The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation.

Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.

Crystallin distribution in Bruch's membrane-choroid complex from AMD and age-matched donor eyes.

Crystallins were consistently found in a recent proteomic analysis of drusen from age-related macular degeneration (AMD) donor eyes. Here we compare the distribution of several crystallins in drusen, Bruch's membrane and choroid from AMD and non-AMD age-matched control eyes. Immunohistochemistry and Western blots of tissue samples were performed using antibodies to alphaA- and alphaB-crystallins. Bruch's membrane, drusen and the subjacent choroidal connective tissue from AMD tissues showed greater immunoreactivity for alphaA- and alphaB-crystallins than were observed in normal age-matched control tissues. Western blots also demonstrated more intense alphaA- and alphaB-crystallin signals from AMD tissues than were present in age-matched controls. These data indicate that alphaA- and alphaB-crystallins accumulate in Bruch's membrane and choroidal connective tissues to a greater degree in AMD than in normal aging. These findings suggest that the accumulation of these small heat shock proteins at this critical interface below the RPE reflects a disease-related stress response manifested during the progression of AMD.

Characterization of alphaA-crystallin from high molecular weight aggregates in the normal human lens.

PURPOSE: Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare alphaA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization. METHODS: HMW and LMW alphaA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in alphaA-crystallin from HMW and LMW fractions. RESULTS: The chaperone activity of HMW alphaA-crystallin was lower than that of LMW alphaA-crystallin. Fluorescence spectra indicated that HMW alphaA-crystallin was more hydrophobic than that of LMW. Isomerization of normal alpha-linkage to abnormal beta-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW alphaA-crystallin compared with that of LMW alphaA-crystallin. The D/L ratio of beta-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW alphaA-crystallin was significantly lower than that of the LMW form. CONCLUSIONS: These results were qualitatively the same as those previously found in alphaA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of alphaA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.

Immunological detection of D-beta-aspartate-containing protein in lens-derived cell lines.

PURPOSE: Although the presence of biologically uncommon D-beta-aspartate (D-beta-Asp) in lens protein is thought to be related to aging, we recently found this isomer in lens alphaA-crystallin from human newborns. The objective of this study was to examine whether D-beta-Asp occurs in protein from lens-derived cell lines. METHODS: We examined the expression of D-beta-Asp-containing protein in the lens-derived cell lines alphaTN4-1 and N/N1003A, by western blot and immunoprecipitation analysis using a polyclonal antibody against Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr (peptide 3R), which corresponds to three repeats of positions 149-153 in human alphaA-crystallin. The anti-peptide 3R antibody, prepared in a previous study, is a useful tool for investigating D-beta-Asp-containing peptides. RESULTS: Western immunoblot and immunoprecipitation analysis showed that a 50 kDa protein in N/N1003A cells was strongly immunoreactive with the anti-peptide 3R antibody. Antibodies against alphaA- and alphaB-crystallin also stained this protein. On the other hand, the alphaTN4-1 cell line only expressed proteins of about 20 kDa, which also reacted to antibodies against alphaA-crystallin and alphaB-crystallin. CONCLUSIONS: The results indicate that the N/N1003A cell line expressed a 50 kDa D-beta-Asp-containing protein, which may share a common amino acid sequence with alphaA- and alphaB-crystallin.

Quantification of the isomerization of Asp residue in recombinant human alpha A-crystallin by reversed-phase HPLC.

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange.

ATP interaction with lens alpha-crystallins leading to enhanced chaperone activity is not yet well understood. One model for chaperone activity of small heat shock proteins proposes that ATP causes small heat shock proteins to release substrates, which are then renatured by other larger heat shock proteins. A similar role has been proposed for ATP in alpha-crystallin chaperone activity. To evaluate this model, ATP-induced structural changes of native human alpha-crystallin assemblies were determined by hydrogen-deuterium exchange. In these experiments, hydrogen-deuterium exchange, measured by mass spectrometry, gave direct evidence that ATP decreases the accessibility of amide hydrogens in multiple regions of both alphaA and alphaB. The surface encompassed by these regions is much larger than would be shielded by a single ATP, implying that multiple ATP molecules bind to each subunit and/or ATP causes a more compact alpha-crystallin structure. Such a conformational change could release a bound substrate. The regions most affected by ATP are near putative substrate binding regions of alphaA and alphaB and in the C-terminal extension of alphaB. The widespread decrease in hydrogen-deuterium exchange with particularly large decreases near substrate binding regions suggests that ATP releases substrates via both direct displacement and a global conformational change.