|Year : 1972 | Volume
| Issue : 2 | Page : 55-62
The subunit structure of chick lens crystallins and its relationship to their antigenic properties
D.E.S Truman, RM Clayton, A.T.H Burns, JC Campbell
M. R. C. Epigenetics Research Group, Institute of Animal Genetics, West Mains Road, Edinburgh EH9, 3JN, United Kingdom
M. R. C. Epigenetics Research Group, Institute of Animal Genetics, West Mains Road, Edinburgh EH9, 3JN
Source of Support: None, Conflict of Interest: None
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Truman D, Clayton R M, Burns A, Campbell J C. The subunit structure of chick lens crystallins and its relationship to their antigenic properties. Indian J Ophthalmol 1972;20:55-62
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Truman D, Clayton R M, Burns A, Campbell J C. The subunit structure of chick lens crystallins and its relationship to their antigenic properties. Indian J Ophthalmol [serial online] 1972 [cited 2021 Jan 25];20:55-62. Available from: https://www.ijo.in/text.asp?1972/20/2/55/34670
Immunglogical methods have long played an important role in studies of the crystallins, and because of the absence of enzymatic function from these structural proteins it is probable that antigenic reactions will continue tc predominate among the methods available for research on crystallins. As techniques available have improved, so the complexity of the immunological data has increased. We may hope eventually to understand this complexity in terms of the detailed molecular structure of the crystallins, but this goal still remains somewhat distant. Nevertheless it is now becoming possible to relate immunological results obtained with crystallins of some species to the quaternary structure of the protein molecules and so to move towards a molecular understanding of crystallin structure and function.
Our studies of the crystallins have concentrated mainly on those of the chick as this species is most convenient for work on embryonic development, but we have benefited from the large amount of work that has been done on other species, particularly the chemical work on bovine crystallins. It is our belief that results obtained with chick lenses may ultimately be of value in understanding developmental processes in other species and so eventually contribute to clinical ophthalmology.
The particular value of the lens as a system for studying cell differentiation has been emphasised by Clayton (1970), and much of our data discussed in the context of background knowledge on crystallin structure, function and development. This article seeks to bring together our own work on the immunological behaviour of chick crystallins and the way in which this may be understood in terms of protein structure. Unfortunately the nomenclature of the chick crystallins has been unsettled for some time and in our earlier papers we have followed different schemes of nomenclature from that now used, which is based on that used by Zwaan and Ikeda (1968).
It was shown by Bloemendal. Bent, Jongkind and Wisse (1962) that bovine α- crystallin molecules, with a molecular weight of 810000. could be broken down into much smaller subunits by treatment with reagents such as 7Murea, which breaks hydrogen bonds but does not break the covalent bonds that hold together protein molecules. It thus appeared that the large molecules of α-crystallin were composed of many smaller subunits held into aggregates by hydrogen bonds. Subsequent work has shown that each of these subunits is composed of a single polypeptide chain, and that more than one type of subunit is present in the total α-crystallin molecule. There has been some controversy about the molecular weight of the subunits, and a value of less than 20000 has been suggested by Hoenders. De Groot. Gerding and Bloemendal (1969) Some work has been done on the determination of the amino acid sequences of the α-crystallin subunits and there are apparently very great similarities between the different types of subunit (Corran and Walley, 1969).
Largely on the basis of studies of antigenic specificity we proposed in 1967 that all of the three major classes of crystallin in the chick lens were probably composed of subunits and that within each class more than one type of polypeptide chain was found (Clayton and Truman, 1967). Subsequent studies have confirmed these views and given us more information about the relationship between the antigenic properties and protein structure of these crvstallins
Chick α-crystalin. Hoenders (1965) found by analytical ultracentrifugation that the proteins of this group had a molecular weight of 470000, while Zwaan (1968), using a method dependent on electrophoresis in polyacrylamide gel, found three separate molecular species in this class with molecular weights of 400.000. 350,000 and 60.000 Three major components in the α- crystallin group were also shown by Truman (1968), though the resolution of these components becomes less clear as the lens ages (Truman, Brown and Campbell, 1972). Corresponding to these three size classes of α-crystal.lin, we also find that immunoelectrophoresis may reveal three concentric precipitin arcs (Truman, Clayton and Campbell, 1967). The three components may also be revealed in quantitative immunoelectrophoresis and in the Osserman tests when embryonic lens extracts are compared with adult crystallins (Truman et al., 1972). During embryonic development there appears to be a change either in the proportions or in the properties of these three classes of α-crystallin.
There has in the past been some controversy about changes in the physical properties of α-crystallin of various species during embryonic development (reviewed by Zwaan 1963), and a number of workers have shown that the subunit composition of bovine α-crystallin changes during development (Schoenmakers and Bloemendal, 1968; Plamer and Papaconstantinou, 1968, 1969). Direct evidence of the subunit structure of chick α- crystalline and its change in composition during development has been put forward by Rana and Maisel (1969), and by Clayton (1969) who separated the different subunits of α-crystallin from adult chicks and from the lenses of 13 day and 18 day embryos. While the adult lens showed three different major types of subnit, the embryonic lenses showed only two.
We see, then, that during the development of the chick the composition of the α-crystallin changes and that there is also evidence that the proportions of the different molecular populations of the α-crystallin aggregates may also undergo change. There is, however, no evidence of the appearance of any different antigenic determinants after the first appearance of α-crystallin antigens at about embryonic stage 25 (Truman et al., 1972). This implies that the different subunits are probably antigenically very similar. Such immunological similarity is seen between the subunits of bovine α- crystallin, in which several subunits appear to be immunologically identical (Bjork, 1964), in agreement with the similarities of amino acid sequence reported by Corran and Waley (1969). It is probable that there is a general structural similarity between the α- crystallins of mammals and birds.
β-crystallins. The β-crystallin class in the chick is that with the lowest molecular weight and with a wide range of electrophoretic mobility. The class is so named because of their immunological cross-reaction with mammalian β-crystallins (Zwaan and Ikeda, 1968). In terms of molecular weight these proteins are heterogeneous, resolving by gel filtration into two main groups Truman, 1968) and probably ranging in molecular weight up to about 60,000 (Truman, Brown and Rao, 1971). When studied by electrophoresis on polyacrylamide gel as many as fifteen components have been found among the β-crystallins of the chick, with mobilities more extreme then either the α-or β crystallins (Truman, 1968).
On treatment with 8M-urea the β-crystallins show a reduction in sedimentation coefficient from about 6.44S to 1.28S. and estimates of the molecular weight of the subunits by gel filtration give a value of about 16.500 (Truman et. al. 1971). This result. and the data of Rana and Maisel, (1970), confirm the prediction of a subunit structure of the β-crystallins which had been proposed earlier from indirect evidence (Clayton and Truman, 1967).
Experiments in which β-crystallins separated by electrophoresis on polyacrylamide gels were then dissociated in urea solutions and re-electrophoresed have shown that there are several different subunits which form the aggregate β-crystallin molecules; that most β-crystallins contain more than one type of subunit and that the electrophoretic mobility of the aggregate is a function of the constituent subunits (Clayton, 1969). These experiments indicated the presence of six major types of subunit and five minor types among the β-crystallins. Subsequent experiments in which β-crystallin preparations have been subjected to isoelectric focussing on columns in the presence of 7M-urea have also shown 6 major and 5 minor components with isoelectric points ranging from pH 7.72 to pH 5.50. A further confirmation has come from experiments in which the β-crystallins were first fractionated by isoelectric focussing in the absence of urea, to yield 9 different /3-crystaliin preparatons. These were each dissociated and analysed; by polyacrylamide electrophoresis in urea at pH 8.9. When allowance was made for any possible cross-contamination, two fractions were found to contain only one major type of subunit, others contained two types, while two fractions each contained three types of subunit. Some of the subunit types were found in more than one aggregate molecule.
In immunoelectrophoresis the β-crystallins of the chick give rise to an extended series of precipitin arcs, known sometimes as the long line' (Zwaan, 1963). This itself implies that there are a series of immunologically related proteins which vary in their electrophoretic mobility. The spurs which are found in the `long line' also indicate that some antigens are not distributed over the entire range of electrophoretic mobility of the β-crystallins. The pattern of occurrence of the various subunits in these crystallins provides a basis for understanding the immunoelectrophoretic pattern, since it can be shown that many of the subunits which differ in their mobility also differ in their antigenicity. However we cannot say that `one subunit is equivalent to one antigen'. Firstly, no one subunit appears to occur throughout the entire electrophoretic range of the `long line', and yet immunoelectrophoresis indicates that one. and possibly two, antigens are common to the entire length of the precipitin line. Thus one determinant must be associated with more than one type of subunit. This has been confirmed by tests on isolated subunits. Such tests also show that a single subunit may possess more than one antigenic determinant (Truman and Clayton, in prepn.).
The correlation between subunits and antigenic determinants is also to be seen as a result of studies of the β-crystallins during development, since the increase in complexity of the `long line' arcs is paralleled by an increase in the number of different types of subunit in the β-crystallins of the chick
A feature comparable to the 'long line' of the chick crystallins is seen in immunoelectrophoresis of crystallins of the toad Xenopus laevis (Campbell, Clayton and Truman, 1968).
stallin. That fraction of the chick crystallins intermediate in molecular weight between the α- and β-crystallins has been called γ-crystallin to emphasise that it is not immunologically cross-reactive with any of the major classes of crystallin of the mammalian lens (Zwaan and Ikeda, 1968). However, it should not be supposed that this group of proteins is analogous with the Y- crystallins of the fish (Bon, Swanborn, Ruttenberg and Dohrn, 1964). The molecular weight of these proteins has been estimated at about 150,000 by Hoenders (1965) and. Truman et al. (1971). but Zwaan 1968 has given evilins with molecular weights rangdence of five classes of Y-crystaling from 155,000 to 460,000. This polydispersity in size may account for the thickness of the precipitin arc which is usually found when γ-crystallin is examined by immunoelectrophoresis, and which has been resolved into at least three components (Truman et al., 1.967).
There is evidence that the γ-crystallins are also composed of subunits, since treatment with urea reduces the molecular weight to about 25.600 . Six such subunits would give an aggregate molecule of weight 154,000 (Truman et al., 1971). Clayton (1969) carried out electrophoresis on polyacrylamide gel at pH 8.9 of purified γ-crystaltin and detected five different components, while studies with starch gel in urea at pH 5.6 have shown at least four different subunits. While the starch gel is less sensitive and its resolution at this pH is poorer than the acrylamide, the avoidance of alkaline urea diminishes the possibility of artefacts caused by the carbamylation of proteins (Stark, Stein and Moore, 1960.)
A further method of fractionation of crystallin subunits which is both sensitive and with a high degree of resolution is isoelectric focussing on polyacrylamide gels. In this system the gel stabilises the pH gradient set up by the ampholytes, but does not act as a molecular sieve (Wrigley, 1958). If total adult crystallin is incorporated into gels containing 6Murea a total of 14 major subunits can be detected using ampholytes with a pH range of 3 to 10. This agrees well with the results of other methods applied to total lens and with the sum of major subunits from the various crystallin fractions. The isoelectric focussing technique in gels is now being used to investigate the subunit composition of purified crystallins and its resolution is being increased by varying the pH range of ampholytes used.
By the use of these different methods we feel confident that the γ-crystallins of the chick are also aggregations of different type of polypeptide chains. Studies of the ontogeny of these crystallins indicate that the relative proportions of the differents subunits in the aggregate molecules may change during development, just as there are changes in the α-and β-crystallins.
Dissociation and reassociation.
The result described above have involved the dissociation of crystallin molecules by treatment with urea solutions which weaken hydrogen bonds between the constituent subunits of the aggregate molecule. When the urea is removed the subunits may reaggregate, at least partially. We showed that dissociated crystallin subunits sedimenting at about 1.9s formed, on removal of the urea, an aggregate sedimenting at 5.9s and which retained an immunological reaction with antiserum to crystallins. It was also possible to show that aggregate molecules could be formed in this way which involved combinations of subunits not present among the native crystallins (Clayton and Truman, 1967).
Other hydrogen bond reagents. such as 6M-guanadinium chloride and formamide, can cause reversible dissociation of crystallins, and there is also evidence of a reversible dissociation to some extent in the absence of such reagents (Truman. Clayton and Campbell, 1968). The relationship between sedimentation rate and protein concentration provides an illustration of this. Normally sedimentation rate diminishes as protein concentration rises due to viscosity effects, but when proteins dissociate upon dilution there is also a reduction in sedimentation rate at low concentrations. Both these effects are seen when the sedimentation velocity of the γ-crystallin peak is measured in preparations of total crystallins at various concentrations, (Clayton, 1970). Further evidence of a spontaneous reversible dissociation comes from studies of the behaviour of β-crystallins during gel filtration (Truman et al., 1971), while there is also an indication that the subunits may reassociate in different combinations by the formation of hybrid molecules between the γ-crystallins of different species of birds (Clayton, 1969).
If dissociation and reaggregation can occur spontaneously in physiological saline solutions, the question arises of whether the crystalline aggregate molecules as we know them may not themselves be the result of changes involved in the isolation of proteins from the lens. Maisel and Perry (Personal communication) have shown that in the chick lens there are filamentous aggregates of what are apparently globular units of α-and β-crystallins, and that these filaments may retain their structure when isolated by very gentle procedures, where as crystallin preparations purified by even so mild a method as gel filtration no longer have this form. This work implies that in the lens there are strong interactions between protein molecules which must be broken in order to isolate the crystallin fractions which have for so long been the material used by protein chemists and immunologists working on the lens. We must be aware that these crystallin preparations may be to some extent artefacts of isolation.
The immunological specificity of crystallins. The question of the organ specificity of the antigens of the lens has been discussed in some detail in an earlier paper (Clayton, Campbell and Truman, 1968. Using a variety of tests and studying tissues from both the chick and from Xenopus laevis we found some cross-reaction with antisera to lens proteins in extracts both of eye tissues and of other organs such as brain, liver and kidney. By means of the Osserman test we were able to show that crystallins in all classes showed cross-reactions with some other tissues. Many of the cross-reactions involved were reactions of partial indentity or reactions with limited portions of the precipitin arcs of the crystallins. It seemed from these results that only certain subunits of the crystallins might have antigenic determinants which were common to other tissues.
A somewhat similar situation obtains when we study the species specificity of chick crystallins. Comparison of the chick crystallins with those of the mouse and of Xenopus laevis again show many reactions of partial identity. For example chick α- crystallin showed one antigen which was shared with the amphibian and with the mammalian species and another antigen which was restricted to the chick. In studies made on isolated subunits it has been found that some cross react with antisera to other species, while some do not. Six out of eleven subunits of chick β-crystallin reacted with antiserum to 4 amphibian lens while two subunits reacted with antiserum to mammalian lens (Clayton and Truman. 1968: Clayton, 1969). This limited cross-reaction can explain the patterns that we obtain in immunoelectrophoresis when chick crystallins are tested with an antiserum to Xenopus laevis lens. We find that the entire length of the precipitin arc of the chick β-crystallin reacts with the antiserum to the amphibian lens, but that no spurs are found of the type that occur when homologous antisera are used. We must conclude that the subunits which react show antigenic indentity and that they are distributed over the entire electrophoretic range of the β-crystallins.
Subunits and antigens during ontogeny. Since the subunits of chick crystallins appear to consist of single polypeptide chains, while the crystallins are aggregates of a number of different subunits, it follows that if we wish to consider the differentiation of the lens fibre as a system involving the activity of a number of different genes, then it is the synthesis of the individual subunits which must be followed. The value of this approach to the question of cell differentiation has been discussed by Clayton (1970). While there is evidence from work on bovine α- crystallin that the different subunits may not necessarily be the products of different genes since one can be converted to another, possibly by mechanisms such as the deamidation of glutamine and asparagine residues (Palmer and Papacenstantinou, 1969; Delcour and Papaconstantinous, 1970, it is nevertheless possible to discover whether such conversions take place by following the kinetics of amino acid incorporation into the subunits. In the chick lens we have as yet no reason for supposing that each subunit is not the product of a separate gene. The value of immunological techniques in these studies of differentiation lies in their ability to characterise and discriminate between the different subunits, and in certain techinques to actually participate in the separation of individual subunits. The sensitivity and specificity of immunological methods has already been applied to studies of lens development in terms of the synthesis of specific subunits (Clayton, 1970; Truman et al., 1972). An example of the indispensabiity of the immunological approach to problems of subunit ontogeny is to be found in the application of immunofluorescence to the studies of the localisation of subunits within the lens (Clayton, 1970).
Immunological methods of the identification of crystallin subunits can also be extended to the isolation of those polysomes involved in the synthesis of particular subunits, making use of the presence of antigenic determinants on the polypeptide chain while it is still being synthesised (Clayton, Truman and Campbell, 1970). Such a fractionation technique can be used to yield a preparation of nolysomes with a particular role in protein biosynthesis, which will contain the messenger RNA specific for that polypeptide to which the antibody is directed. We have already begun to apply this method to studies of the stability of the messenger RNA molecules which specify particular crystallin subunits. and to the problem of the way in which this messenger RNA becomes stabilised during the differeniation of the lens fibre (Clayton, Truman and Campbell, 1972).
We believe that the work which has been done to relate the structure of the crystallins to their immunological properties can throw light on some of the immunological problems of the lens, and that, in turn, the immunologists' techniques can then be directed towards the elucidation of some of the molecular problems of cell differentiation.
Acknowledgments . We wish to thank Mrs. A. G. Brown, Mr. A. G. Gillies, Mrs. A. H. Hannah and Mrs. H. J. Mackenzie for their skilled technical assistance. We are grateful to Sterling Poultry Products Ltd., and D. B. Marshall (Newbridge) Ltd. for their continuing co-operation in the supply of material for our experiments. This work has been made possible by the financial support of the Medical Research Council, the Cancer Research Campaign and the W. H. Ross Foundation to Combat Blindness.
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