Cell Stress and Chaperones

, Volume 22, Issue 4, pp 541–552 | Cite as

An alternative splice variant of human αA-crystallin modulates the oligomer ensemble and the chaperone activity of α-crystallins

  • Waldemar Preis
  • Annika Bestehorn
  • Johannes Buchner
  • Martin Haslbeck


In humans, ten genes encode small heat shock proteins with lens αA-crystallin and αB-crystallin representing two of the most prominent members. The canonical isoforms of αA-crystallin and αB-crystallin collaborate in the eye lens to prevent irreversible protein aggregation and preserve visual acuity. α-Crystallins form large polydisperse homo-oligomers and hetero-oligomers and as part of the proteostasis system bind substrate proteins in non-native conformations, thereby stabilizing them. Here, we analyzed a previously uncharacterized, alternative splice variant (isoform 2) of human αA-crystallin with an exchanged N-terminal sequence. This variant shows the characteristic α-crystallin secondary structure, exists on its own predominantly in a monomer–dimer equilibrium, and displays only low chaperone activity. However, the variant is able to integrate into higher order oligomers of canonical αA-crystallin and αB-crystallin as well as their hetero-oligomer. The presence of the variant leads to the formation of new types of higher order hetero-oligomers with an overall decreased number of subunits and enhanced chaperone activity. Thus, alternative mRNA splicing of human αA-crystallin leads to an additional, formerly not characterized αA-crystallin species which is able to modulate the properties of the canonical ensemble of α-crystallin oligomers.


Alpha-crystallin Alternative splicing Protein folding Chaperone function sHsp 



We thank Gina Feind for excellent experimental assistance, Robert Pesch and Ralf Zimmer for discussion of bioinformatics data on alternative splicing, and Evgeny Mymrikov for αB-crystallin. The Deutsche Forschungsgemeinschaft (SFB 1035) and CIPSM are acknowledged for financial support.

Supplementary material

12192_2017_772_MOESM1_ESM.pdf (1 mb)
ESM 1 (PDF 1044 kb)


  1. Bassnett S, Shi Y, Vrensen GF (2011) Biological glass: structural determinants of eye lens transparency. Philos Trans R Soc Lond Ser B Biol Sci 366:1250–1264. doi: 10.1098/rstb.2010.0302 CrossRefGoogle Scholar
  2. Bepperling A et al (2012) Alternative bacterial two-component small heat shock protein systems. Proc Natl Acad Sci U S A 109:20407–20412. doi: 10.1073/pnas.1209565109 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Berengian AR, Parfenova M, McHaourab HS (1999) Site-directed spin labeling study of subunit interactions in the alpha-crystallin domain of small heat-shock proteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP 27, and HSP 16.3. J Biol Chem 274:6305–6314. doi: 10.1074/jbc.274.10.6305 CrossRefPubMedGoogle Scholar
  4. Bhat SP, Nagineni CN (1989) αB subunit of lens-specific protein α-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun 158:319–325. doi: 10.1016/S0006-291X(89)80215-3 CrossRefPubMedGoogle Scholar
  5. Bhattacharyya J, Das KP (1998) Alpha-crystallin does not require temperature activation for its chaperone-like activity. Biochem Mol Biol Int 46:249–258. doi: 10.1080/15216549800203762 PubMedGoogle Scholar
  6. Bloemendal H, de Jong W, Jaenicke R, Lubsen NH, Slingsby C, Tardieu A (2004) Ageing and vision: structure, stability and function of lens crystallins. Prog Biophys Mol Biol 86:407–485. doi: 10.1016/j.pbiomolbio.2003.11.012 CrossRefPubMedGoogle Scholar
  7. Bohm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5:191–195. doi: 10.1093/protein/5.3.191 CrossRefPubMedGoogle Scholar
  8. Bonaldo MF, Lennon G, Soares MB (1996) Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res 6:791–806. doi: 10.1101/gr.6.9.791 CrossRefPubMedGoogle Scholar
  9. Bova MP, Ding LL, Horwitz J, Fung BK (1997) Subunit exchange of alphaA-crystallin. J Biol Chem 272:29511–29517. doi: 10.1074/jbc.272.47.29511 CrossRefPubMedGoogle Scholar
  10. Bova MP, McHaourab HS, Han Y, Fung BK (2000) Subunit exchange of small heat shock proteins. Analysis of oligomer formation of alphaA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations. J Biol Chem 275:1035–1042. doi: 10.1074/jbc.275.2.1035 CrossRefPubMedGoogle Scholar
  11. Braun N et al (2011) Multiple molecular architectures of the eye lens chaperone alphaB-crystallin elucidated by a triple hybrid approach. Proc Natl Acad Sci U S A 108:20491–20496. doi: 10.1073/pnas.1111014108 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Carver JA, Aquilina JA, Truscott RJ, Ralston GB (1992) Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin. FEBS Lett 311:143–149. doi: 10.1016/0014-5793(92)81386-Z CrossRefPubMedGoogle Scholar
  13. Clark AR, Lubsen NH, Slingsby C (2012) sHSP in the eye lens: crystallin mutations, cataract and proteostasis. Int J Biochem Cell Biol 44:1687–1697. doi: 10.1016/j.biocel.2012.02.015 CrossRefPubMedGoogle Scholar
  14. Cohen LH, Westerhuis LW, de Jong WW, Bloemendal H (1978a) Rat alpha-crystallin A chain with an insertion of 22 residues. Eur J Biochem 89:259–266. doi: 10.1111/j.1432-1033.1978.tb20921.x CrossRefPubMedGoogle Scholar
  15. Cohen LH, Westerhuis LW, Smits DP, Bloemendal H (1978b) Two structurally closely related polypeptides encoded by 14-S mRNA isolated from rat lens. Eur J Biochem 89:251–258. doi: 10.1111/j.1432-1033.1978.tb20920.x CrossRefPubMedGoogle Scholar
  16. de Jong WW, Cohen LH, Leunissen JAM, Zweers A (1980) Internally elongated rodent α-crystallin A chain: resulting from incomplete RNA splicing? Biochem Biophys Res Commun 96:648–655. doi: 10.1016/0006-291X(80)91404-7 CrossRefPubMedGoogle Scholar
  17. Delaye M, Tardieu A (1983) Short-range order of crystallin proteins accounts for eye lens transparency. Nature 302:415–417. doi: 10.1038/302415a0 CrossRefPubMedGoogle Scholar
  18. Delbecq SP, Klevit RE (2013) One size does not fit all: the oligomeric states of alphaB crystallin. FEBS Lett 587:1073–1080. doi: 10.1016/j.febslet.2013.01.021 CrossRefPubMedGoogle Scholar
  19. Derham BK et al (2001) Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites. Eur J Biochem 268:713–721. doi: 10.1046/j.1432-1327.2001.01929.x CrossRefPubMedGoogle Scholar
  20. Dubin R, Wawrousek E, Piatigorsky J (1989) Expression of the murine alpha B-crystallin gene is not restricted to the lens. Mol Cell Biol 9:1083–1091. doi: 10.1128/MCB.9.3.1083 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Eifert C, Burgio MR, Bennett PM, Salerno JC, Koretz JF (2005) N-terminal control of small heat shock protein oligomerization: changes in aggregate size and chaperone-like function. Biochim Biophys Acta 1748:146–156. doi: 10.1016/j.bbapap.2004.12.015 CrossRefPubMedGoogle Scholar
  22. Fagerholm PP, Philipson BT, Lindström B (1981) Normal human lens—the distribution of protein. Exp Eye Res 33:615–620. doi: 10.1016/S0014-4835(81)80101-7 CrossRefPubMedGoogle Scholar
  23. Fleming TP, Song Z, Andley UP (1998) Expression of growth control and differentiation genes in human lens epithelial cells with extended life span. Invest Ophthalmol Vis Sci 39:1387–1398PubMedGoogle Scholar
  24. Graw J (2009) Genetics of crystallins: cataract and beyond. Exp Eye Res 88:173–189. doi: 10.1016/j.exer.2008.10.011 CrossRefPubMedGoogle Scholar
  25. Haslbeck M, Franzmann T, Weinfurtner D, Buchner J (2005) Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Mol Biol 12:842–846. doi: 10.1038/nsmb993 CrossRefPubMedGoogle Scholar
  26. Haslbeck M, Peschek J, Buchner J, Weinkauf S (2016) Structure and function of alpha-crystallins: traversing from in vitro to in vivo. Biochim Biophys Acta 1860:149–166. doi: 10.1016/j.bbagen.2015.06.008 CrossRefPubMedGoogle Scholar
  27. Heirbaut M et al (2016) The preferential heterodimerization of human small heat shock proteins HSPB1 and HSPB6 is dictated by the N-terminal domain. Arch Biochem Biophys 610:41–50. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  28. Hendriks W, Sanders J, de Leij L, Ramaekers F, Bloemendal H, de Jong WW (1988) Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript. Eur J Biochem 174:133–137. doi: 10.1111/j.1432-1033.1988.tb14072.x CrossRefPubMedGoogle Scholar
  29. Hoehenwarter W, Klose J, Jungblut PR (2006) Eye lens proteomics. Amino Acids 30:369–389. doi: 10.1007/s00726-005-0283-9 CrossRefPubMedGoogle Scholar
  30. Horwitz J (1992) Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 89:10449–10453CrossRefPubMedPubMedCentralGoogle Scholar
  31. Horwitz J (1993) Proctor lecture. The function of alpha-crystallin. Invest Ophthalmol Vis Sci 34:10–22PubMedGoogle Scholar
  32. Iwaki T, Kume-Iwaki A, Goldman JE (1990) Cellular distribution of alpha B-crystallin in non-lenticular tissues. J Histochem Cytochem 38:31–39. doi: 10.1177/38.1.2294148 CrossRefPubMedGoogle Scholar
  33. Iwaki T, Kume-Iwaki A, Liem RKH, Goldman JE (1989) αB-crystallin is expressed in non-lenticular tissues and accumulates in Alexander’s disease brain. Cell 57:71–78. doi: 10.1016/0092-8674(89)90173-6 CrossRefPubMedGoogle Scholar
  34. Jaenicke R, Slingsby C (2001) Lens crystallins and their microbial homologs: structure, stability, and function. Crit Rev Biochem Mol Biol 36:435–499. doi: 10.1080/20014091074237 CrossRefPubMedGoogle Scholar
  35. Jakob U, Gaestel M, Engel K, Buchner J (1993) Small heat shock proteins are molecular chaperones. J Biol Chem 268:1517–1520PubMedGoogle Scholar
  36. Jaworski CJ, Piatigorsky J (1989) A pseudo-exon in the functional human alpha A-crystallin gene. Nature 337:752–754. doi: 10.1038/337752a0 CrossRefPubMedGoogle Scholar
  37. Jehle S et al (2011) N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc Natl Acad Sci U S A 108:6409–6414. doi: 10.1073/pnas.1014656108 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Kato K, Shinohara H, Kurobe N, Goto S, Inaguma Y, Ohshima K (1991a) Immunoreactive αA crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochim Biophys Acta 1080:173–180. doi: 10.1016/0167-4838(91)90146-Q CrossRefPubMedGoogle Scholar
  39. Kato K, Shinohara H, Kurobe N, Inaguma Y, Shimizu K, Ohshima K (1991b) Tissue distribution and developmental profiles of immunoreactive αB crystallin in the rat determined with a sensitive immunoassay system. Biochim Biophys Acta 1074:201–208. doi: 10.1016/0304-4165(91)90062-L CrossRefPubMedGoogle Scholar
  40. King CR, Piatigorsky J (1983) Alternative RNA splicing of the murine αA-crystallin gene: protein-coding information within an intron. Cell 32:707–712. doi: 10.1016/0092-8674(83)90056-9 CrossRefPubMedGoogle Scholar
  41. Kundu M, Sen PC, Das KP (2007) Structure, stability, and chaperone function of alphaA-crystallin: role of N-terminal region. Biopolymers 86:177–192. doi: 10.1002/bip.20716 CrossRefPubMedGoogle Scholar
  42. Laganowsky A et al (2010) Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function. Protein Sci 19:1031–1043. doi: 10.1002/pro.380 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Laganowsky A, Eisenberg D (2010) Non-3D domain swapped crystal structure of truncated zebrafish alphaA crystallin. Protein Sci 19:1978–1984. doi: 10.1002/pro.471 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Lenstra JA, Hukkelhoven MWAC, Gròeneveld AA, Smits RAMM, Weterings PJJM, Bloemendal H (1982) Gene expression of transformed lens cells. Exp Eye Res 35:549–554. doi: 10.1016/S0014-4835(82)80069-9 CrossRefPubMedGoogle Scholar
  45. Liang JN, Andley UP, Chylack LT Jr (1985) Spectroscopic studies on human lens crystallins. Biochim Biophys Acta 832:197–203CrossRefPubMedGoogle Scholar
  46. Mainz A et al (2015) The chaperone alphaB-crystallin uses different interfaces to capture an amorphous and an amyloid client. Nat Struct Mol Biol 22:898–905. doi: 10.1038/nsmb.3108 PubMedGoogle Scholar
  47. Merck KB, De Haard-Hoekman WA, Oude Essink BB, Bloemendal H, De Jong WW (1992) Expression and aggregation of recombinant alpha A-crystallin and its two domains. Biochim Biophys Acta 1130:267–276. doi: 10.1016/0167-4781(92)90439-7 CrossRefPubMedGoogle Scholar
  48. Merck KB, Horwitz J, Kersten M, Overkamp P, Gaestel M, Bloemendal H, de Jong WW (1993) Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein. Mol Biol Rep 18:209–215. doi: 10.1007/BF01674432 CrossRefPubMedGoogle Scholar
  49. Mymrikov EV, Daake M, Richter B, Haslbeck M, Buchner J (2016) The chaperone activity and substrate spectrum of human small heat shock proteins. J Biol Chem. doi: 10.1074/jbc.M116.760413 PubMedGoogle Scholar
  50. Peschek J, Braun N, Franzmann TM, Georgalis Y, Haslbeck M, Weinkauf S, Buchner J (2009) The eye lens chaperone alpha-crystallin forms defined globular assemblies. Proc Natl Acad Sci U S A 106:13272–13277. doi: 10.1073/pnas.0902651106 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Peschek J et al (2013) Regulated structural transitions unleash the chaperone activity of alphaB-crystallin. Proc Natl Acad Sci U S A 110:E3780–E3789. doi: 10.1073/pnas.1308898110 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Raman B, Ramakrishna T, Rao CM (1995) Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Lett 365:133–136. doi: 10.1016/0014-5793(95)00440-K CrossRefPubMedGoogle Scholar
  53. Raman B, Rao CM (1994) Chaperone-like activity and quaternary structure of α-crystallin. J Biol Chem 269:27264–27268PubMedGoogle Scholar
  54. Reddy GB, Reddy PY, Suryanarayana P (2001) αA- and αB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation. Biochem Biophys Res Commun 282:712–716. doi: 10.1006/bbrc.2001.4642 CrossRefPubMedGoogle Scholar
  55. Robinson ML, Overbeek PA (1996) Differential expression of alpha A- and alpha B-crystallin during murine ocular development. Invest Ophthalmol Vis Sci 37:2276–2284PubMedGoogle Scholar
  56. Salerno JC, Eifert CL, Salerno KM, Koretz JF (2003) Structural diversity in the small heat shock protein superfamily: control of aggregation by the N-terminal region. Protein Eng 16:847–851. doi: 10.1093/protein/gzg102 CrossRefPubMedGoogle Scholar
  57. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619. doi: 10.1016/S0006-3495(00)76713-0 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Schuck P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320:104–124. doi: 10.1016/S0003-2697(03)00289-6 CrossRefPubMedGoogle Scholar
  59. Siezen RJ, Wu E, Kaplan ED, Thomson JA, Benedek GB (1988) Rat lens γ-crystallins. J Mol Biol 199:475–490. doi: 10.1016/0022-2836(88)90619-5 CrossRefPubMedGoogle Scholar
  60. Srinivasan AN, Nagineni CN, Bhat SP (1992) Alpha A-crystallin is expressed in non-ocular tissues. J Biol Chem 267:23337–23341PubMedGoogle Scholar
  61. Tardieu A (1988) Eye lens proteins and transparency: from light transmission theory to solution X-ray structural analysis. Annu Rev Biophys Biophys Chem 17:47–70. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  62. van den Heuvel R, Hendriks W, Quax W, Bloemendal H (1985) Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling. J Mol Biol 185:273–284. doi: 10.1016/0022-2836(85)90403-6 CrossRefPubMedGoogle Scholar
  63. Wistow G et al (2002) Expressed sequence tag analysis of human retina for the NEIBank project: retbindin, an abundant, novel retinal cDNA and alternative splicing of other retina-preferred gene transcripts. Mol Vis 8:196–204PubMedGoogle Scholar
  64. Wistow GJ, Piatigorsky J (1988) Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem 57:479–504. doi: 10.1146/ CrossRefPubMedGoogle Scholar

Copyright information

© Cell Stress Society International 2017

Authors and Affiliations

  • Waldemar Preis
    • 1
  • Annika Bestehorn
    • 1
  • Johannes Buchner
    • 1
  • Martin Haslbeck
    • 1
  1. 1.Department Chemie, Center for Integrated Protein ScienceTechnische Universität MünchenGarchingGermany

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