Exp. Eye Rrs. (1977) 25, 473-481
On the Existence of pcrystallin DAVID
1’~ iwrsity
in the Bird Lens
S. IICDEVITT
Depurtursent of Anirnnl Biology, Rchool of iveterinary Medicine, of Pmrasylvanin, 3800 Spruw St., Philadelphin, Pa. 19104, 7’.S.,il. Imtitute of Animal Genetics, I:nirersity qf Er~idxtr~~h
LAURIE
(Received 11 February
R.
ad
CROFT
1977 and in revised form
7 April
1977. Xew
York)
Sauropsidans until recently were acknowledged to possess the lens-specific proteins. M. and /&crystallins, while lacking y-crystallins. The y-crystallins have been employed as specific indicators of normal and regenerating lens fiber cell differentiation in several other organisms. It has been reported, however, that indeed, birds do possess y-crystallins, and that fllrthermore this protein is not lens-fiber specific, since it could be found in annular pad and epithelium as well as fibers. Several physical characteristics of this protein cited by those suggesting it,s existence and at odds with those known for y-cystallins, led the authors t,o further investigate t,he putative y-crystallin. Pigeon “y“-rrystallm was prepared exactly according to previously published methods, and subjected to amino acid and N-terminal analyses. and peptide mapping. The fraction isolated satisfied none of the commonly-accepted criteria fi,r y-crystallins. Thus the pigeon “y”-crystallin did not exhibit low values for alanine and lysine; a free N-terminal amino acid could not be detected: and peptide mapping did not reveal the distinctive peptide ofy-crystallins, the fastest moving orange spot in t’he electrophoresis direction. The bird lens, then, does not contain y-crystallin: t’he y-wystallins. when present, remain as reliable indicators of lens fiber differentiation. Kry WO&: lens; bird; y-crystallin; differentiation; amino arid composition; ,T-terminal amino acid; peptide mapping.
1. Introduction Non-sauropsidan vertebrate lensesare considered t’o contain the structural prot’eins X-, p- and y-crystallins, all but the latter heteropolymers, with the broad criteria. (Jf decreasing molecular weight and electrophoretic mobility, K-crystallin + y-crystalliu (for reviews see Clayton, 1970, 1974). The y-crystallins, of interest here, have been reported as having a molecular weight of approximately 20 000, and have been used as indicators of normal and regenerating lens fiber cell differentiation in several organisms (Papaconstantinou, 1965; Takata, Alhright and Yamada, 1966; NcDevitt, Meza and Yamada, 1969; Shubert, Trevithick and Hollenherg, 1970; McDevitt, an11 Brahma~ 1973; Brahma and McDevitt, 1974a,b). Sauropsidansuntil recently were considered to possessE and p crystallins as well RS an immunologically unique crystallin, (non-cross reactive with other vertebrate lens proteins) &crystallin, while lacking y-crystallin (Zwaan and Ikeda, 1968; (‘laytfon, 1970, 1974). There have appeared reports, however, that indeed, birds do possess y-crystallins (Rabaey, Rikkers and de Mets, 1972; Brahma, Rabaey and van Doorenmaalen, 1972; Brahma and van Doorenmaalen, 1973). This protein was Ilot found to be lens-fiber specific, as y-crystallins had been in other, non-sauropsidan species;since it could be found in annular pad and epithelium as well as in fibers (Brahma et al., 1972). 473
1). s. hl- gel liltrat
eon. /;:l.r/). BIJ~ I/r.v. 3, 2iS
53. Bjork.
T. (1970). Studies of y-crystallin from c.alf lens. 111. Comparison of thus main protein (‘onponents by peptide mapping. h’.rp. Eye Elrs. 9, 1.52-7. BOWS. J. (1973). Free isoelectric focusing of bovine lens crystallins. E:‘sp. Eyr f&s. 16, :i()L- 15. Brahms, S. K. and ROWS. J. (1972). Thin layer isoelectric focusing of the soluble lens extracts from larval stages and adult Senopw Irreris. E.rp. Eye h’rs. 13, 309-3314. Brahms, S. K. and McDevitt,, D. S. (1974a). Ontogeny and localizatio:r of gamma-~~~st~bllins in Krtncr tempomrh. Amby.~fomn rn~.rir~~nut~ and I’leurodrles wctlilii normal lens development. Exp. fcye Res. 19, 379-87. Brahma. S. K. and MrDevitt. D. S. (1974b). Ontogeny and localization of the lens crystallins in Senopus laecis lens regeneration. J. Embryol. Exp. Jlor$l. 32, 78%94. Brahms, S. K., Rabaey. M. and van Doorenmaalen. I\‘. J. (1972). Ontogeny and localizat,ion of y-crystallin antigen in the developing pigeon (Coluntba Eiritr) lens. Is’rp. Eye Res. 14, 13&34. Brahnm, S. K. and van Doorenmaalen, W. .J. (1973). Ontogeny of y-crystallin in the c3ltic.k lens. Kxp. Bye 1~e.s. 17, M&6. Campbell. J. C.. Clayto:r, K. M. and Truman. D. E. S. (19fiX). Antigens of the lens of X~nopvs Ineris. hY.zp. Eye Reg. 7, 4-10. Clayton, R. ;\I. (1970). Problems of differentiation in the vertebrate lens. (‘WT. Top. Oerelop. Hiol. 5, 115. Clayt)on, IL. M. (1974). Comparative aspects of lens proteins. In The Bye (Ed. Davson, H.). 1’01. 5. pp. 399494. Academic Press, New York. Clayton, R. M. and Truman, D. E. S. (1974). The ant,igenic st,ructure of (hickB-c~ryst,allin snbunits. Exp. Bye Res. l&495-506. Corran, P. H. and Waley. S. Q. (1969). S-terminal sequenres of x-crystallin. Hiochevt. J. 115, 789996. Croft, L. R. (1972). The amino acid sequence ofy-crystaltin (fraction 11) from calf lens. Hiochen/. J. 128, 96-70. Croft, L. R. (1973a). LOW molecular weight prot.eins of the lens. In ‘I’& ilu,tm?h Lel~s in Kelntion to Cataract. Ciba Foundation Syrr~posiu~~ 19. Pp. 207-26. Amsterdam. Croft, L. R. (197313). Amino and carboxy terminal sequence of y-crystallin from haddoctk lens. Biochim. Biophys. Attn. 295, 1747. Gray. W. R. (1967). Dansyl rhloride procedure. In Methods in En;y~ology (Ed. Hirs. (‘. H. \V.). Volume Il. pp. 139-51. Academir Press. New York. Heathcote, J. G.. Ivashington, R. J.. Keogh. B. J. and Glansville, 15. IV. (1972). An improved technique for t.he analysis of amino acids and relat.ed compounds on thin layers of cellulose. VI. characterization of small peptides by thin-layer and ion-exchange chromatography. J. Chromatoy. 65, 397405. Heilmann. J., Barrollier, J. and Watzke, E. (1957). Beitrag zur aminosaurebestimming au papierchromatogrammen. Hoppe-Seyler‘s Z. Physiol. C’hem. 309, 219-21). Hirs, C. H. \V. (1967). Performic acid oxidation. In Methods in En;ymoZoyy (Ed. Hirs, C’. H. CV.). Volume 11. pp. 19779. Academic Press, New York. Hoenders. H. L. and Bloemendal. H. (1967). The N-terminus of the lens protein, r-crystallin. Bioehrnr. Biophys. Actn 147, 183-5. Mason, C!. V. and Hines, M. (1966). x-. /3- and y-Crystallins in the ocular lens of rabbits: preparation and partial characterization. Incest. Ophthnlmol. 5, 601--g. McDevitt. D. S. and Brahma, S. K. (1973). Ontogeny and localization of the crystallins during embryonic lens development in Xenopus Znecis. J. Ezp. Zool. 186, l?‘i-ZU. McDevitt. D. S.. Meza, I. a.nd Yamada. T. (1969). lmmunofluorescence localization ofthe crystallins in amphibian lens development. with special reference to the y-crystallins. 1)eveZop. Biol. 19, 581-607. Metzger, H., Shapiro, M. B.. Mosimann, J. E. and Vinton, J. E. (1968). Assessment of compositional relatedness between proteins. hrature. Land. 219, 11668. Papaconstantinou, J. (1965). Biochemistry of bovine lens proteins. 11. The y-crystallins of adult bovine, calf, and embryonic lenses. Biochk. Biophys. Acta 107, X1-90. Rabaey, 1.. Rikkers and de Mets, M. (1972). Low molecular weight. protein (y-cryst.allin) in the lens of birds. E3cp. h’ye Res. 14,208-13.
y-C’RYSTALLIN
IS
BIRD
LENS
1SI
Sargent. J. R. (1965). Methods in Zone Electrophoresis. British Drug Houses Ltd., Poole. Dorset. Shubert, E. E., Trevithick, J. R. and Hollenberg, 11. J. (1970). Localization of gamma rrystallins in the developing lens of the rat. Can. J. O$thnb~oZ. 5, X3-6s. Slingsby. C. and Croft,, L. R. (1973). Developmental changes in the low molecular \veight proteins of the bovine lens. Exp. Eye Res. 17, 369-76. Takata, C’.. Albright’, J. F. and Yamada. T. (1966). C+amma crystallins in 1Volfhan lens regeneration drmonstrated by immunofluorescence. Develop. Biol. 14, 382400. Truman. D. E. 8. and Clayton, It. &I. (1974). The subunit structure of c,hick p-crystallins. K.rp, l;‘qp KPS. 18, %-E-74. van IJam. A. F. (I 966). Purifiration and composition studies of Ps-c,rystallin. K.rfj. &qp 12r.y. 5. “.55%X. \Valey, S. C:. arid IVatson, J. (1953). The action of trypsin on polylysine. Is’iorhr),/. ./. 55, XX 39. \Voods. I
On the Existence of pcrystallin DAVID
1’~ iwrsity
in the Bird Lens
S. IICDEVITT
Depurtursent of Anirnnl Biology, Rchool of iveterinary Medicine, of Pmrasylvanin, 3800 Spruw St., Philadelphin, Pa. 19104, 7’.S.,il. Imtitute of Animal Genetics, I:nirersity qf Er~idxtr~~h
LAURIE
(Received 11 February
R.
ad
CROFT
1977 and in revised form
7 April
1977. Xew
York)
Sauropsidans until recently were acknowledged to possess the lens-specific proteins. M. and /&crystallins, while lacking y-crystallins. The y-crystallins have been employed as specific indicators of normal and regenerating lens fiber cell differentiation in several other organisms. It has been reported, however, that indeed, birds do possess y-crystallins, and that fllrthermore this protein is not lens-fiber specific, since it could be found in annular pad and epithelium as well as fibers. Several physical characteristics of this protein cited by those suggesting it,s existence and at odds with those known for y-cystallins, led the authors t,o further investigate t,he putative y-crystallin. Pigeon “y“-rrystallm was prepared exactly according to previously published methods, and subjected to amino acid and N-terminal analyses. and peptide mapping. The fraction isolated satisfied none of the commonly-accepted criteria fi,r y-crystallins. Thus the pigeon “y”-crystallin did not exhibit low values for alanine and lysine; a free N-terminal amino acid could not be detected: and peptide mapping did not reveal the distinctive peptide ofy-crystallins, the fastest moving orange spot in t’he electrophoresis direction. The bird lens, then, does not contain y-crystallin: t’he y-wystallins. when present, remain as reliable indicators of lens fiber differentiation. Kry WO&: lens; bird; y-crystallin; differentiation; amino arid composition; ,T-terminal amino acid; peptide mapping.
1. Introduction Non-sauropsidan vertebrate lensesare considered t’o contain the structural prot’eins X-, p- and y-crystallins, all but the latter heteropolymers, with the broad criteria. (Jf decreasing molecular weight and electrophoretic mobility, K-crystallin + y-crystalliu (for reviews see Clayton, 1970, 1974). The y-crystallins, of interest here, have been reported as having a molecular weight of approximately 20 000, and have been used as indicators of normal and regenerating lens fiber cell differentiation in several organisms (Papaconstantinou, 1965; Takata, Alhright and Yamada, 1966; NcDevitt, Meza and Yamada, 1969; Shubert, Trevithick and Hollenherg, 1970; McDevitt, an11 Brahma~ 1973; Brahma and McDevitt, 1974a,b). Sauropsidansuntil recently were considered to possessE and p crystallins as well RS an immunologically unique crystallin, (non-cross reactive with other vertebrate lens proteins) &crystallin, while lacking y-crystallin (Zwaan and Ikeda, 1968; (‘laytfon, 1970, 1974). There have appeared reports, however, that indeed, birds do possess y-crystallins (Rabaey, Rikkers and de Mets, 1972; Brahma, Rabaey and van Doorenmaalen, 1972; Brahma and van Doorenmaalen, 1973). This protein was Ilot found to be lens-fiber specific, as y-crystallins had been in other, non-sauropsidan species;since it could be found in annular pad and epithelium as well as in fibers (Brahma et al., 1972). 473
1). s. hl- gel liltrat
eon. /;:l.r/). BIJ~ I/r.v. 3, 2iS
53. Bjork.
T. (1970). Studies of y-crystallin from c.alf lens. 111. Comparison of thus main protein (‘onponents by peptide mapping. h’.rp. Eye Elrs. 9, 1.52-7. BOWS. J. (1973). Free isoelectric focusing of bovine lens crystallins. E:‘sp. Eyr f&s. 16, :i()L- 15. Brahms, S. K. and ROWS. J. (1972). Thin layer isoelectric focusing of the soluble lens extracts from larval stages and adult Senopw Irreris. E.rp. Eye h’rs. 13, 309-3314. Brahms, S. K. and McDevitt,, D. S. (1974a). Ontogeny and localizatio:r of gamma-~~~st~bllins in Krtncr tempomrh. Amby.~fomn rn~.rir~~nut~ and I’leurodrles wctlilii normal lens development. Exp. fcye Res. 19, 379-87. Brahma. S. K. and MrDevitt. D. S. (1974b). Ontogeny and localization of the lens crystallins in Senopus laecis lens regeneration. J. Embryol. Exp. Jlor$l. 32, 78%94. Brahms, S. K., Rabaey. M. and van Doorenmaalen. I\‘. J. (1972). Ontogeny and localizat,ion of y-crystallin antigen in the developing pigeon (Coluntba Eiritr) lens. Is’rp. Eye Res. 14, 13&34. Brahnm, S. K. and van Doorenmaalen, W. .J. (1973). Ontogeny of y-crystallin in the c3ltic.k lens. Kxp. Bye 1~e.s. 17, M&6. Campbell. J. C.. Clayto:r, K. M. and Truman. D. E. S. (19fiX). Antigens of the lens of X~nopvs Ineris. hY.zp. Eye Reg. 7, 4-10. Clayton, R. ;\I. (1970). Problems of differentiation in the vertebrate lens. (‘WT. Top. Oerelop. Hiol. 5, 115. Clayt)on, IL. M. (1974). Comparative aspects of lens proteins. In The Bye (Ed. Davson, H.). 1’01. 5. pp. 399494. Academic Press, New York. Clayton, R. M. and Truman, D. E. S. (1974). The ant,igenic st,ructure of (hickB-c~ryst,allin snbunits. Exp. Bye Res. l&495-506. Corran, P. H. and Waley. S. Q. (1969). S-terminal sequenres of x-crystallin. Hiochevt. J. 115, 789996. Croft, L. R. (1972). The amino acid sequence ofy-crystaltin (fraction 11) from calf lens. Hiochen/. J. 128, 96-70. Croft, L. R. (1973a). LOW molecular weight prot.eins of the lens. In ‘I’& ilu,tm?h Lel~s in Kelntion to Cataract. Ciba Foundation Syrr~posiu~~ 19. Pp. 207-26. Amsterdam. Croft, L. R. (197313). Amino and carboxy terminal sequence of y-crystallin from haddoctk lens. Biochim. Biophys. Attn. 295, 1747. Gray. W. R. (1967). Dansyl rhloride procedure. In Methods in En;y~ology (Ed. Hirs. (‘. H. \V.). Volume Il. pp. 139-51. Academir Press. New York. Heathcote, J. G.. Ivashington, R. J.. Keogh. B. J. and Glansville, 15. IV. (1972). An improved technique for t.he analysis of amino acids and relat.ed compounds on thin layers of cellulose. VI. characterization of small peptides by thin-layer and ion-exchange chromatography. J. Chromatoy. 65, 397405. Heilmann. J., Barrollier, J. and Watzke, E. (1957). Beitrag zur aminosaurebestimming au papierchromatogrammen. Hoppe-Seyler‘s Z. Physiol. C’hem. 309, 219-21). Hirs, C. H. \V. (1967). Performic acid oxidation. In Methods in En;ymoZoyy (Ed. Hirs, C’. H. CV.). Volume 11. pp. 19779. Academic Press, New York. Hoenders. H. L. and Bloemendal. H. (1967). The N-terminus of the lens protein, r-crystallin. Bioehrnr. Biophys. Actn 147, 183-5. Mason, C!. V. and Hines, M. (1966). x-. /3- and y-Crystallins in the ocular lens of rabbits: preparation and partial characterization. Incest. Ophthnlmol. 5, 601--g. McDevitt. D. S. and Brahma, S. K. (1973). Ontogeny and localization of the crystallins during embryonic lens development in Xenopus Znecis. J. Ezp. Zool. 186, l?‘i-ZU. McDevitt. D. S.. Meza, I. a.nd Yamada. T. (1969). lmmunofluorescence localization ofthe crystallins in amphibian lens development. with special reference to the y-crystallins. 1)eveZop. Biol. 19, 581-607. Metzger, H., Shapiro, M. B.. Mosimann, J. E. and Vinton, J. E. (1968). Assessment of compositional relatedness between proteins. hrature. Land. 219, 11668. Papaconstantinou, J. (1965). Biochemistry of bovine lens proteins. 11. The y-crystallins of adult bovine, calf, and embryonic lenses. Biochk. Biophys. Acta 107, X1-90. Rabaey, 1.. Rikkers and de Mets, M. (1972). Low molecular weight. protein (y-cryst.allin) in the lens of birds. E3cp. h’ye Res. 14,208-13.
y-C’RYSTALLIN
IS
BIRD
LENS
1SI
Sargent. J. R. (1965). Methods in Zone Electrophoresis. British Drug Houses Ltd., Poole. Dorset. Shubert, E. E., Trevithick, J. R. and Hollenberg, 11. J. (1970). Localization of gamma rrystallins in the developing lens of the rat. Can. J. O$thnb~oZ. 5, X3-6s. Slingsby. C. and Croft,, L. R. (1973). Developmental changes in the low molecular \veight proteins of the bovine lens. Exp. Eye Res. 17, 369-76. Takata, C’.. Albright’, J. F. and Yamada. T. (1966). C+amma crystallins in 1Volfhan lens regeneration drmonstrated by immunofluorescence. Develop. Biol. 14, 382400. Truman. D. E. 8. and Clayton, It. &I. (1974). The subunit structure of c,hick p-crystallins. K.rp, l;‘qp KPS. 18, %-E-74. van IJam. A. F. (I 966). Purifiration and composition studies of Ps-c,rystallin. K.rfj. &qp 12r.y. 5. “.55%X. \Valey, S. C:. arid IVatson, J. (1953). The action of trypsin on polylysine. Is’iorhr),/. ./. 55, XX 39. \Voods. I
