Exp. Eye Res. (1990) 50, 429437

Differential

Synthesis

of Crystallins Rat Eye Lens

CHRISTINA E. M.VOORTER*, MONIQUE M. P. HERMANS, HANS Department

of Biochemistry,

(Received

7 August

University

in the

Developing

WILLEKE A. DE HAARD-HOEKMAN, BLOEMENDAL AND WILFRIED W. DE JONG of Nijmegen, The Netherlands

1989 and accepted

P.O. Box 9101, 6500 HB Nijmegen,

in revised

form 3 October

1989)

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [36S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of aA and /?A3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of /3B2 and ys shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six y-crystallins (yA-yF). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency. Key words: lens: protein synthesis; rat: development.

and Rink, 1983) and protein synthesis (Carper et al., 1985) during development of the rat lens, the use of

1. Introduction The bulk of the soluble proteins in the mammalian eye lens is made up by the products of some 15 different crystallin genes (Wistow and Piatigorsky, 1988 ; Lubsen, Aarts and Schoenmakers, 1988). The limited number of crystallin polypeptide chains and the detailed knowledge of their gene and protein structures make the lens an ideal model to study developmental changes in protein composition. The differential synthesis of crystailins during development, combined with the specific growth pattern of the lens, results in a gradient in protein concentration and composition of the successive lens layers (Piatigorsky, 198 1; Carper et al., 1985; Hejtmancik et al., 1985; Thomson and Augusteyn, 1985; Zlgler et al., 1985; Bessems et al.. 1986; Murer-Orlando et al., 1987; Van Leen et al., 198 7). Subsequent post-translational modifications generate additional diversity of protein composition (Zigler and Goosey, 1981; Hoenders and Bloemendal, 1983; de Jong et al., 1988; Chiesa et al., 1988). The resulting spatial distribution of the crystallins most probably contributes to the optical properties of the ocular lens, and gene expression in the lens might therefore require a carefully balanced regulation. In rat lenses the regulation of crystallin expression has been studied at the transcriptional level by determination of mRNA levels for different crystallins during development (van Leen et al., 1987; Aarts, Lubsen and Schoenmakers, 1989). However, these mRNA levels do not necessarily reflect actual protein synthesis, due to variations in translational efficiency. Although several studies have dealt with changes in protein composition (Ramaekers et al., 1982; Uchiumi, Kimura and Ogata, 198 3 ; Vornhagen, * For correspondence. 00144835/90/040429+09

$03.00/O

Bours

different

nomenclatures

for the observed

proteins,

without any link to the corresponding genes, makes it impossible to correlate the observed changes with the alterations at the mRNA level. Recently, Siezen et al. (1988) inferred the differential synthesis of ycrystallins during development from the amounts present in the outer layer of rat lenses of diierent ages. These authors conclude that the proportions of synthesized y-crystallins did not correlate very well with the reported levels of the corresponding mRNAs (Van Leen et al., 1987). We approached the problem more directly by determining the in situ synthesis of crystallins in rat lenses of different ages. We therefore incubated intact rat lenses in medium containing [35S]methionine, and compared the incorporation of radioactivity in the different crystallin chains. We studied not only the synthesis of the y-crystallins, but also that of the CL-and P-crystallms. The identification of the de novo synthesized crystallin gene products enables us to compare the developmental changes of protein synthesis with the previously measured mRNA levels (Van Leen et al., 1987: Aarts et al.. 1989). 2. Materials and Methods Rat Lens Incubations

Wistar rats of different ages were obtained from the Central Animal Facilities of the University of Nijmegen, School of Medicine. Rat eye lenses were immediately removed after death by decapitation, and directly placed in incubation medium of 37°C. The lenses were not decapsulated. One to three lenses, depending

on the age of the animal, containing of medium

were incubated in 05 ml 2 5 ,&i [35S]methionine

0 1990 Academic Press Limited

430

C E. M.VOORTER

(Amersham Corp.), as described previously (de Jong et al., 1986). After labeling for 6 hr at 37°C. incubations were stopped by rinsing twice with cold phosphatebuffered saline (PBS). Lenses were homogenized in 2.5% SDS. This homogenization was complete for lenses from rats younger than 2 months, whereas from older lenses the nucleus remained after homogenization. Protein synthesis in 2-month-old rat lenses is, however, known to be essentially restricted to the lens cortex (Young and Fulhorst, 1966). yA-yF

Crystallin

lsolation and ldentijcation

Eye lenses of 3-month-old rats were homogenized in 2 volumes of buffer (50 mM Tris-HCl, 50 mM NaCl and 1 mM EDTA, pH 76), and y-crystallins were isolated by gel permeation chromatography on Ultrogel AcA34 (LKB). To avoid contamination with ys-crystallin, the second part of the y-peak was used for further separation on an LKB HPLC-system equipped with a MonoS cation-exchange column (Pharmacia). Proteins were eluted at a flow rate of 1 ml min-* and a gradient of sodium acetate as indicated in Fig. 3, using a starting buffer of 20 mM Tris-acetate (pH 5.0) and an endbuffer of O-5 M sodium acetate in 20 mM Tris-acetate (pH 6.6). For identification of the different peaks, fractions were pooled and subjected to both two-dimensional gel electrophoresis and tryptic digestion. Peptide mapping of these tryptic digests was performed by high voltage paper electrophoresis at pH 6.5 and descending chromatography as described earlier (Van der Ouderaa, De Jong and Bloemendal. 19 73). Fluorescamine-stained spots were subjected to amino acid analysis. Because of the coelution of yA and yD on the MonoS column, these two y-crystallins were separated by two-dimensional gel electrophoresis and blotted onto nitrocellulose (Aebersold et al., 1987). The separated y-crystallins from six gels were combined, digested with trypsin, and the tryptic peptides were separated by reversed-phase HPLC on an RP-18 column (Merck), as described previously (Voorter et al., 1986). A linear gradient of 040% acetonitrile in 40 min was used. Some well-separated peaks were used for amino acid analysis. Nomenclature

Identification of the different crystallins is in accordance with a recently published guideline (Bloemendal, Piatigorsky and Spector, 1989). yscrystallin was previously called /?s. Miscellaneous Methods

Two-dimensional gel electrophoresis was performed according to O’Farrell (1975). The isofocusing gels contained ampholines (LKB) in the pH ranges 3.5-10 and 7-9, mixed in a ratio of 1:4. This specific ampholine mixture was chosen for optimal separation

ET AL.

of the y-crystallins in the more basic region. Slight variations in isoelectric focusing migration behaviour of PBl and yF can be attributed to a slight difference in pH range of the isofocusing gels. as these two proteins, especially, showed an aberrant migration if ampholines of 6-8 instead of 7-9 were used. Gels were stained with Coomassie Brilliant Blue (CBB) and subsequently processed for fluorography. dried and exposed to Kodak X-Omat AR films. For comparison of the fluorographs, equal amounts of protein were loaded on the two-dimensional gels, whereas differences in label incorporation were compensated by exposure times. To identify the radioactive spots, notably the y-crystallins in older rats. fluorographs were superimposed on the corresponding Coomassie blue-stained gels. Similar patterns were obtained from three independent experiments. For immunoblot purposes, 2-month-old rat lenses were used. Immunoblotting of two-dimensional gels was performed as described previously (Mulders et al.. 198 7). The antibodies used were prepared against fiB2 (Mulders et al., 198 7 ; dilution 1: 2000). PBl (Mulders et al., 1987; dilution 1: SOO), aA (Hendriks et al., 1988; Cr. I-1, dilution 1: 50000), aB (dilution 1: 2000) and ys (dilution 1: SO). For the preparation of the latter two, the immunization scheme as described by Mulders et al. (1988) was used. As second antibodies peroxidase-conjugated swine-antirabbit (SWARPO, Dakopatts, dilution 1: 200) and rabbit-anti-mouse (RAMPO, Dakopatts, dilution 1: 200) immunoglobulins were used for the polyclonal (pB2, pS1, aB and ys) and monoclonal (aA) antibodies, respectively. Amino acid analyses were performed on an LKB Alpha Plus amino acid analyzer.

3. Results Changes of Crystallin Eye Lens

Synthesis in the Developing Rat

In order to determine the changes in crystallin synthesis with development, rat eye lenses of different ages were incubated in the presence of [35S]methionine. After homogenization of the incubated rat lenses, and two-dimensional gel electrophoresis of the lens extracts, fluorographs were obtained, as shown in Fig. 1. The radioactive spots were identified as indicated in Fig. l(A, G), in accordance with their genes of origin (for identification see the next section). Although, with this method, crystallins lacking a methionine residue will be overlooked, this is unlikely to occur, since all hitherto sequenced mammalian crystallins contain one or more methionine residues. By comparing the radioactive protein patterns of rat lenses of different ages, several conspicuous features can be observed. Compared to aA, the synthesis of aB shows a slight increase with age. In agreement with the ratio of aA to aB (3 : 1) in x-crystallin (De Jong

DIFFERENTIAL

SYNTHESIS

OF

CRYSTALLINS

431

432

C. E. M. VOORTER

ET AL.

FIG. 1. Patterns of proteins synthesized in rat lenses of different ages. Rat lenses were incubated in the presence of [36S]methionine, and total lens extracts were analyzed by two-dimensional gel electrophoresis and fluorography. Rat lenses of age 0 (A), 2 days (B), 7 days (C), 14 days (D), 1 month (E), 2 months (F) and 4 months (G) were used. Identification of the indicated polypeptides is described in the text. Square brackets mark some overlapping polypeptides. among which at least aAins and PAl. The arrow points to a spot most probably corresponding with @lb. Note that panel D is missing part of 7ABC. To give an indication of the &I,-range presented on the two-dimensional gels: it varies between 2 7 88 1 (PSI) and 19 832 (aA). as calculated from the amino acid sequences. et al., 1975), the amount of [35S]methionine incorporated in aB is always lower as compared to crA, even

though rat aB contains three methionine residues as opposed to two in rat aA (W. Hendriks, H. Bloemendal and W. W. De Tong, unpubl. res.). Furthermore, Fig. 1 clearly shows a pronounced increase iu the relative synthesis of ,&i2 and ys with age. @B2-synthesis is already detectable in newborn rats, whereas incorporation of label in ys-crystallin appears at 14 days [Fig. l(D)]. That the synthesis of

pB2 and ys is almost exclusively post-natal is further conilrmed by comparison of the CoomassieBrilliant Blue-stained gels of newborn and l-month-old rat lenses [Fig. 2(A. B)]. In newborn rat lenses, both pB2 and ys are not present in detectable amounts, whereas

rat lenses of 1 month contain abundant amounts of both crystallins. This post-natal pattern of synthesis makes it very plausible that the 27-kDa protein, mentioned by Carper et al. (198 S), corresponds with pS2, whereas their ~3, also previously mentioned by Vornhagen et al. (1983) resembles ys-crystallin. In contrast to ys, a differential decline of the relative synthesis of the other y-crystallins is observed in incubated rat lenses from 14 days on [Fig. l(D-G)]. Rat eye lenses of 2 months do not synthesize yA, yE and yF anymore in detectable amounts [Fig. l(F)], whereas the labeling of yD has vanished almost completely at the age of 4 months [Fig. 1 (G)]. Synthesis of yB and yC is still detectable at this age, albeit at a very low level compared to, for example, aA. Fur-

FIG. 2. Two-dimensionalgel electrophoreticpatterns, stainedwith Coomassie Brilliant Blue. of lensproteinsfrom newborn (A) and l-month-old (B) rats. For the indicationsseeFig. 1.

DIFFERENTIAL

SYNTHESIS

OF

433

CRYSTALLINS

thermore, the CBB-stained gel of a newborn rat lens [Fig. 2(A)] clearly shows the presence of only small amounts of ?/ABC, compared to 7DEE. This indicates that, among total y-crystallins, the synthesis of yDEF dominates in the prenatal stage, as was previously found by Siezen et al. (1988). The relative incorporation of [35S]methionine in PA3 stays fairly constant with increasing age, whereas in comparison pS1 shows some fluctuations in label incorporation. The changes in protein synthesis of ,l3AI and aAins cannot be distinguished with this method, since both proteins show an almost identical mobility on the two-dimensional gel system used (Fig. 1, square bracket). The presence of other crystallins as well in this rather crowded area cannot be excluded, In addition to the clearly labeled spots in Fig. 1, slightly labeled spots, which correspond with rather heavily stained spots on the CBB-pattern, can sometimes be seen on the fluorographs as well (cf. Figs 1 and 2). The most basic one [Figs l(A) and 2(A), arrow] corresponds most likely with /?Blb, as it shows (-

ho

immunoreactivity with the anti-@Bl serum (data not shown). The presence of [35S]methionine in this protein after 6 hr of incubation indicates that the posttranslational modification, by which @lb arises from flla (Berbers et al., 1983), can be a rather fast process. The other slightly labeled spots might also be post-translational modification products of certain crystallins. However, it is also possible that some of them correspond with the orthologues of bovine PA2, PA4 and /3B3 (Berbers et al., 1984). These three proteins could not positively be identified on our twodimensional gels, although the mBNA of pB3 is present in rat lenses (Aarts et al., 1989). Comparison of Fig. 2(A, B) reveals that, apart from pS2 and ys, a large number of spots appear on the gel of l-monthold rat lenses compared with newborn rats. Furthermore, by comparing this CBB-stained pattern with the corresponding fluorograph [Figs 2(B) and l(E)], it is obvious that considerable posttranslational modifications must occur in rat eye lenses within 1 month after birth. M NaAc

1

( ----I

A+D

0.1

0.1

E

ox

C __-

___--1

Time

II

-Ai

--

___---

__--

----

F

o.,

Al

(min)

FIG. 3. Ehtion profile of rat y-crystallins A-F. The y-crystallins were fractionated by HPLC on a MonoS cation-exchange column with a linear gradient of sodium acetate (---), as described under Materials and Methods. y-crystaltins are designated according to their corresponding genes. The insert shows the -separation of pooled peak fractions by two-dimensional gel electrophoresis. To facilitate localization of the spots on the two-dimensional gels, a small amount of total water soluble rat lens

proteins was added for a background.

434

C. E. M. VOORTER

ET AL

TABLE I

Amino acid compositions(mol amino acid per mol peptide) of tryptic peptidesof yA-yF. Peptideswere isolated either by high voItage electrophoresisand chromatography (yB, yC, yE, YF) or by reversed-phaseHPLC (yA, yD). Amino acidsdiagnosticfor the identification of thesey-crystallins are in bold (seealso Fig. 4) T8 Amino acid Asp Thr Ser Glu Pro ‘JY Ala Val Met Ile Leu Tyr Phe His LYS Arg

T9b*

YB YC

YD

YE P

3.1

2.8

3.1

3.0

2.9

2.0 2.2 1.0 2.2

2.1 2.2 0.9 2.2

1.8 2.3 1.1 2.0

2.0 2.2 1,o 2.2

1.9 2.3 1.0 2.2

06 1.0

1.2 1.2

1.0 0.8

1.1 1.0

1.2 0.9

Asp Thr Ser Glu Pro GUY Ala Val Met Ile Leu ‘JhPhe His LYS Arg

YB YD

YE

YB

YC

Tll YE

YF

YB

YC

___---. ..~ YE YF

1.1

1.2

1.2

0.9

0.9

1.1

0.9

1.0

1.0

1.0

0.9

1.5 1.0

1.6 1.1

1.7 1.0

1.8 1.1

1.1

0.9

3.1

1.0 1.2

0.8 1.1 1.1

1.0

0.9

1.2 1.1

1.0 1.0

1.2

0.8 0.9 0.8

1.6 1.0

0.9 0.9

0.9

08 0.9

0.9

1.7

2.1

1.0

1.0

0.8

1.0 0.9

0.9

1.0

0.8

1.2

@8

0.9

T12 Amino acid

TlO

T16

YB

YD

YE

YF

YA

2.0

1.1

1.1

l..l

1.2

1.1

1.1

1.0

1.3 1.0 1.0

YB

2.1 1.1 1.2

1.2

1.1

1.1

T16a T16bt YE

2.2 0.8 1.3

YF

1.1 1.0

YF

1.1

1.2

T18 YA

YB

.____ YE YF

1.9

2.1

2.2

T19 YB

YC

YE

YF

1.0

1.1

0.9

0.9

1.2

1.2 0.9

1.3

1.1

0.8

0.8

0.9

0.9

1.0

0.9

1.0

1.2

2.0

1.0 1.2 1.8

1.2 2.9

1.2 1.8

1.2 2.0

1.1

1.1

0.8

0.8

0.9

0.9

1.1

1.0

0.9 1.2 0.9

08

1.9 1.8

0.8

2.1 1.6

2.2 1.6

1.9 0.8

0.8

1.1

1.0 0.8

0.9

1.0 0.9 1.0 1.0

1.0

1.1

1.1

1.8

2.0

1.7

2.2

1.1

1.1 1.0

* TYb is the second part of TY. resulting from tryptic digestion at Arg-79 in yA-$ and comprising residues 80-89. t This peptide has an additional cleavage at Arg-149 in yF. resulting in two tryptic fragments T16a and T16b.

Identification of the De Nova Synthesized Crystallins

Ramaekers et al. (1982) already gave a nomenclature for the rat lens crystallins, based on their twodimensional gel electrophoretic mobility. However, this nomenclature needs to be revised in the light of the present detailed knowledge of the corresponding bovine

proteins

and

of the

rat

crystallin

genes.

Therefore, we attempted to identify all de novo synthesized crystallins present on the fluorographs of Fig. 1. A number of polypeptides could readily be identified by immunoblotting of rat lens proteins on twodimensional gels with antisera against /?F%l, fl2, ys, ccA and olB (data not shown). Their relative positions

resemble those of the corresponding bovine crystallins (Berbers et al., 1984). Selective hybridization of rat lens mRNA with bovine cDNA clones, followed by in vitro translation, had previously enabled the identification of rat /?Al/A3 (Y. Quax-Jeuken, H. Bloemendal and W. W. De Jong, pers. commun.). The individual y-crystallins (yA-)/F) were identified after isolation by gel permeation chromatography (AcA34) and subsequent separation by MonoS cation exchange chromatography. The resulting elution profile is shown in Fig. 3. In order to correlate the peaks of this elution pattern with spots on the twodimensional gels, peak fractions were pooled and subjected to two-dimensional gel electrophoresis. A small amount of total water-soluble rat lens proteins

DIFFERENTIAL

SYNTHESIS

OF

435

CRYSTALLINS

T8 4 60

TlO

‘I’9 b4

YA

GDYPDYQQWMGFSDSIRSCR@IP@

YB'

GDYPDYQQWMGFSDSIRSCRLIP@)@SG~RMR~YE;DD@R

YC

GDYPDYQQWMGFSDSIRSCRLIPH

YD

GDYPDYQQWMGFSDSVRSCRLIPH + GDYPDYQQWMGFSDSVRSCRLIPH

YE YF

TSSHRIRLYERDDYR

TGSH:,:,Ep;~~ BGSHRIRLYEREDYR

4

SSSHRIRIYEREDYR

l

-‘Me---,

l

SSSHRIRIYEREDYR 4-b-M

GDYPDYQQWMGFSDSVRSC@LIPH -T16

T18

T17

T12

+4-b+----,* 90

80

70

Tll

‘I’19

v*-150

160

YA

QYLLRPGOY

RiHDWGAMDAiVGSLR

YB

$YLLRPGEY;R

YC

QYLLRP@EYRRYHDWGA@DAK

YD

QYLLRPGEYRRYHDWGAMNARVGSLR

YE

;YLLRPGEY;RYHDWGAMNARVGSLR -'f-------, EYRRYHDWGAMNARVGSLR

~DwGA@NAKVGS@)R +;GSLi

FIG. 4. Partial amino acid sequences enablingthe discriminationof the six rat y-crystallins, yA-yF, alignedaccordingto den Dunnen et al. (1986). Tryptic peptidesare numberedaccording to their position in the sequenceof the protein. Peptides, resulting from tryptic digestionof the six different y-crystallins, and usedfor their positiveidentification, are underlined(see alsoTable I). Circlesencloseunique residuesat a given position(DenDunnen et al., I986), which simplified&e i&&&&ion of the differenty-crystallins. In T9, an additionalcleavagesitefor trypsin occursat kg-79 in yA-@, and in TI6, at Arg-149 in yF.

was added, to obtain a background pattern of all ycrystallins to facilitate identification. The results, as shown in Fig. 3 (insert) reveal that the Erst eluting ycrystallins, yA and yD, are poorly resolved, whereas the other four, yB, yC, yE and yF, are well separated. For further identification of the four well separated y-crystallins, tryptic digestion was performed, followed by peptide mapping and amino acid analyses. It was possible to identify these four y-crystallins as yB, yC!, yE and yF (as indicated in Fig. 3), by comparing the amino acid compositions of tryptic peptides with the deduced amino acid sequence of the six rat ycrystallins (Den Dunnen et al., 1986). In Table I and Fig. 4 the amino acid compositions and the alignment of the peptides in the region of residues 60-99 and 143-168 are indicated, which were most diagnostic to distinguish the different y-crystallins. Although yA and yD are well separated on twodimensional gels, these two proteins could not sat30

isfactorily be resolved on the MonoS column. Therefore, we decided to identify yA and yD by tryptic digestion of these proteins after blotting onto nitrocellulose, as described in Materials and Methods. In Table I and Fig. 4 the peptides are indicated by which the identity of yA and yD is unambiguously established. As Ramaekers et al. (1982) used a comparable twodimensional gel electrophoretic system, it can be concluded that the polypeptides identified as ~1-6 in their nomenclature correspond with the y-gene products yC, yB, yA, yF, yE and yD, respectively. This correlation between yl-y6 and the corresponding ycrystallin genes is in accordance with predictions of van Leen et al. (1987) and Siezen et al. (1988) which were based on developmental changes and p1 differences, respectively. All identified crystallins are indicated as such in Fig. l(A, G) and Fig. 2(A, B). EER 50

436

4. Discussion Siezen et al. (1988) were recently able to separate the seven rat y-crystallins and assign them to their corresponding genes, yA-yF and ys. By determining the amounts of these y-crystallins present in the outer cortex they deduced the differential synthesis of ycrystallins in the developing rat lens. In the present study we determined more directly the developmental changes in the de novo synthesis of rat y-crystallins, and, moreover, extended this investigation to aA- and aB-crystallin and several pcrystallins. This enables us to correlate the relative synthesis of these crystallins with previously published developmental changes in mRNA levels (Van Leen et al., 1987; Aarts et al., 1989). The correlation is basedon the comparison of the changes in synthesis of the different crystallins relative to aA, with the alterations in the amounts of transcripts compared to aA as well. Except for ml, these examined crystallins showed a good correlation between the changes in synthesis and in mRNA levels, although small differences cannot be excluded with this coarsemethod of comparison. With respect to the yA--yF crystallins, Van Leen et al. (198 7) already establishedthat the expression of these genes is differentially switched off during the post-natal stages of development of the lens. The changes in the relative post-natal synthesis observed in our study parallel the differential decline of mRNA levels of rat y-crystallins, as measured by Van Leen et al. (1987). Between 1 and 2 months of age, labeling of yA, yE and yF has vanished completely [cf. Fig. l(E, F)], whereas synthesis of yB, yC and yD is still detectable in 2-month-old rat lenses.These results are in agreement with the measured ratio of the corresponding mRNAs. Although a rapid decline in the levels of yE and yF has also been found by Siezen et al. (1988), some discrepancies exist between our results and their deduced yA-yD synthesis values. Presumably, these discrepancies are due to differences in the methods used. Our method enables us to determine actual [35S]methionine incorporation in y-crystallins at different ages, whereas Siezen et al. (1988) inferred the y-crystallin synthesis from percentual weight differences of the individual y-crystallins between rat lensesof different ages.With the latter method it is not possibleto determine the time at which synthesis of a specific protein stops, becausenot all protein present in the outer cortex is necessarily synthesized at that specific moment. The observed differences in synthesis of the different y-crystallins, and even within one ycrystallin class,can, in view of their physico-chemical properties, contribute to the optical quality of the lens. In contrast to the other y-crystallins, the synthesis of ys in rat lensesis detectable only some time after birth, and this perfectly reflects the absence of its corresponding mRNA in fetal lenses and the rapid

C. E. M. VOORTER

ET AL

postnatal rise of the number of transcripts (Aarts et al., 1989). The post-natal increase of ys-crystallin synthesis in human and bovine lenses has already been described (Slingsby and Croft, 1973 ; Thomson and Augusteyn, 1985 ; Pierscionek and Augusteyn, 1988). A comparable gradual increase in the amount of ys-crystallin in rat lenseswith age was observed by Siezen et al. (1988). The synthesis of aA is higher than that of aB at all investigated ages,whereas the post-natal lens contains significantly more aB- than aA-transcripts (Aarts et al., 1989). This discrepancy clearly reflects a lower translational efficiency for the aB mRNA as compared to that of aA. The sameholds true for /3B2 in relation to pS1. Until the age of 14 days, the methionine incorporation is higher in pS1 than in /3B2, although the number of methionines and the amount of transcripts are both higher for the PB2-crystallin. Although translational efficiencies may be different for various mRNAs, the changes in relative protein synthesis are generally in good agreement with the changing levels of mRNA. Thus, the present results do not indicate that differential regulation at the translational level plays a major role in crystallin gene expression in the lens,

Acknowledgments The authorsthank Dr J. Horwitz, LosAngeles,for the /3B2antiserum,andG. van Rens,Nijmegen,for the ys-antiserum. Valuable discussions with Drs N. Lubsenand R. Siezenare gratefully acknowledged.This work was supportedby the NetherlandsFoundationfor ChemicalResearch(SON)with financial aid from the NetherlandsOrganizationfor Scientific Research(NWO). References Aarts, H. J. M., Lubsen,N. H. and Schoenmakers,J. G. G. (1989). Crystallin gene expressionduring rat lens development.Eur. J. Biochem. 183, 31-6. Aebersold,R. H., Leavitt, J., Saavedra,R. A., Hood,L. E.and Kent, S.B. H. (1987). Internal amino acid sequence analysis of proteins separated by one- or twodimensionalgel electrophoresisafter in situ protease digestionon nitrocellulose.Proc. Nutl. Acad. Sci. U.S.A. 84, 69704.

Berbers.G. A. M., Hoekman.W. A., Bloemendal,H.. DeJong. W. W., Kleinschmidt, T. and Braunitzer. G. (1983). Proline- and alanine-rich N-terminal extension of the basic bovine /%crystallin Bl chains. FEBS Mt. 161, 225-9.

Berbers. G. A. M., Hoekman, W. A., Bloemendal.H., De Jong, W. W., Kleinschmidt, T. and Braunitzer, G. (1984). Homology betweenthe primary structures of the major bovine @rystallin chains. Eur. J. Biochem. 139, 467-79. Bessems, G. J. H., De Man, B. M.. Bours,J. and Hoenders,

H. J. (1986). Age-relatedvariations in the distribution of proteins from bovine lens parts. Exp. Eye Res. 43. 1019-30.

Bloemendal,H., Piatigorsky, J. and Spector, A. (1989). Recommendationsfor crystaiiin nomenclature. Exp. Eye Res. 48, 465-6.

DIFFERENTIAL

SYNTHESIS

OF

437

CRYSTALLINS

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Piatigorsky, J. (1981). Lensdifferentiation in vertebrates.A review of cellular and molecularfeatures.DifjPrPntiation 19. 134-53. Pierscionek. B. and Augusteyn, R. C. (1988). Protein distribution patterns in concentric layers from single bovine lenses:changeswith developmentand ageing. Curr. Eye Res. 7, 11-23.

Ramaekers, F., Dodemont, H., Vorstenbosch, P. and Bloemendal, H. (1982). Classification of rat lens crystallinsand identification of proteinsencodedby rat lensmRNA. Eur. J. Biochem.128, 503-8. Siezen, R. J., Wu, E.. Kaplan. E.D.. Thomson, J. A. and Benedek, G. B. (1988). Rat lens y-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis.1. Mol. Biol. 199. 475-90. Slingsby,C. andCroft, C. R. (1973). Developmentalchanges in the low molecularweight proteinsof the bovine lens. Exp. Eye Res. 17, 369-76. Thomson,J. A. and Augusteyn, R. C. (1985). Ontogeny of the human lenscrystallins. Exp. Eye Res. 40. 393-410. Uchiumi, T., Kimura. S. and Ogata, K. (1983). Protein componentsof rat lens and their age-relatedchanges observed with two-dimensional polyacrylamide gel electrophoresis.Elcp.Eye Res. 36, 125-34. Van der Ouderaa,F. J.. De Jong,W. W. and Bloemendal,H. (1973). The amino acid sequenceof the itA chain of bovine a-crystallin. Eur. 1. Biochem.39, X17-22. Van Leen,R. W.. Van Roozendaal,K. E.P., Lubsen,N. H. and Schoenmakers,J. G. G. (1987). Differential expression of crystallin genesduring developmentof the rat eye lens. Dev. Biol. 120, 457-64. Voorter, C. E.M.. Mulders,J. W. M., Bloemendal,H. and De Jong, W. W. (1986). Someaspectsof the phosphorylation of a-crystallin A. Eur. 1. Biochem. 160. 203-10. Vornhagen, R.. Bours, J. and Rink. H. (1983). Immunological properties of rat lens gamma-crystallins.III. Changesduring developmentand ageing. Ophthalmic Res. 15. 126-30. Wistow, G. J. andPiatigorsky.J. (1988). Lenscrystallins: the evolution and expressionof proteins for a highly specializedtissue.Annu. Rev.Biochem.57, 479-504. Young, R. W. and Fulhorst, H. W. (1966). Regional differencesin protein synthesiswithin the lens of the rat. Invest. Ophthalmol.5. 288-97. Zigler. Jr. J. S. and Goosey J. (1981). Aging of protein molecules:lens crystallins as a model system.Trrnds Biochem.Sci. 6. 133-6. Zigler, Jr. J. S.. Russell,P.. Takemoto, L. J.. Schwab, S.J.. Hansen,J. S.. Horwitz. J. and Kinoshita. J. H. (1985). Partial characterization of three distinct populationsof human y-crystallins. Invest. Ophthalmol. Vis. Sci. 26, 525-31.