Sequence Analysis of βA3, βB3, and βA4 Crystallins Completes the Identification of the Major Proteins in Young Human Lens

Abstract

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as ␣A, ␣B, ␤A1, ␤A3, ␤A4, ␤B1, ␤B2, ␤B3, ␥S, ␥C, and ␥D-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human ␤A1, ␤A3, ␤A4, and ␤B3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of ␤A3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.Elucidating the structure and function of crystallins, the major proteins of human lens, is important, because alterations in these proteins may contribute to cataract formation. The goal of our laboratories is to determine what changes occur in human crystallins during aging and cataract formation. To accomplish this goal it is first necessary to perform the following in young, normal human lenses: 1) determine which crystallin subunits are present in the young lens; 2) map the positions where these crystallins migrate on two-dimensional electrophoretic gels; and 3) deduce and confirm the amino acid sequences of these proteins.The sequences of human ␣A, ␣B, ␤B1, ␤B2, ␥S, ␥C, and ␥D-crystallins have already been determined and their presence in the human lens has been demonstrated (1-11). Furthermore, the positions where human ␣A, ␣B, ␤B1, ␤B2, and ␥S-crystallins migrate on two-dimensional gels have been determined (12-14). In the present study, we completed the identification of all major crystallins resolved by two-dimensional electrophoresis, and add ␤A1, ␤A3, ␤B3, and ␤A4 to the list of major ␤-crystallins in young, normal human lens. We also correct the sequence of human ␤A3, complete the sequence determination of human ␤B3, and for the first time, report the sequence of human ␤A4. These deduced amino acid sequences were confirmed by mass spectrometry. MATERIALS AND METHODS Identification of Major HumanCrystallins-The posterior poles of human eyes from organ donors 7-months of age or less were obtained from the Lions Eyebank of Oregon within 48 h post-mortem. Following decapsulation, the lenses were homogenized in 1.0 ml of 20 mM phosphate buffer (pH 7.0), and 0.1 mM EGTA. Water-soluble proteins were isolated by centrifugation at 10,000 ϫ g for 30 min. Protein content was assayed by the BCA assay (Pierce) according to the manufacturer's protocol using bovine serum albumin as a standard. Water-soluble fractions were then dried by vacuum centrifugation and stored at Ϫ70°C prior to electrophoretic or chromatographic separation.Two-dimensional electrophoresis of water-soluble lens proteins, transfer to polyvinylidene difluoride membranes, and direct sequence analysis were carried out as described previously (15). Non-equilibrium pH gradient...

Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q

“…The gene for CRYAA (CRYAA) is a member of the sHSP family of molecular chaperones and encodes B20% of the soluble protein present in the newborn human lens, 48 accounting for a substantial portion of its refractive power. 40 Previously, missense (R116C) and nonsense (W9X) mutations in CRYAA have been linked with autosomal dominant 15 and recessive 16 forms of nonsyndromic cataract, respectively.…”

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

“…Human cD-crystallin (cD-crys), one of the most abundant c-crystallins in the lens, 15 is comprised of 173 amino acids. As shown in Figure 1(a), the single domains of cD-crystallin, each consisting of two intercalated antiparallel b-sheet Greek key motifs, termed N-td 1 , N-td 2 , C-td 3 , and C-td 4 , are connected by a six residue linker peptide.…”

Genetic and epigenetic mechanisms of gene regulation during lens development

“…αA-crystallin mRNA appears first in the invaginating lens placode followed by uniform expression in the lens vesicle (Robinson and Overbeek, 1996). Dramatic upregulation of both αA-and αB-crystallin expression is associated with primary lens fiber cell differentiation although, in mammalian lenses, αA-crystallin represents from 20-40% of total lens crystallin while αB-crystallin is present at only 5% of total crystallins (Lampi et al, 1997;Ueda et al, 2002). In contrast, αB-crystallin is the most abundant crystallin found in the lens epithelium.…”