Human βB1-crystallin is partially degraded at its N-terminal extension during lens maturation

Purpose. To determine the sequence of human βB1-crystallin cDNA; confirm the deduced amino acid sequence of the protein by mass spectrometric analysis, and analyze both water-soluble and water-insoluble fractions of human lens for partially degraded forms of βB1. Methods. Human lens βB1-crystallin cDNA was amplified using 5′ and 3′ RACE PCR, resulting products cloned, and the cDNA sequence determined. Intact and partially degraded forms of βB1-crystallin were isolated from donors ranging in age from newborn to 27 years using either two-dimensional electrophoresis or gel filtration. Whole or trypsinized proteins were then separated by reverse phase chromatography and analyzed by either fast atom bombardment mass spectrometry or electrospray ionization mass spectrometry. Results. Human βB1-crystallin cDNA encoded a protein of 251 amino acids in length. The deduced amino acid sequence was confimed by mass analysis of whole βB1 and tryptic fragments of βB1. The intact protein contained no N-terminal methionine and appeared acetylated (measured mass of 27,941 Da). Newborn lenses also contained a partially degraded form of βB1 missing 15 residues from its N-terminus. Further analysis of lens proteins from donors up to 27 years of age suggested that a progressive shortening of the N-terminal extension of βB1 occurs in both water-soluble and water-insoluble fractions. Additional forms of βB1 missing 33, 34, 35, 36, 39, 40, and 41 amino acid residues were detected. Conclusions. The appearance of partially degraded forms of βB1 quite early in life suggested that this crystallin is uniquely susceptible to proteolysis, and that the cleavage may play an important role in lens maturation.