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Comparative studies of lens α-crystallin from eight species
Camp. Biochem. Physiol., 1971, Vol. 38A, pp. 637 to 643. Pergamon Press. Printed in Great Britain
COMPARATIVE
STUDIES OF LENS a-CRYSTALLIN FROM EIGHT SPECIES*
P. D. MEHTA Department
and
S. LERMAN
of Ophthalmology Research, McIntyre Medical McGill University, Montreal, Quebec (Received
30 July
Sciences
Centre,
1970)
Abstract-l.
The cross-reactions of or-crystallins from bovine, sheep, rabbit, human, monkey, rat, chicken and dogfish lens were studied by the quantitative complement fixation test using rabbit antisera specific for bovine cu-crystallin. 2. By this method, the bovine, sheep, and rabbit or-crystallins appeared to be identical, while the human, monkey and rat showed a somewhat lower degree of complement fixation with the antibovine or-crystallin sera. The chicken 01crystallin showed approximately 40 per cent cross-reactivity with the bovine protein, and there was no detectable cross-reaction in the case of dogfish ol-crystallin. 3. Excluding dogfish, the electrophoretic patterns of different ol-crystallins in 8 M urea starch gel electrophoresis at pH 3.0 were similar.
INTRODUCTION THE
COMPARISON
of the structure of homologous proteins from different species can contribute towards an understanding of the relationship between protein structure and function as well as the nature of the evolutionary processes. It has been established that cross-reactions occur within all three of the main groups of lens proteins, namely the OL-,/I- and y-crystallins (Manski et al., 1960). Of the different lens proteins, cy-crystallin generally shows the greatest degree of organ specificity. Many reports have been published on immunochemical comparisons of whole lens proteins from different species (Halbert & Fitzgerald, 1958; Manski et al., 1960; Maisel, 1963; Mehta et al., 1964; Manski et al., 1967), whereas little work has been carried out on the immunological cross-reactions of isolated lens protein such as ar-crystallin. Maisel (1964) compared a-crystallin by the gel diffusion technique and found differences between five vertebrate classes. Bjiirk (1968) reported that mammalian cz-crystallin behaved identically in the quantitative precipitin test and in gel diffusion experiments, but using the quantitative complement fixation technique, we have been able to differentiate between these closely related proteins. * This investigation Council, Canada.
was supported
by Grant
637 22
No. MA-3208
from the Medical
Research
P. D. MEHTA
638
AND S. LERMAN
In the present article ol-crystallin of seven species are compared with that of bovine ol-crystallin, in reaction with antiserum to the bovine protein. Immunological methods are employed with particular emphasis on the quantitative complement fixation technique. This method has been shown to be sensitive to small differences in amino acid sequence (Reichlin et al., 1964) and capable of providing an approximate measure of structural relationship between homologous proteins (Wilson et al., 1964; Sarich & Wilson, 1966). In addition, the polypeptide chains of these ol-crystallins are compared in urea starch gel electrophoresis at acidic pH.
MATERIALS
AND
METHODS
Lenses
from eight species were used. These included bovine (Bos taurus), sheep (Otis (Homo sapiens), monkey (Macuca irus),rabbit (OryctoZagus cuniculus), chicken (Gallus domesticus), rat (Rattus norvegicus) and dogfish (Mustellis cunis). Normal adult human lenses were obtained within 12 hr after death. The other lenses were obtained immediately after the animal’s death and were carefully dissected free from the capsule in an iced container and homogenized in 0.15 M NaCl with 50 to 100 mg wet wt. tissue per ml. The homogenate was centrifuged at 12,000 x g for 30 min at 4°C and the clear supernatant was stored at -20°C until required for the subsequent isolation procedures. avies),human
Isolation
of ol-crystullin
a-crystallin was initially purified by zone electrophoresis on starch at a potential gradient 20 V/in. in a barbital buffer, pH 8.6, ionic strength 0.05 (Kunkel, 1954). The material was then further purified by gel filtration using Sephadex G-200 which was equilibrated with 0.1 M Tris-HCl+ 1 M NaCl buffer, pH 7.8. Determination
of protein
Protein concentrations (Lowry et al., 1951). Preparation
concentration were
determined
by
the
modified
Folin-Ciocalteu
method
of antisera
Antisera to total soluble lens proteins and isolated bovine or-crystallin were prepared in rabbits by weekly injections of the antigen in complete Freund’s adjuvant. The antisera to bovine cu-crystallin were rendered specific by adding fi-crystallins (dry protein) in a suitable amount and removing the resulting antigen-antibody precipitate by centrifugation (Mehta & Lerman, 1970). Immunoelectrophoresis
and immunodiflusion
Double diffusion in agar was performed according to the method of Ouchterlony (1958) and immunoelectrophoresis on glass microscopic slides was employed as described by Scheideggar (1955). CompZement fixation
test
The complement fixation test was done according (1948), with the modification that the test was done reactants remaining the same (Nisonoff et uZ., 1967). Starch
gel electrophovesis
to the technique of Mayer in 1.0 ml, the concentration
et al. of all
in urea
Vertical starch gel electrophoresis (1959) with the formate-urea buffer
was performed according to the method system as described by Edelman & Poulik
of Smithies (1961).
STUDIES OF LENS O+CRYSTALLIN
Amino
FROM
EIGHT
639
SPECIES
acid composition
Samples of or-crystallin were hydrolyzed in sealed evacuated Pyrex tubes containing 6 N HCl at 110°C for 24 hr. Amino acid analyses were done with a Beckman 120C autoanalyzer according to the procedures described by Moore & Stein (1963). RESULTS
ol-crystallins from eight species were tested with either homologous or heterologous antitotal soluble lens sera by immuno-electrophoresis and Ouchterlony analysis. Each preparation showed a single precipitin arc which was at a position characteristic for a-crystallin (Fig. 1). The immunological interrelationships between cu-crystallins of the different species were studied by the gel diffusion technique using two different rabbit In order to augment the results of antisera made specific for bovine a-crystallin. the gel diffusion technique, these proteins were also compared by the quantitative complement fixation test using the same antisera. Figure 2 shows the results of gel diffusion test of rabbit antiserum to bovine oc-crystallin with the ol-crystallin of eight species. By this method, the reaction of identity was observed between bovine, sheep, monkey, human, rabbit and rat oc-crystallins. A reaction of partial identity was observed between bovine and chicken ol-crystallins, while the dogfish a-crystallin showed no precipitin line. In order to study the cross-reactions by the complement fixation test, the maximum amount of complement that can be fixed at each antiserum concentration was determined (Mayer et al., 1948). Cross-reactions of antisera with heterologous a-crystallins were then studied by using a fixed antiserum concentration with Thus, purified bovine cu-crystallin reacted to varying concentrations of antigen. fix 60 units of complement with a 1 : 110 dilution of the second serum. Twenty pg of the a-crystallin was required for the maximum fixation. The results of the quantitative complement fixation test between different (IIcrystallins and rabbit antisera to bovine cu-crystallin are shown in Table 1. The TABLE
~-CROSS-REACTIONS
OF
BOVINEa-CRYSTALLINSERA
EIGHT
DIFFERENT
ASDETERMINBD
a-CRYSTALLINS
BYQUANTITATIVE
o/0of homologous
maximum
a-Crystallin Bovine Sheep Rabbit Monkey Human Rat Chicken Dogfish
antiserum
100 100 94 80 80 72 42 0
WITH
TWO
RABBIT
COMPLEMENTFIXATION
reaction in complement fixation
at
1
2
antiserum
100 100 100 84 82 78 44 0
ANTI-
P. D.
640
MEHTA
AND
S. LERMAN
maximum amount of complement fixed at the constant antiserum concentration was first determined and this is calculated and expressed as a percentage of the maximal amount of complement fixed by the homologous antigen and is listed in Table 1. The bovine, sheep and rabbit a-crystallins fixed 100 per cent of complement, whereas the monkey, human and rat fixed about 80-84 per cent of complement. On the other hand, the chicken oc-crystallin fixed 40 per cent complement while the dogfish a-crystallin showed no fixation. The two antisera showed hardly any quantitative differences in the extent of cross-reaction. Thus, the order of decreasing maximal complement fixation by various a-crystallins is much the same for both antisera. The amino acid composition of bovine, sheep, monkey, chicken and dogfish a-crystallins are shown in Table 2. These species were chosen on the basis of the
TABLE ~-AMINO ACID COMPOSITION OF DIFFERENTO+CRYSTALLINS.VALUESEXPRESSEDAS RESIDUES PER 1000 MOLES
Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Bovine
Sheep
Monkey
Chicken
Dogfish (Lerman, 1969)
51 35 74 94 36 106 107 75 61 42 52 17 51 90 33 77
50 42 75 92 34 114 105 75 58 46 59 12 43 88 33 76
49 37 75 97 49 96 109 70 57 37 62 14 44 93 32 78
49 35 77 90 38 125 117 81 49 30 46 25 65 93 21 57
48 30 76 103 34 71 129 107 58 36 56 29 53 64 39 66
results obtained by the complement fixation test. In general, the amino acid composition of a-crystallin from bovine, sheep and monkey were similar, although some minor discrepancies in the amino acid composition of bovine and monkey can be seen. On the other hand, the composition of ol-crystallin for bovine differs in several amino acid residues from that of the chicken and dogfish ol-crystallins. Figure 3 shows the results of urea starch gel electrophoresis of the different a-crystallins. The eight purified a-crystallins resolved into three components at pH 3.0. All the proteins displayed remarkably similar behaviour in starch gel electrophoresis except that of dogfish which was significantly slower.
f Bavine
lens
Bovine
ff
Sheep
a - cry
Bovine
lens
Human
o -cry
Monkey
a - cry
A -CT
y
A
+ B
A
Chicken
lens
Chicken
a-
Bovine
cry
lens
Rabbit
a -cry
Dogfish
lens
Dogfish
a -cry
C
D
Rat
lens
Rat
a-cry
FIG. 1. Imu~oelectrophoretic anaIysis of total soluble lens and purified acrystallins. Troughs contain : A, rabbit antibovine lens serum; B, rabbit antichicken lens serum; C, rabbit antidogfish lens serum; and D, rabbit antirat lens serum.
STUDIES
OF LENS
WCRYSTALLIN
FROM
EIGHT
SPECIES
641
DISCUSSION
The results from the gel diffusion test indicate that except for chicken and dogfish, all cu-crystallins appeared to be identical. The precipitin line of bovine ol-crystallin spurs over the chicken line, indicating that the chicken or-crystallin is antigenically deficient to bovine cr-crystallin. Furthermore, the dogfish a-crystaliin did not react at all under these conditions. The results obtained from the quantitative complement fixation technique showed that the bovine and sheep or-crystallins One of the striking findings is that the exhibit identical immunological reactions. rabbit ol-crystallin is quantitatively indistinguishable from the bovine ol-crystallin with antibovine a-crystallin serum produced in rabbits. Although the difference in the degree of complement fixation between human, monkey or rat ol-crystallins compared to bovine protein is small, the difference is greater than the error of duplicate analysis and therefore it signifies antigenic differences. Many examples of this type of behaviour in both the haemoglobin and cytochrome-c have been demonstrated by Reichlin et al. (1964) and Margoliash et al. (1970). One of the conclusions from this study is that the structures of a11 the mammalian a-crystallins are not identical. These results at first glance appear contradictory to those reported by Bjijrk (1968), but two points of difference must be considered. Firstly, the relative insensitivity of the immunodiffusion technique to small differences in protein structure has been shown before in studies on haemoglobin (Reichlin et al., 1964) and serum albumin (Sarich & Wilson, 1966). Secondly, the complement fixation test is more sensitive than the quantitative precipitin test as shown by Levine & van Vunakis (1967). In the present study the concentrations of antigen and antibody used for complement fixation were about l/l00 of that used for the precipitin test; thus, at high dilutions, the test emphasizes the stronger antigen-antibody interactions and detects differences between antigens, which are not apparent when the same two antigens are compared in the quantitative precipitin test. However, Bjerk (1968) stated that human, bovine and rat ol-crystallins are identical as judged by the quantitative precipitin test and in gel diffusion experiments. Both of our antisera clearly distinguish bovine from human, monkey and rat in the complement fixation test, Although the amino acid composition is only a very rough guide, the immunological data parallel the variation in the amino acid composition of these cw-crystallins. The amino acid compositions of a-crystaIlins from bovine and sheep are quite similar, whereas those of chicken and dogfish are considerably different. Bjijrk (1968) compared the different mammalian a-crystallins in 8 M urea polyacrylamide gel electrophoresis at alkaline pH and observed about 12-15 bands. The electrophoretic pattern of these a-crystallins showed minor differences in mobilities. However, he did not compare the electrophoretic patterns at acidic pH. Recent experiments of Schoenmakers & Bloemendal (1968) indicate that ol-crystallin dissociates into sub-units or polypeptide chains at acidic pH in the presence of 8 M urea. Our results on starch gel electrophoresis at acidic pH showed that the mobilities of the polypeptide chains of the a-crystallins were
P. D. MEHTA AND S. LERMAN
642
remarkably similar except those of dogfish which showed a significantly slower mobility. The reason for this is not known. Previous workers have suggested that there is a strong relationship between the extent of immunological cross-reactions and the relatedness in primary structure of proteins of known amino acid sequence such as cytochromes-c (Margoliash et al., 1970) and haemoglobins (Reichlin et al., 1964). The complement fixation test has also predicted the degree of sequence differences among proteins such as lysozyme (Arnheim et al., 1969) and ribonuclease (Brown et al., 1960). How strong the correlation is between the amino acid sequence and extent of crossreactions in ol-crystallin will be decided only by further studying the chemical and immunological aspects of closely related species. REFERENCES ARNHEIM N., PRAGER E. M. & WILSON A. C. (1969) Immunological prediction of sequence differences among proteins. J. biol. Chem. 244, 2085-2094. BJ~~RK I. (1968) Comparative studies of ol-crystallin from lenses of different mammalian species. Exptl Eye Res. 7, 129-133. BROWN R. K., TACEY B. C. & ANFINSEN C. B. (1960) The reaction of porcine and ovine ribonucleases with antibody to bovine ribonuclease. Biochim. biophys. Acta 39, 528-530. ELXLMAN G. M. & POULIK M. D. (1961) Studies on structural units of y-globulins. J. exp. Med. 113, 861-884. HALBERT S. P. & FITZGERALD P. L. (1958) Studies on the immunologic organ specificity of ocular lens. Am.J. Ophthal. 46, 187-196. In Methods of biochemical analysis 1, 141-170. KUNKEL H. G. (1954) Zone electrophoresis. Interscience, New York. LERMAN S. (1969) Characterization of the insoluble protein fraction in the ocular lens. Can. J. Biochem. 47, 1115-1119. LEVINE L. & VAN VUNAKIS H. (1967) Micro complement fixation. In Methods in Enzymology (Edited by HIRS C. H. W.), Vol. 11, 928-936. Academic Press, New York. LOWRY 0. H., ROSEBROUCH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAISEL H. (1963) The immunologic specificity of lens antigens. Am. J. Ophthal. 55,
1208-1216. Arch. Ophthal. 72,829-83 1. MAISEL H. (1964) The serological specificity of lens a-crystallin. MANSKI W. J., AUERBACH T. P. & HALBERT S. P. (1961) The evolutionary significance of lens organ specificity. Am. J. Ophthal. 50, 985-990. MANSKI W., HALBERT S. P., JAVIER P. & AUERBACH T. P. (1967) On the use of antigenic relationships among species for the study of molecular evolution. Int. Avch. Allergy 31, 475-489. MARCOLIASH E., NISONOFF A. & REICHLIN M. (1970) Immunological activity of cytochrome-c. J. Biol. them. 245, 931-939. MAYER M. M., OSLER A. G., BIER 0. G. & HEIDELBERCERM. (1948) Quantitative studies of complement fixation. J. Immunol. 59, 195-206. MEHTA P. D., COOPER S. N. & RAO S. S. (1964) The identification of species specific and organ specific antigens in lens proteins. Exptl Eye Res. 3, 192-197. MEHTA P. D. & LERMAN S. (1970) Immunochemical relationship between soluble and insoluble lens proteins. Ophthal. Res. 1, 10-20. MOORE S. & STEIN W. II. (1963) Chromatographic determination of amino acids by the use In Methods in Enzymology (Edited by COLWICK S. P. of automatic recording equipment. & KAPLAN N. O.), Vol.
6, 819-831.
Academic
Press, New York.
STUDIES OF LENSa-CRYSTALLINFROMEIGHTSPBCIES
643
NISONOFFA., MARCOLIASHE. & BRICHLINM. (1967) Antibodies to rabbit cytochromes-c arising in rabbits. Science 155, 1273-1275. OUCHTRRLONY 0. (1958) Diffusion in gel methods for immunological analysis. Progress of Allergy 5, l-78. Karger, BaseI/New York. BRICHLINM., HAY M. & LEVINEL. (1964) Antibodies to human Ai hemoglobin and their reaction with Az, S, C and H hemoglobins. I~~unoc~e~. 1, 21-30. SARICHV. M. & WILSON A. C. (1966) Q uantitative immunochemistry of the evolution of primate albumins : Micro complement fixation. Science 154, 1563-l 566. SCHEIDECGER J. J. (19.55) Une micro methode de l’immunoelectrophorese. Int. Arch. Allergy 7, 103-I 10. SCHOENMAKERS J. G. G. & BLOEMENDAL H. (1968) Non-identified subunits in iu-crystallin. Biochem. biophys. Res. ~omrnu~. 31, 257-261. SMITKIES0. (1959) An approved procedure for starch gel electrophoresis. Further variations in the serum proteins of normal individuals. Biochem. J. 71, 585-587. WILSON A. C., KAPLANN. O., LEVINEIi., PESCEA., BRICHLINM. & ALLISONW. S. (1964) Evolution of lactic dehydrogenase. Fed. Proc. 23, 1258-1266. Key Word Index-Lens proteins; a-crystallin; starch gel electrophoresis; antiserum; cross-reaction.
complement
fixation;
gel diffusion;
COMPARATIVE
STUDIES OF LENS a-CRYSTALLIN FROM EIGHT SPECIES*
P. D. MEHTA Department
and
S. LERMAN
of Ophthalmology Research, McIntyre Medical McGill University, Montreal, Quebec (Received
30 July
Sciences
Centre,
1970)
Abstract-l.
The cross-reactions of or-crystallins from bovine, sheep, rabbit, human, monkey, rat, chicken and dogfish lens were studied by the quantitative complement fixation test using rabbit antisera specific for bovine cu-crystallin. 2. By this method, the bovine, sheep, and rabbit or-crystallins appeared to be identical, while the human, monkey and rat showed a somewhat lower degree of complement fixation with the antibovine or-crystallin sera. The chicken 01crystallin showed approximately 40 per cent cross-reactivity with the bovine protein, and there was no detectable cross-reaction in the case of dogfish ol-crystallin. 3. Excluding dogfish, the electrophoretic patterns of different ol-crystallins in 8 M urea starch gel electrophoresis at pH 3.0 were similar.
INTRODUCTION THE
COMPARISON
of the structure of homologous proteins from different species can contribute towards an understanding of the relationship between protein structure and function as well as the nature of the evolutionary processes. It has been established that cross-reactions occur within all three of the main groups of lens proteins, namely the OL-,/I- and y-crystallins (Manski et al., 1960). Of the different lens proteins, cy-crystallin generally shows the greatest degree of organ specificity. Many reports have been published on immunochemical comparisons of whole lens proteins from different species (Halbert & Fitzgerald, 1958; Manski et al., 1960; Maisel, 1963; Mehta et al., 1964; Manski et al., 1967), whereas little work has been carried out on the immunological cross-reactions of isolated lens protein such as ar-crystallin. Maisel (1964) compared a-crystallin by the gel diffusion technique and found differences between five vertebrate classes. Bjiirk (1968) reported that mammalian cz-crystallin behaved identically in the quantitative precipitin test and in gel diffusion experiments, but using the quantitative complement fixation technique, we have been able to differentiate between these closely related proteins. * This investigation Council, Canada.
was supported
by Grant
637 22
No. MA-3208
from the Medical
Research
P. D. MEHTA
638
AND S. LERMAN
In the present article ol-crystallin of seven species are compared with that of bovine ol-crystallin, in reaction with antiserum to the bovine protein. Immunological methods are employed with particular emphasis on the quantitative complement fixation technique. This method has been shown to be sensitive to small differences in amino acid sequence (Reichlin et al., 1964) and capable of providing an approximate measure of structural relationship between homologous proteins (Wilson et al., 1964; Sarich & Wilson, 1966). In addition, the polypeptide chains of these ol-crystallins are compared in urea starch gel electrophoresis at acidic pH.
MATERIALS
AND
METHODS
Lenses
from eight species were used. These included bovine (Bos taurus), sheep (Otis (Homo sapiens), monkey (Macuca irus),rabbit (OryctoZagus cuniculus), chicken (Gallus domesticus), rat (Rattus norvegicus) and dogfish (Mustellis cunis). Normal adult human lenses were obtained within 12 hr after death. The other lenses were obtained immediately after the animal’s death and were carefully dissected free from the capsule in an iced container and homogenized in 0.15 M NaCl with 50 to 100 mg wet wt. tissue per ml. The homogenate was centrifuged at 12,000 x g for 30 min at 4°C and the clear supernatant was stored at -20°C until required for the subsequent isolation procedures. avies),human
Isolation
of ol-crystullin
a-crystallin was initially purified by zone electrophoresis on starch at a potential gradient 20 V/in. in a barbital buffer, pH 8.6, ionic strength 0.05 (Kunkel, 1954). The material was then further purified by gel filtration using Sephadex G-200 which was equilibrated with 0.1 M Tris-HCl+ 1 M NaCl buffer, pH 7.8. Determination
of protein
Protein concentrations (Lowry et al., 1951). Preparation
concentration were
determined
by
the
modified
Folin-Ciocalteu
method
of antisera
Antisera to total soluble lens proteins and isolated bovine or-crystallin were prepared in rabbits by weekly injections of the antigen in complete Freund’s adjuvant. The antisera to bovine cu-crystallin were rendered specific by adding fi-crystallins (dry protein) in a suitable amount and removing the resulting antigen-antibody precipitate by centrifugation (Mehta & Lerman, 1970). Immunoelectrophoresis
and immunodiflusion
Double diffusion in agar was performed according to the method of Ouchterlony (1958) and immunoelectrophoresis on glass microscopic slides was employed as described by Scheideggar (1955). CompZement fixation
test
The complement fixation test was done according (1948), with the modification that the test was done reactants remaining the same (Nisonoff et uZ., 1967). Starch
gel electrophovesis
to the technique of Mayer in 1.0 ml, the concentration
et al. of all
in urea
Vertical starch gel electrophoresis (1959) with the formate-urea buffer
was performed according to the method system as described by Edelman & Poulik
of Smithies (1961).
STUDIES OF LENS O+CRYSTALLIN
Amino
FROM
EIGHT
639
SPECIES
acid composition
Samples of or-crystallin were hydrolyzed in sealed evacuated Pyrex tubes containing 6 N HCl at 110°C for 24 hr. Amino acid analyses were done with a Beckman 120C autoanalyzer according to the procedures described by Moore & Stein (1963). RESULTS
ol-crystallins from eight species were tested with either homologous or heterologous antitotal soluble lens sera by immuno-electrophoresis and Ouchterlony analysis. Each preparation showed a single precipitin arc which was at a position characteristic for a-crystallin (Fig. 1). The immunological interrelationships between cu-crystallins of the different species were studied by the gel diffusion technique using two different rabbit In order to augment the results of antisera made specific for bovine a-crystallin. the gel diffusion technique, these proteins were also compared by the quantitative complement fixation test using the same antisera. Figure 2 shows the results of gel diffusion test of rabbit antiserum to bovine oc-crystallin with the ol-crystallin of eight species. By this method, the reaction of identity was observed between bovine, sheep, monkey, human, rabbit and rat oc-crystallins. A reaction of partial identity was observed between bovine and chicken ol-crystallins, while the dogfish a-crystallin showed no precipitin line. In order to study the cross-reactions by the complement fixation test, the maximum amount of complement that can be fixed at each antiserum concentration was determined (Mayer et al., 1948). Cross-reactions of antisera with heterologous a-crystallins were then studied by using a fixed antiserum concentration with Thus, purified bovine cu-crystallin reacted to varying concentrations of antigen. fix 60 units of complement with a 1 : 110 dilution of the second serum. Twenty pg of the a-crystallin was required for the maximum fixation. The results of the quantitative complement fixation test between different (IIcrystallins and rabbit antisera to bovine cu-crystallin are shown in Table 1. The TABLE
~-CROSS-REACTIONS
OF
BOVINEa-CRYSTALLINSERA
EIGHT
DIFFERENT
ASDETERMINBD
a-CRYSTALLINS
BYQUANTITATIVE
o/0of homologous
maximum
a-Crystallin Bovine Sheep Rabbit Monkey Human Rat Chicken Dogfish
antiserum
100 100 94 80 80 72 42 0
WITH
TWO
RABBIT
COMPLEMENTFIXATION
reaction in complement fixation
at
1
2
antiserum
100 100 100 84 82 78 44 0
ANTI-
P. D.
640
MEHTA
AND
S. LERMAN
maximum amount of complement fixed at the constant antiserum concentration was first determined and this is calculated and expressed as a percentage of the maximal amount of complement fixed by the homologous antigen and is listed in Table 1. The bovine, sheep and rabbit a-crystallins fixed 100 per cent of complement, whereas the monkey, human and rat fixed about 80-84 per cent of complement. On the other hand, the chicken oc-crystallin fixed 40 per cent complement while the dogfish a-crystallin showed no fixation. The two antisera showed hardly any quantitative differences in the extent of cross-reaction. Thus, the order of decreasing maximal complement fixation by various a-crystallins is much the same for both antisera. The amino acid composition of bovine, sheep, monkey, chicken and dogfish a-crystallins are shown in Table 2. These species were chosen on the basis of the
TABLE ~-AMINO ACID COMPOSITION OF DIFFERENTO+CRYSTALLINS.VALUESEXPRESSEDAS RESIDUES PER 1000 MOLES
Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Bovine
Sheep
Monkey
Chicken
Dogfish (Lerman, 1969)
51 35 74 94 36 106 107 75 61 42 52 17 51 90 33 77
50 42 75 92 34 114 105 75 58 46 59 12 43 88 33 76
49 37 75 97 49 96 109 70 57 37 62 14 44 93 32 78
49 35 77 90 38 125 117 81 49 30 46 25 65 93 21 57
48 30 76 103 34 71 129 107 58 36 56 29 53 64 39 66
results obtained by the complement fixation test. In general, the amino acid composition of a-crystallin from bovine, sheep and monkey were similar, although some minor discrepancies in the amino acid composition of bovine and monkey can be seen. On the other hand, the composition of ol-crystallin for bovine differs in several amino acid residues from that of the chicken and dogfish ol-crystallins. Figure 3 shows the results of urea starch gel electrophoresis of the different a-crystallins. The eight purified a-crystallins resolved into three components at pH 3.0. All the proteins displayed remarkably similar behaviour in starch gel electrophoresis except that of dogfish which was significantly slower.
f Bavine
lens
Bovine
ff
Sheep
a - cry
Bovine
lens
Human
o -cry
Monkey
a - cry
A -CT
y
A
+ B
A
Chicken
lens
Chicken
a-
Bovine
cry
lens
Rabbit
a -cry
Dogfish
lens
Dogfish
a -cry
C
D
Rat
lens
Rat
a-cry
FIG. 1. Imu~oelectrophoretic anaIysis of total soluble lens and purified acrystallins. Troughs contain : A, rabbit antibovine lens serum; B, rabbit antichicken lens serum; C, rabbit antidogfish lens serum; and D, rabbit antirat lens serum.
STUDIES
OF LENS
WCRYSTALLIN
FROM
EIGHT
SPECIES
641
DISCUSSION
The results from the gel diffusion test indicate that except for chicken and dogfish, all cu-crystallins appeared to be identical. The precipitin line of bovine ol-crystallin spurs over the chicken line, indicating that the chicken or-crystallin is antigenically deficient to bovine cr-crystallin. Furthermore, the dogfish a-crystaliin did not react at all under these conditions. The results obtained from the quantitative complement fixation technique showed that the bovine and sheep or-crystallins One of the striking findings is that the exhibit identical immunological reactions. rabbit ol-crystallin is quantitatively indistinguishable from the bovine ol-crystallin with antibovine a-crystallin serum produced in rabbits. Although the difference in the degree of complement fixation between human, monkey or rat ol-crystallins compared to bovine protein is small, the difference is greater than the error of duplicate analysis and therefore it signifies antigenic differences. Many examples of this type of behaviour in both the haemoglobin and cytochrome-c have been demonstrated by Reichlin et al. (1964) and Margoliash et al. (1970). One of the conclusions from this study is that the structures of a11 the mammalian a-crystallins are not identical. These results at first glance appear contradictory to those reported by Bjijrk (1968), but two points of difference must be considered. Firstly, the relative insensitivity of the immunodiffusion technique to small differences in protein structure has been shown before in studies on haemoglobin (Reichlin et al., 1964) and serum albumin (Sarich & Wilson, 1966). Secondly, the complement fixation test is more sensitive than the quantitative precipitin test as shown by Levine & van Vunakis (1967). In the present study the concentrations of antigen and antibody used for complement fixation were about l/l00 of that used for the precipitin test; thus, at high dilutions, the test emphasizes the stronger antigen-antibody interactions and detects differences between antigens, which are not apparent when the same two antigens are compared in the quantitative precipitin test. However, Bjerk (1968) stated that human, bovine and rat ol-crystallins are identical as judged by the quantitative precipitin test and in gel diffusion experiments. Both of our antisera clearly distinguish bovine from human, monkey and rat in the complement fixation test, Although the amino acid composition is only a very rough guide, the immunological data parallel the variation in the amino acid composition of these cw-crystallins. The amino acid compositions of a-crystaIlins from bovine and sheep are quite similar, whereas those of chicken and dogfish are considerably different. Bjijrk (1968) compared the different mammalian a-crystallins in 8 M urea polyacrylamide gel electrophoresis at alkaline pH and observed about 12-15 bands. The electrophoretic pattern of these a-crystallins showed minor differences in mobilities. However, he did not compare the electrophoretic patterns at acidic pH. Recent experiments of Schoenmakers & Bloemendal (1968) indicate that ol-crystallin dissociates into sub-units or polypeptide chains at acidic pH in the presence of 8 M urea. Our results on starch gel electrophoresis at acidic pH showed that the mobilities of the polypeptide chains of the a-crystallins were
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remarkably similar except those of dogfish which showed a significantly slower mobility. The reason for this is not known. Previous workers have suggested that there is a strong relationship between the extent of immunological cross-reactions and the relatedness in primary structure of proteins of known amino acid sequence such as cytochromes-c (Margoliash et al., 1970) and haemoglobins (Reichlin et al., 1964). The complement fixation test has also predicted the degree of sequence differences among proteins such as lysozyme (Arnheim et al., 1969) and ribonuclease (Brown et al., 1960). How strong the correlation is between the amino acid sequence and extent of crossreactions in ol-crystallin will be decided only by further studying the chemical and immunological aspects of closely related species. REFERENCES ARNHEIM N., PRAGER E. M. & WILSON A. C. (1969) Immunological prediction of sequence differences among proteins. J. biol. Chem. 244, 2085-2094. BJ~~RK I. (1968) Comparative studies of ol-crystallin from lenses of different mammalian species. Exptl Eye Res. 7, 129-133. BROWN R. K., TACEY B. C. & ANFINSEN C. B. (1960) The reaction of porcine and ovine ribonucleases with antibody to bovine ribonuclease. Biochim. biophys. Acta 39, 528-530. ELXLMAN G. M. & POULIK M. D. (1961) Studies on structural units of y-globulins. J. exp. Med. 113, 861-884. HALBERT S. P. & FITZGERALD P. L. (1958) Studies on the immunologic organ specificity of ocular lens. Am.J. Ophthal. 46, 187-196. In Methods of biochemical analysis 1, 141-170. KUNKEL H. G. (1954) Zone electrophoresis. Interscience, New York. LERMAN S. (1969) Characterization of the insoluble protein fraction in the ocular lens. Can. J. Biochem. 47, 1115-1119. LEVINE L. & VAN VUNAKIS H. (1967) Micro complement fixation. In Methods in Enzymology (Edited by HIRS C. H. W.), Vol. 11, 928-936. Academic Press, New York. LOWRY 0. H., ROSEBROUCH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAISEL H. (1963) The immunologic specificity of lens antigens. Am. J. Ophthal. 55,
1208-1216. Arch. Ophthal. 72,829-83 1. MAISEL H. (1964) The serological specificity of lens a-crystallin. MANSKI W. J., AUERBACH T. P. & HALBERT S. P. (1961) The evolutionary significance of lens organ specificity. Am. J. Ophthal. 50, 985-990. MANSKI W., HALBERT S. P., JAVIER P. & AUERBACH T. P. (1967) On the use of antigenic relationships among species for the study of molecular evolution. Int. Avch. Allergy 31, 475-489. MARCOLIASH E., NISONOFF A. & REICHLIN M. (1970) Immunological activity of cytochrome-c. J. Biol. them. 245, 931-939. MAYER M. M., OSLER A. G., BIER 0. G. & HEIDELBERCERM. (1948) Quantitative studies of complement fixation. J. Immunol. 59, 195-206. MEHTA P. D., COOPER S. N. & RAO S. S. (1964) The identification of species specific and organ specific antigens in lens proteins. Exptl Eye Res. 3, 192-197. MEHTA P. D. & LERMAN S. (1970) Immunochemical relationship between soluble and insoluble lens proteins. Ophthal. Res. 1, 10-20. MOORE S. & STEIN W. II. (1963) Chromatographic determination of amino acids by the use In Methods in Enzymology (Edited by COLWICK S. P. of automatic recording equipment. & KAPLAN N. O.), Vol.
6, 819-831.
Academic
Press, New York.
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NISONOFFA., MARCOLIASHE. & BRICHLINM. (1967) Antibodies to rabbit cytochromes-c arising in rabbits. Science 155, 1273-1275. OUCHTRRLONY 0. (1958) Diffusion in gel methods for immunological analysis. Progress of Allergy 5, l-78. Karger, BaseI/New York. BRICHLINM., HAY M. & LEVINEL. (1964) Antibodies to human Ai hemoglobin and their reaction with Az, S, C and H hemoglobins. I~~unoc~e~. 1, 21-30. SARICHV. M. & WILSON A. C. (1966) Q uantitative immunochemistry of the evolution of primate albumins : Micro complement fixation. Science 154, 1563-l 566. SCHEIDECGER J. J. (19.55) Une micro methode de l’immunoelectrophorese. Int. Arch. Allergy 7, 103-I 10. SCHOENMAKERS J. G. G. & BLOEMENDAL H. (1968) Non-identified subunits in iu-crystallin. Biochem. biophys. Res. ~omrnu~. 31, 257-261. SMITKIES0. (1959) An approved procedure for starch gel electrophoresis. Further variations in the serum proteins of normal individuals. Biochem. J. 71, 585-587. WILSON A. C., KAPLANN. O., LEVINEIi., PESCEA., BRICHLINM. & ALLISONW. S. (1964) Evolution of lactic dehydrogenase. Fed. Proc. 23, 1258-1266. Key Word Index-Lens proteins; a-crystallin; starch gel electrophoresis; antiserum; cross-reaction.
complement
fixation;
gel diffusion;