Ontogeny of γ-crystallin in the chick lens

Exp. Eye Res. (1973) 17, 341-346

Ontogeny of y-Crystallin S. K.

BRAHMA

AND

W. J.

in the Chick Lens VAX

DOORENMAALEK

Department of Medical Anatomy and Enabryology, The State University, Janskerlclzof 3A, Utrecht, The Netherlands (Received 25 June 1973, London) Lyophilized were tested methods in lens. Results line against lens. This delay crystallin is

extracts of embryonic, post-embryonic and adult chick total Iens proteins against pigeon lens y-crystallin antiserum by different immunochemical order to determine the time of appearance of this crystallin antigen in chick obtained from the experiments agree in showing that the immunoprecipitin pigeon lens y-crystallin antiserum first begins to appear from the l-week-old in appearance so low in adult

could possibly chick lens.

be one of the

reasons

why

the

amount

of y-

1. Introduction lens y-crystallin or low molecular weight protein (LM) shows an intermediate mobility. This is found in the P-crystallin region and as a result it is difIi.cult to detect (Rabaey, Rikkers and De Mets, 1972). According to these authors, in chick lens this protein is present in extremely low amounts (1% of the total protein content), while in other birds it can be detected easily. Isolated avian lens y-crystallin shows immunological cross-reactivity with different birds, and with members of other classes too (Rabaey et al., 1972). This crystallin has an elution volume similar to myoglobin on G-200 plate and shows one symmetrical peak in ultracentrifugation analysis (Rabaey et al., 1972). But when subjected to isoelectric focusing on polyacrylamide gel it shows four components, two major and two minor (Brahma, Rabaey and van Doorenmaalen, 1972). In a previous study, Brahma et al. (1972) detected y-crystallin antigen in the adult chick total lens protein extract by immunoelectrophoresis and tandem antigenantibody crossed electrophoresis against pigeon lens y-crystallin antiserum. But when embryonic chick lenses from various developmental stages were exposed to the same antiserum for indirect immunofluorescent staining no positive reactions could be obtained.. This prompted us to examine chick lens extracts from embryonic, postembryonic and adult animals against pigeon lens y-crystallin antiserum by immunodiffusion, immunoelectrophoresis and tandem antigen-antibody crossed electrophoresis in order to determine the time of appearance of the y-crystallin in the chick lens. Thin-layer isoelectric focusing on polyacrylamide gel was also performed to determine the isoelectric points of the four different components of the isolated pigeon lens y-crystallin antigen reported earlier (Brahma et al., 1972). Avian

2. Materials

and Methods

Lenses were isolated from 8-, lo-, 14-, 16-, and H-day embryonic and l- and a-week-old c,hicks and also from adult animals. These were homogenized in cooled deionized water at 4°C. The homogenate was then centrifuged at 49 500 g for 20 min a.t 4°C in a Sorvall 341

342

S.

K. BRAHMA

AND

W.

J.

VAN

DOORENMAALEN

RCS-B centrifuge. The clear supernatants were lyophilized and stored at -20°C until used. Two per cent solutions in saline were prepared from all these extracts. Immunodiffusion analysis (Ouchterlony, 1953) was carried out on 7.5 x2.5cm glass slides in 1.5% B&o agar (Difco Laboratories) prepared with high-resolution buffer (HRB Aronsson and Griinwall, 1957) at pH 8.9. Gelman immunodiffusion gel puncher was used to punch the holes. We used 10 ~1 of all the lens extracts and 10 $ of the pigeon lens y-crystallin antiserum. The gels were incubated during 48 hr at room temperature in a humid chamber, washed in saline, dried and stained with Amido black. Immunoelectrophoresis (Scheidegger, 1955) was carried out at 4°C also in 1.5% Bacto agar prepared with HRB at pH 8.9. The run was performed in a Shandon electrophoretic tank for 90 min with a constant current of 25 mA. Reaction against the antiserum was allowed to continue overnight in a humid chamber at room temperature. Slides were then washed in saline, dried, and stained with Amido black. In this experiment we used 10 ~1 of all the antigens and 100 ~1 of the antiserum. Tandem antigen-antibody crossed electrophoresis (Krell, 1968) was performed at 4°C for a direct comparison of the y-crystallin antigens in all t.hese total lens protein extracts. We followed the two-dimensional crossed-electrophoresis method of Clarke and Freeman (1968) as modified by Truman, Brown and Campbell (1972). The wells containing the lens antigens were at a distance of 0.8 cm from one another. The electrophoresis in the first dimension was for 45 min with 300 V and 19 mA, and in the second dimension for 22 hr with 150 V and 9 mA. The well on the cathodic side of all the gels always contained adult chick total lens protein extract for comparison. In the control experiment adult chick and pigeon total lens protein extracts were used. The gels were washed in saline at 40°C for 3 hr, dried and stained with Amido black. In this series we used 4 ~1 of all the lens extracts and 0.8 ml of the antiserum for each experiment. Thin-layer isoelectric focusing of the isolated pigeon lens y-crystallin antigen along with the adult chick and pigeon total lens protein extracts was carried out on 10 x 15-cm plate according to the method described by Bours (1971). The electrodes were placed at a distance of 13 cm. We used 15 ~1 from each of the 2% total lens extracts, and 10 ~1 from 1% solution of the pigeon lens y-crystallin antigen all dissolved in 2% Ampholine (pH 3.0-10.0). These samples were placed over 0.2~1 cm Whatman glass fibre papers. Eleetrofocusing was carried out for 20 hr at 4°C with an initial current of 4 mA and 80 V; which at the end of the experiment recorded 0.9 mA and 106 V. pH gradient along the gel was measured with a flat membrane glass electrode (Radiometer G 242C) at 4°C. Protein components on the gel were fixed at 70°C with 14% trichloroacetic acid (TCA) for 1 hr. This was replaced by 10 and 5% TCA at an interval of 2 hr and subsequently the gel was kept overnight at 3% TCA. It was then stained, destained, reswollen and photographed according to Bours (1971).

3. Results and Discussion Immunodiffusion analysis showed that the immunoprecipitin. line begins to appear with l-week-old chick lens [Fig. l(F)]. Th e reaction of identity between 1 and 2 weeks and the adult chick lens y-crystallin antigens showed that their antigenic properties are the same [Fig. l(F), (G), (H)]. Immunoelectrophoretic analysis also showed that the first appearance of the precipitin line for y-crystallin antigen starts with the l-week-old lens. In the beginning it is rather faint appearing near the antigen well and, with the age of the lens, it becomes prominent. In adult lens, the precipitin line forms an arc on the anodic side of the gel as reported earlier (Brahma et al., 1972).

y-CRYSTALLIN

IN

THE

CHICK

LENS

343

Tandem antigen-antibody crossed electrophoresis also showed that the immunoprecipitin line for the y-crystallin antigen begins to appear from the l-week-old lens [Fig. 2(b)]. In the beginning, the peak height is low but continuous with the peak resulting from adult chick total lens protein extract. Lens extracts from l&lay embryonic chicks did not show any such peak [Fig. 2(a)]. In 2-week-old chick total lens protein extract, the height of the peak for y-crystallin was prominent and continuous with the peak obtained from total lens protein extract [Fig. 2(c)]. In [Fig. 2(a), (b), (c)], well A contained total lens protein extract from adult chick, while well B contained total lens protein extracts from different stages (see legend of Fig. 2). In a control experiment, fusion of the two peaks obtained by total lens protein extracts from adult chick and pigeon was very prominent [Fig. 2(d)], but the peak height from chick lens was lower.

FIG;. 1. Immunodiffusion analysis of the embryonic, post-embryonic and adult chick total lens protein extracts against pigeon lens y-crystallin antiserum. Immunodiffusion was continued for 48 hr at room temperature in a humid chamber. The gel was then washed in saline, dried and stained with Amide black. Well A = 8 days embryonic total lens protein; well B = 10 days embryonic total lens protein; well C = 14 days embryonic total lens protein; well D = 16 days embryonic total lens protein; well I2 = 18 days embryonic total lens protein; well F = l-week-old total lens protein; well G = Z-week-old total lens protein; well H = adult total lens protein; well X = pigeon lens y-crystallin antiserum. Precipitin line starts from well F.

As reported earlier (Brahma et al., 1972), pigeon lens T/-crystallin antigen showed four components, and the isoelectric points of these components numbered one through to four determined in this series of experiments are 6.54, 6.29, 6.06 and 5.92; respectively [Fig. 3(b)]. Th e arrows indicate the position of the components 3 and 4 which are not visible in the photograph. The positions of these components in relation to the total protein patterns from pigeon and chick lenses are shown in Fig. 3(a), (c), and these fall within the “long line material” (Zwaan, 1963) or pcrystallin (Zwaan, 1966) regions whose isoelectric points extend from 5.85-7.58 (Bours, 1971). Results obtained from different immunochemical experiments corroborate one another with respect to the time of appearance of y-crysfallin antigen in the chick lens. This explains why Brahma et al. (1972) failed to get any positive immuno-

344

S. K.

BRAHMA

AND

W.

J.

VAN

DOORENMAALEN

fluorescent reaction in the developing embryonic chick lenses exposed to pigeon lens y-crystallin antiserum, and the concentration of the antigen does not seem to be the cause for negative results as suggested earlier (Brahma et al., 1972).

(a)

B

A

I3

A

B

A

B

A

‘”

(d)

FIG. 2. Tandem antigen-antibody crossed electrophoresis patterns obtained by specific antibodies against pigeon lens y-crystallin antigen. (a) In wells A and B adult and 1%day embryonic total lens protein extracts were used. No precipitin line developed with the antigens in well B. A peak due to antigens in well A is distinct. (b) In wells A and B, adult and l-week-old total lens protein extracts were used. A small hump due to antigens in well B (arrow) is seen and it is continuous with the peak obtained due to antigens in well A. (G) In well A and B adult and a-week-old total lens protein extracts were used. The small peak due to antigens in well B (arrow) is prominent and it is continuous with the peak obtained due to antigens in well A. (d) In wells A and B total lens protein extracts from adult chick and pigeon were used. The peak obtained due to antigens in well A (arrow) is prominent and continuous with the peak obtained from antigens in well B. It is apparent from this series of experiments that adult chick lens contains less y-crystallin antigen than adult pigeon lens.

From tandem antigen-antibody crossedelectrophoresis it is evident that the concentration of y-crystallin, which appears in the l-week-old lens, increaseswith age, but the amount is much lower in comparison to the y-crystallin in the pigeon lens. Our immunofluorescent experiments (in preparation) show that antigenically

y-CRY8TALLIN

IN

THE

CHICK

LENS

346

y-crystallin and PISC (First Important Soluble Crystallin, Rabaey; 1962) or “delta”crystallin (Zwaan and Ikeda, 1965) are different, and the latter appears much earlier than the former during lens development. pHt4*C)

9

a

(of

(cl

FIG. 3. Thin-layer isoelectric focusing of pigeon lens y-crystallin antigen, adult pigeon and chick total lens protein extracts on 5% polyacrylamide gel containin g 2% Ampholine carrier ampholyt,e (LKBProdukter, Sweden), pH range 3.0-10.0. Electrodes E+ and E- were at a distance of 13 cm. (a) Adult pigeon lens total protein extract. (b) Isolated pigeon lens y-crystallin antigen. (c) Adult chick lens total protein extract. Xumbers 1-4 indicat,e t.he components of the y-crystallin. The arrows show the position of components 3 and 4, which are not visible in the photograph. The samples were dissolved in 2% ampholine (pH 3.0-10 0).

n

346

S. K.

BRAHNA

AND

W.

J.

VAX

DOORENMAALEN

From t.hin-layer isoelectric focusing, it appearsthat pigeon lens y-crystallin antigen has isoelectric points higher than chick lens “delta”-cryetallin (5.10-5.35, Bours and van Doarenmaalen, 1972). In chick lens all the other crystallins, e.g. “delta’‘-crystallin, a-, and /3-crystallin, appear during early embryonic life (Rabaey, 1962; Zwaan, 1963; Zwaan and Ikeda, 1965; Ikeda and Zwaan, 1967; Zwaan and Ikeda, 1968; Zwaan, 1968; Brahma and van Doorenmaalen, 1971; Katoh and Yoshida, 1973), only the y-crystallin is the exception appearing in post-embryonic life. This delay could possibly be one of the reasonswhy its amount is so low in an adult lens. ACKNOWLEDGMENTS

We are extremely grateful to Professor31. Rabaey for the pigeon lensy-crystallin antigen fraction part of which wasusedin our previous experiment. We are alsothankful to Mr J. v. Ramshorstand Mr A. Wachter for their excellent technical help and to Mr Th. Hulskes and Mr A. M. v. Egeraat for the illustrations. REFERENCES Aronsson, T. and Grijnwall, A. (1957). &and. J. Cl&z. Lab. Invest. 9, 338. Bours, J. (1971). J. Chromatogr. 60, 225. Bours, J. and van Doorenmaalen, W. J. (1972). Exp. Eye Res. 13, 236. Brahma, S. K. and van Doorenmaalen, W. J. (1971). Ophthul. Res. 2, 344. Brahma, S. K., Rabaey, M. and van Doorenmaalen, W. J. (1972). Exp. Eye Res. 14, 103. Clarke, H. G. M. and Freeman, T. (1968). Clin. Sci. 35,403. Ikeda, A. and Zwaan, J. (1967). Develop. Biol. 15, 348. Katoh, A. and Yoshida, K. (1973). Exp. Eye Res. 15, 333. Krall, J. (1968). Scan& J. Clin. Lab. Invest. 22, 79. Ouchterlony, 0. (1953). Actu Pathol. Nicrobiol. Stand. 32, 231. Rabaey, &I. (1962). Exp. Eye Res. 1, 310. Rabaey, M. Rikkers. I. and De Mets, N. (1972). E’xp. Eye Res. 14, 208. Scheidegger, J. J. (1955). Id. Arch. AZZergy Appi. Immunol. 7, 103. Truman, D. E. S., Brown, A. 0. and Campbell, J. C. (1972). Exp. Eye Res. 13, 58. Zwaan, J. (1963). Immunochemical analysis of the eye lens during development. Thesis, University of Amsterdam. Zwaan, J. and Ikeda, -4. (1965). In !Z’he Second Syn~posium on the Structure of the Eye (Ed. Rohen, J. W.). P. 419. Schattauer-Verlag, Stuttgart. Zwaa.n, J. and Ikeda, A. (1968). Exp. Eye Res. 7,301. Zwaan, J. (1968). J. Cell. Physiol. (Suppl.), 72, 47.