The Sequence of Human B1-Crystallin cDNA Allows Mass Spectrometric Detection of B1 Protein Missing Portions of Its N-terminal Extension (*)

The sequence of human βB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact βB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of βB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human βB1 to the cleavage occurring in bovine βB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human βB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of βB1 amoung species. Results from animal models suggest that cleavage of the N-terminal extension of βB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded βB1-crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed βB1-crystallins become water-insoluble is relatively slow in humans.

[1]  T. Blundell,et al.  Eye‐lens proteins: The three‐dimensional structure of β‐crystallin predicted from monomeric γ‐crystallin , 1981 .

[2]  W. D. de Jong,et al.  Proline‐ and alanine‐rich N‐terminal extension of the basic bovine β‐crystallin B1 chains , 1983, FEBS letters.

[3]  Colin R. Janssen,et al.  Bovine beta-crystallin complementary DNA clones. Alternating proline/alanine sequence of beta B1 subunit originates from a repetitive DNA sequence. , 1984, Journal of molecular biology.

[4]  J. T. Dunnen,et al.  Rat lens β-crystallins are internally duplicated and homologous to γ-crystallins , 1985 .

[5]  L. Tsui,et al.  Characterization of the human beta-crystallin gene Hu beta A3/A1 reveals ancestral relationships among the beta gamma-crystallin superfamily. , 1986, The Journal of biological chemistry.

[6]  A. Spector,et al.  Changes in lens protein in concentric fractions from individual normal human lenses. , 1986, Current eye research.

[7]  B. Groth-Vasselli,et al.  A critical maturation period in neonatal-rat-lens development. , 1986, Experimental eye research.

[8]  M. McFall-Ngai,et al.  Age-dependent changes in the heat-stable crystallin, βBp, of the human lens , 1986 .

[9]  M. Thompson,et al.  cDNA and deduced protein sequence for the beta B1-crystallin polypeptide of the chicken lens. Conservation of the PAPA sequence. , 1986, The Journal of biological chemistry.

[10]  J. Piatigorsky,et al.  Recruitment of enzymes as lens structural proteins. , 1987, Science.

[11]  H. Aarts,et al.  The evolution of lenticular proteins: the β- and γ-crystallin super gene family , 1988 .

[12]  H. Maisel,et al.  Human beta crystallins: regional and age related changes. , 1988, Current eye research.

[13]  Zhongrui Zhou,et al.  Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry , 1990, Journal of protein chemistry.

[14]  W. W. Jong,et al.  Isolation and characterization of cDNAs encoding βA2- and βA4-crystaIlins: heterologous interactions in the predicted βA4-/JB2 heterodimer , 1991 .

[15]  C. G. Edmonds,et al.  Mass spectrometric analysis of lens β-crystallins , 1991 .

[16]  C. Slingsby,et al.  Structural studies on βH-crystallin from bovine eye lens , 1992 .

[17]  Manuel B. Datiles III,et al.  Two-dimensional gel electrophoretic analysis of human lens proteins. , 1992, Current eye research.

[18]  R. Farrell.  RNA methodologies : a laboratory guide for isolation and characterization , 1993 .

[19]  Jean B. Smith,et al.  Amino acid sequence of human lens βB2‐crystallin , 1993 .

[20]  P. Russell,et al.  Sequence of the human lens βB2-crystallin-encoding cDNA , 1993 .

[21]  T. Shearer,et al.  Sequence analysis of lens beta-crystallins suggests involvement of calpain in cataract formation. , 1993, The Journal of biological chemistry.

[22]  T. Shearer,et al.  β‐Crystallins insolubilized by calpain II in vitro contain cleavage sites similar to β‐crystallins insolubilized during cataract , 1993 .

[23]  Hao‐Chia Chen,et al.  βA3/Al-crystallin association: role of the N-terminal arm , 1994 .

[24]  Y. Sun,et al.  Post-translational modifications of water-soluble human lens crystallins from young adults. , 1994, The Journal of biological chemistry.

[25]  R. Glockshuber,et al.  Dimerization of βB2‐crystallin: The role of the linker peptide and the N‐ and C‐terminal extensions , 1994, Protein science : a publication of the Protein Society.

[26]  D. Moss,et al.  Close packing of an oligomeric eye lens beta-crystallin induces loss of symmetry and ordering of sequence extensions. , 1994, Journal of molecular biology.

[27]  J. Carver,et al.  Supramolecular order within the lens: 1H NMR spectroscopic evidence for specific crystallin-crystallin interactions. , 1994, Experimental eye research.

[28]  J. Carver,et al.  α-Crystallin: molecular chaperone and protein surfactant , 1994 .

[29]  T. Shearer,et al.  Cataract and the acceleration of calpain-induced beta-crystallin insolubilization occurring during normal maturation of rat lens. , 1994, Investigative ophthalmology & visual science.

[30]  G. S. Elliott,et al.  The role of the sequence extensions in beta-crystallin assembly. , 1994, Protein engineering.

[31]  S. Patterson.  From electrophoretically separated protein to identification: strategies for sequence and mass analysis. , 1994, Analytical biochemistry.

[32]  Kaili Wu,et al.  Analysis of normal human fetal eye lens crystallins by high‐performance liquid chromatography/mass spectrometry , 1995 .

[33]  Jean B. Smith,et al.  Glutathione adducts, not carbamylated lysines, are the major modification of lens α-crystallins from renal failure patients , 1995, Journal of protein chemistry.

[34]  J. Piatigorsky,et al.  Sequence and Expression of Chicken βA2- and βB3-crystallins , 1996 .