of 120000. In addition a number of less prominent, faster-moving peaks were also present. When gels were stained for protein the two membrane bands S 1 and S 2 were found to contain a different spectrum of polypeptides as shown in Fig.6. A prominently staining band of apparent molecular weight 120000 was present in membrane band S1 (Fig.6A) and represents the major glycoprotein of this fraction. No similar band staining for protein was found in membrane fraction S 2 (Fig. 6B). However, it cannot be excluded that a glycoprotein of electrophoretic mobility similar to that of the major radio-labelled band of fraction S 1 is present in fraction S 2 although a proportionately greater amount of the total glucosamine label in this fraction was present in materials of lower molecular weight running more quickly during electrophoresis. Glycoprotein Secretion by Cultured Cells

Culture fluids obtained from lens epithelial cells grown in the presence of [3H]glucosamine as described previously were dialysed extensively against water in the cold. The non-diffusible fraction contained radioactive substances of relatively large molecular size. These glycoproteins were chromatographed on Sepharose 6 B in an eluting buffer containing sodium dodecylsulphate to ensure disaggregation of the labelled materials. The result is shown in Fig. 4. A broad peak with a pronounced leading shoulder was eluted in the included region of the column. Only a small amount of radioactivity was present in the salts peak showing that most of the radioactive precursor had been removed during dialysis. The approximate size of the main radioactive peak was estimated by reference to the points of elution from the column under exactly the same conditions of immunoglobulin (mol. wt 150000) and the adenovirus fibre protein (mol. wt 620000). The comparison suggested that the radioactively labelled glycoproteins secreted into the medium by cultured lens epithelial cells were Eur. J. Biochem. 52 (1975)

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Fig. 6. Sodium dodecylsut'phate-polyacrylamide gel electrophoresis of ( A ) lens epithelial cell membrane band S I ;( B ) lens epithelial cell membrane band S 2 ; ( C ) and ( E ) sodium dodecylsulphate 2-mercaptoethanol extract, lens capsule contaminated with cellular material: ( D ) and (F) sodium dodecylsulphate 2-mercapioethanol extract, lens capsuleuncontaminated with cellular material. All gels were stained with amido black except gels E and F. These were stained by a periodate-Schiffs reagent. The arrows in the left-hand margin denote a major band of lens epithelium membranes having a molecular weight of 120000, and two components of capsule extracts having approximate molecular weight respectively of 90 000 and 45000. The fast-moving crystalline proteins are also indicated (Cr)

of similar average size as the adenovirus fibre protein with a broad spread towards both the low and high molecular weight regions of the column. The radioactive peak from Sepharose 6 B was divided arbitrarily into two subfractions. Detergent was removed by dialysis, the non-diffusible fractions were lyophilized and dissolved in water. The recovery of radioactivity from the column was 68 %. Radioactive lens glycoproteins from culture fluids and fractionated by Sepharose 6B (Fig.4) was examined directly by acrylamide electrophoresis in buffers containing sodium dodecylsulphate. The samples, each containing 10000 cpm counts/min 3H, were heated beforehand for 5 min at 90 "C in 1 % sodium dodecylsulphate / 0.1 % 2-mercaptoethanol / 0.01 M phosphate, pH 7. After electrophoresis the gels were sliced and counted for 3H radioactivity. The profiles obtained are shown in Fig. 5 C and D. Clearly chromatography on Sepharose 6B separated secreted radio-

152

active materials into fractions that could be distinguished after reduction and electrophoresis by different patterns of polypeptides. One explanation possible for the fractionation obtained on Sepharose 6B chromatography is that this proceeded on the basis of size of the unreduced glycoproteins. If this is the case, some overlap in the apparent molecular size of the constituent glycosylated polypeptide subunits as estimated by polyacrylamide-gel electrophoresis in reducing conditions of materials eluted in separate regions of the Sepharose column in the absence of reducing agent is not unexpected. Only the samples taken for electrophoresis were treated with reducing agent. Since it is unlikely that reassociation of subunits would be complete during electrophoresis, the electrophoresis patterns therefore reflect subunit molecular weights. The two profiles fit into a consistent pattern with a number of peaks distributed across the length of the gels. These peaks have been numbered 1- 5 with the following approximate molecular sizes: 1, 120000; 2, 65000; 3, 40000; 4, 20000; 5,14000. In order to tentatively identify collagen-like componcnts in the culture fluids of epithelial cells, the technique of iso-electric focussing in sucrose gradients was used. It is known that collagen has a relatively high isoionic point (pl 6.6-6.8). By contrast, many glycoproteinsderived from cellular plasma membranes contain a high proportion of sialic acid [29] and would consequently focus to lower p l values. Dialysed culture fluids obtained from cells ( 5 x lo6) cultured in the presence of ['4C]glucosamine (160 pCi, 45 mCi/ mmol) for three days, containing 2.5 x lo6 counts per min of 14C were fractionated by addition of saturated ammonium sulphate solution at 20 "C. The proportions of the total radioactivity found in the 0-33% precipitate, the 33 - 66 % precipitate and the 66 supernatant were 31 %, 63 %, and 6 % respectively. Each fraction was further separated by isoelectric focussing in a 5 - 50 % (w/w) sucrose gradient modified [33] to extend the pH range to low value using an ampholite mixture having a normal pH range of 3 - 10. The profiles of radioactivity recovered from each gradient after focussingshowed that the materials precipitated by ammonium sulphate had relatively high isoelectric points in the region of pH 7, similar to values (6.6-6.8) reported for collagen. By contrast a large proportion (75 - 85 %) of the material soluble in 66% saturated ammonium sulphate was spread throughout the pH gradient at lower pH values. Several well-defined peaks were obtained with apparent isoelectric points of 5.6, 5.2 and 2.8. We therefore conclude that both acidic glycoproteins (minor proportion) and more neutral glycoproteins (major proportion) are shed from epithelial cells in culture

Lens-Membrane Glycoproteins

and a partial fractionation of these components is obtained by salt precipitation. Glycoproteins of Lens Capsule

In order to determine the glycoprotein content of lens capsule for comparison with the membrane glycoproteins of epithelial cells and glycoproteins shed by the cells into the culture medium, materials made soluble by hot sodium dodecylsulphate 2-mercaptoethanol mixtures were examined by sodium dodecylsulphate- polyacrylamide gel electrophoresis under the conditions used for the lens epithelial cell fractions. As seen in Fig.6D the polypeptides extracted were of high molecular weight and almost all of them appeared to be glycosylated (Fig. 6 F). In addition to these high molecular weight glycoproteins, extracts obtained from some preparations of capsule with sodium dodecylsulphate contained a number of faster migrating non glycosylated polypeptides (Fig. 6C). These include a prominent band of apparent molecular weight 45 000 the possible significance of which is discussed later, and three polypeptide bands of lower molecular weights staining intensely for protein that were tentatively identified as the major soluble lens proteins, the cytoplasmic proteins of lens cells a, b and y crystallins [8,9]. The b and y crystallins were unresolved in this system. The two subunits [9] of CI crystallin however were clearly separated. The presence of crystallin proteins in the lens capsule preparation shows that removal of cellular material was incomplete. Others [25,26] have commented on the difficulty of separating basement membranes from adhering epithelium. However, the origins of the glycoproteins of high molecular weight clearly cannot be from a cellular contaminant. This is shown by the finding that the concentration of these components in capsule extracts is entirely unrelated to the degree of contamination of the capsules with the cellular cytoplasmic proteins. Thus, these glycoproteins are prominent in capsule extracts even when only faint amounts of cell proteins such as tl, 8, y crystallins are detected (Fig. 6 D and F). A prominent capsular glycoprotein band with an apparent molecular weight of 120000 was detected, a size similar to the major glycoprotein of the smooth membrane band S 1 of cultured lens epithelial cells. However, it is relevant to note that the final form of lens cr-collagen consists of polypeptides with a molecular weight of 115000 [20]. A large precursor polypeptide of lens collagen has a molecular weight of 140000 [21]. The capsular glycoproteins of apparent high molecular weights and migrating with and more slowly than the 120000 molecular weight band may Eur. J. Biochem. 52 (1975)

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It is clear from the staining patterns that considerable quantities of material in capsule extracts did not penetrate the 7.5% polyacrylamide gel. It is possible that this represents cross-linked collagen since the intensity of stain at the origin is greatly reduced in samples treated with collagenase.

DISCUSSION

Fig. I . Collagenase treatment o j isolated lens capsule. The electrophoretic analysis of the solubilized capsule preparation is shown in Fig.6 gels D and F. In the present experiment reaction mixtures containing (A) capsule and collagenase or (B) collagenase alone were examined after incubation. The gels were stained for protein. The molecular weights of the separated bands were estimated by comparison with structural polypeptides of adenovirus as described in Materials and Methods. Note the absence of bands in both gels moving more slowly than a component of apparent molecular weight 105000. This latter component is derived form the collagenase preparation

therefore derive from collagen-containing components of the basement membrane. This supposition was strengthened by the susceptibility of these bands, having apparent molecular weights of 120000,140000 and 155000, to collagenase. After treatment with the enzyme, no prominent polypeptide bands were detected in these regions of the gels (compare gels Fig.6D and Fig.7A). The collagenase preparation used contains a number of polypeptides migrating during gel electrophoresis in the region of the fourth prominent capsular glycoprotein component with apparent molecular weight 90 000 and the susceptibility of this material cannot be assessed unequivocally by this method. The nature of this material is under study. The collagenase-treated capsules on gel electrophoresis also show several fast-moving polypeptides that are absent in untreated capsules and in the enzyme. These presumably represent degradation fragments produced during collagenase action on the higher molecular weight fractions. Eur. J. Biochem. 52 (1975)

One of the purposes of the present study was to compare the glycoproteins of lens capsule with those synthesized by the lens epithelium. It should be noted that the criteria used in the present paper to establish a relationship between glycoprotein fractions are preliminary and depend largely on the rates of migration during polydcrylamide-gel electrophoresis under dissociating conditions, on the estimated net charge determined by isoelectric focussing and lastly on the sensitivity of the glycoproteins to collagenase. Nevertheless, some relationships are suggested between glycoprotein synthesis carried out by the lens epithelial cells in culture and the dual biological function of these cells in vivo in capsule synthesis and differentiation into fibre cells. The components of the lens capsule, as with all basement membranes, are synthesized in the underlying monolayer of epithelial cells and are subsequently secreted [4]. Collagen biosynthesis in cultured lens epithelium has been studied thoroughly [5,21]. It has been found that only a small proportion (2%) of the total protein synthesized by these cells is basement membrane collagen. The presence of glycoproteins additional to collagen in lens capsule has been repeatedly reported [4,22 - 241 and these components may be assumed provisionally also to be synthesized in the epithelial cells. The carbohydrate composition of basement membranes is quite complex and of uncertain origin. Thus, heterosaccharides present in soluble fractions of lens capsule obtained by treatment with collagenase [22] may derive from collagen itself rather than separate glycoproteins. However several glycoproteins have been described that remain insoluble after collagenase treatment of bovine lens capsule [24]. One glycoprotein accounting for 80 ”/, of the collagenase-insoluble carbohydrate and containing galactose, mannose, glucose, fucose, N-acetylneuraminic acid and N-acetylhexosamines has been reported to have a subunit molecular weight of approximately 35000 on sodium dodecylsulphate electrophoresis and forms larger aggregates (mol. wt. 75 000 - 150000) in the absence of detergent. In the present work, it was reasoned that if separate glycoproteins do indeed exist in lens

Lens-Membrane Glycoproteins

154

capsule then these substances might be synthesized and secreted in vitro by cultured lens epithelial cells. Clearly, however, the situation is not so clear cut. No glycoproteins of low molecular weight were detected in significant amounts in the extracts of lens capsule used in the present work in contrast to other results [24]. Possibly these glycoproteins are present in such small amounts in bovine capsule that they escape detection unless the large excess of collagen is removed first by collagenase. Alternatively, they may be degraded to low molecular weight fragments by collagenase under the conditions used by Dische, and form part of much larger components in the native capsule. It is of some interest, however, to find a major glycoprotein both in dodecylsulphate extracts of capsule and in EDTA extracts of epithelial cells with a similar molecular weight of about 75000- 90000. Attempts to show an antigenic and chemical relationship between these components, respectively of the lens capsule basement membrane or loosely attached to the surface of the lens epithelial cells and detached by chelating agent, are in progress. In this view it is tentatively considered that the loosely attached material may be a capsular glycoprotein in the process of secretion from the lens epithelial cells supporting its biosynthesis. The extracellular fluids of cultured epithelial cells contain a host of glycoproteins differing in molecular weight and ranging from a high apparent size of 120000 down to fast migrating glycoproteins of apparent size 14000.The presence of a minor glycoprotein peak of 120000 among the glycoproteins secreted by lens epithelial cells is especially interesting since this is similar to the major glycoprotein of the smooth membrane fraction S 1. The mechanism of shedding of integral surface membrane components into the medium by cells in culture [27,28,30] is largely unknown. It is interesting to speculate however to what extent the complicated pattern of glycoproteins present in culture fluids of lens epithelial cells results from the turnover of a small number of integral glycoproteins of the epithelial cell surface membrane. The secreted glycoproteins may not of course all derive from surface membrane turnover [30] and could contain substances, or perhaps proteolytic fragments of substances, not directly related to the cell surface membrane components. Indeed secretion of an antigenic glycoprotein component of yolk sac basement membranes occurs during culture of a transplantable parietal-yolk-sac carcinoma of the mouse [31,32]. This process might explain the presence of collagen-like molecules in culture fluids of lens epithelial cells detected in the present work. These

substances have a relatively high isoelectric point and are clearly separated from more acidic secreted glycoproteins by isoelectric focussing. The relation of the more acidic non-collageneous glycoproteins to turnover of the epithelial-cell membrane on the one hand and to the non-collagenous basement membrane components on the other remains to be determined. The authors are grateful to Mr T. B. Butters for the gel electrophoresis and his help in the analyses of enzymes, and to Miss Jacqueline Tassin for expert assistance in cell culture experiments.

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R. C. Hughes, M. Laurent, M. 0. Lonchampt, andY. Courtois 25. Mohos, S. C . & Skoza, L. (1969) Science (Wash. D.C.) 164,1519- 1521. 26. Mohos, S . C. & Skoza, L. (1970) Exp. Mol. Pathol. 12, 316- 321. 27. Hughes, R. C., Sanford, B. H. & Jeanloz, R. W. (1972) Proc. Natl Acad. Sci. U.S.A. 69, 942-945. 28. Chiarugi, V. P. & Urbano, P. (1972) J. Gen. Virol. 14, 133-140. 29. Hughes, R. C. (1973) Progr. Biophys. Mol. Biol. 26, 189-268.

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R. C. Hughes, M.R.C. National Institute for Medical Research, The Ridgeway, Mill Hill, London, Great Britain NW7 1AA M. Laurent and M. 0. Lonchampt, Groupe de Recherches sur les Maladies de la Rktine, Hapita1 Cochin, 27, rue du Faubourg St-Jacques, F-75014 Paris, France Y. Courtois, Unitt de Recherches Gerontologiques de l’I.N.S.E.R.M., 29, rue Wilhelm, F-75016 Paris, France

Eur. J. Biochem. 52 (1975)

Lens glycoproteins: biosynthesis in cultured epithelial cells of bovine lens.

1. Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated iso...
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