|Molecular Vision 2000;
Received 26 July 2000 | Accepted 30 August 2000 | Published 5 September 2000
Increased deamidation of asparagine during human senile cataractogenesis
Larry Takemoto, Daniel Boyle
Division of Biology, Kansas State University, Manhattan, KS
Correspondence to: Larry Takemoto, Ph.D., Division of Biology, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-6811; FAX: (785) 532-6799; email: firstname.lastname@example.org
Purpose: To identify and quantitate deamidation of a specific asparagine residue of gS-crystallin that preferentially undergoes deamidation during the process of human senile cataractogenesis.
Methods: Reverse phase chromatography, together with synthetic peptide standards, was used to resolve the amidated and deamidated forms of asparagine-143 in the gS-crystallin sequence 131-145 from total tryptic digests of the central, nuclear region of human cataractous and normal lenses. Identities of the resolved peptides co-eluting with synthetic peptide standards were confirmed by mass spectral analysis. The synthetic peptide standards were also used to quantitate the amount of deamidation occurring in individual cataractous and normal lenses.
Results: In all lenses analyzed, there was greater deamidation of asparagine-143 in cataractous lenses, as compared with age-matched normal lenses.
Conclusions: The results demonstrate, for the first time, that increased deamidation of a specific asparagine residue is present in proteins from the human cataractous lens.
Probably the most abundant post-translational modification in biological tissue involves the nonenzymatic deamidation of asparagine and glutamine to form aspartate and glutamate residues, respectively . The rate at which this process occurs varies widely, depending upon the specific asparagine and glutamate residue of the protein. Previous studies have demonstrated that both the primary amino acid sequence, as well as the three-dimensional structure of the protein, can be determinants of the deamidation rate . Because deamidation is accompanied by the introduction of a negative charge, it has been hypothesized that this process may have profound effects upon the structural and functional properties of the protein that is being modified [1,3]. For some proteins, deamidation may be a necessary process, serving as a "Biological Clock" to facilitate the rapid turnover of biologically important components of the cell . Alternatively, during the aging process, nonenzymatic deamidation may have a deleterious effect upon the structural and functional properties of biologically important proteins. Therefore, it is possible that nonenzymatic deamidation may play an important role in the biogenesis of many human disorders that are associated with aged tissue.
Among the most prevalent age-related disorders is senile cataractogenesis of the lens. It occurs in patients of approximately 60 years or older, resulting in loss of transparency, especially in the older, central region of the lens. It has been hypothesized that a contributory cause of this opacification process involves changes in the structural properties of the so-called a-, b-, and g-crystallins, that comprise almost all of the dry weight material of the lens. Since there is almost no protein synthesis in this part of the lens during the lifetime of the patient, the crystallins might be subjected to age-related protein modifications for many decades, such as nonenzymatic deamidation of asparagine and glutamine residues.
Previous studies of proteins from the normal human lens have indeed shown that deamidation of specific asparagine and glutamine residues does occur during the aging process [5,6]. A central question to be addressed is whether increased deamidation of these residues and/or deamidation of additional residues is present in proteins from the cataractous lens. Such post-translational modifications may have profound effects upon the structural properties of crystallins which may, in turn, affect the transparent properties of the lens. In the following report, reverse phase chromatography, together with the use of synthetic peptide standards and mass spectral analysis, has been used to identify and quantitate the extent of deamidation of asparagine-143 of gS-crystallin from the central region of human transparent and cataractous lenses. The results demonstrate, for the first time, that increased deamidation of a specific asparagine residue has occurred during the process of human senile cataractogenesis.
Cataractous lenses, containing opacifications in the central nuclear region, were obtained after intracapsular extraction in India. All lenses were classified according to CCRG protocols . Normal lenses were obtained from the National Disease Research Interchange (Philadelphia, PA). In all cases, informed consent was received, and the principle of the Declaration of Helsinki was followed. Lenses were stored at -75 °C until use. The thawed lenses were decapsulated, and the outer cortical material was removed with a spatula. The remaining nuclear material was dissolved anaerobically in 2.0 ml of solution containing 7 M guanidine hydrochloride, 0.3 M Tris, 10 mM EDTA, pH 8.6, then reduced and carboxymethylated as previously described . Following dialysis against distilled water, protein was lyophilized, and 1.5 mg of protein was digested with a total of 3.0 ml of buffer containing 12.5 mg of sequencing grade trypsin (Boehringer Mannheim, Indianapolis, IN), 0.01% (w/v) sodium azide, 0.1 M Tris hydrochloride, pH 7.4, at 37 °C for 20 h. Protein was determined according to Bradford , using bovine serum albumin as standard. Approximately 3% of the tryptic digest was resolved using a 4.6 x 250 mm C18 column (Vydac, Hesperia, CA), using a linear gradient containing 30-40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid, over a period of 60 min, at a flow rate of 1 ml/min.
Synthetic peptides corresponding to the expected tryptic fragment of gS-crystallin corresponding to the amidated (VLEGVWIFYELPNYR) or deamidated form (VLEGVWIFYELPDYR) of sequence 131-145 were synthesized by Research Genetics (Huntsville, AL) using F-Moc chemistry. The synthetic peptides were purified by reverse phase chromatography and quantitated by amino acid analysis. Analysis of peptides by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI) was done at the Department of Chemistry of Kansas State University, using an IonSpec HiRes mass spectrometer (Irvine, CA). Monoisotopic molecular weights of most abundance were calculated using GPMAW software from WindowChem (Fairfield, CA).
Since an earlier study suggested that asparagine-143 of gS-crystallin might be deamidated during cataract formation , peptide standards corresponding to the expected tryptic fragment 131-145 that includes this residue were synthesized and purified. Table 1 shows the molecular weights of the purified synthetic peptides as determined by mass spectrometry. Within experimental error (0.002%), the observed molecular weights correlate with the calculated molecular weights, demonstrating that the purified synthetic peptides do indeed correspond to the amidated and deamidated forms of sequence 131-145 from gS crystallin.
Together with HPLC, these purified peptides were then used as markers to develop the optimal acetonitrile gradient for separation of the two forms from a tryptic digest of total lens proteins. Figure 1 shows that these two forms can be separated using a C18 reverse phase column, with a linear gradient of 30-40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid, over a period of 60 min. This gradient was subsequently used to resolve peptides from a tryptic digest of total proteins from the central, nuclear region of cataractous and normal lenses. Figure 2A shows that two peaks (Peak 1 and Peak 2, solid and open arrows, respectively), eluting at the same time as the purified standards, were obtained from the tryptic digest of the nuclear region of a cataractous lens from a 60 yr old patient. Mass spectral analysis (Figure 3 and Table 1) showed that each peak was comprised of a single component of molecular weight 1,898.003 for Peak 1, and 1,899.001 for Peak 2. Based upon the same elution times as compared with peptide standards, and upon their observed molecular weights, Peak 1 must correspond to the amidated form of gS-sequence 131-145, and Peak 2 must correspond to the deamidate form of the same sequence. As shown in Figure 2A, Peak 2 contained a shoulder that eluted slightly before the main peak. Different acetonitrile gradients as well as different ion-pairing agents in the acetonitrile were unsuccessful in resolving the shoulder from the main peak (results not shown), suggesting that the two components were very similar in sequence. Based upon a single molecular weight of 1,899.001 for Peak 2 (including its shoulder), and upon previous studies of asparagine deamidation, the peak and its shoulder probably contain different forms of the deamidated asparagine residue, present as D- or L- isomers of the aspartate or isoaspartate residue .
The nuclear region of a normal lens from a 64 yr old donor was analyzed in an identical manner. Figure 2B shows that the amidated form of tryptic peptide 131-145 (Peak 1) was the predominant species, with a very small amount of material (<10%) eluting at the expected time of the deamidated form of the peptide (Peak 2). Taken together, the results of Figure 2 suggest that relative to the normal human lens, a larger percentage of asparagine-143 from gS-crystallin has undergone increased deamidation in the cataractous lens.
To verify this conclusion, four other cataractous lenses and four other normal lenses in the same approximate age range were analyzed in an identical manner as shown in Figure 2. Known amounts of the purified synthetic peptides were used as standards to quantitate the amount of asparagine-143 deamidation for each lens. Table 2 shows that all normal lenses had low (<10%) levels of deamidation, while cataractous lenses had levels of asparagine-143 deamidation ranging from 29.6-74.4 percent, clearly showing that increased deamidation of this residue has occurred in cataractous human lenses. Since 4 out of the 5 lenses analyzed contained a fully opaque nucleus with a CCRG classification of N=4 , it was impossible to make a correlation between the degree of opacification and percent deamidation of asparagine-143.
Since almost all the dry weight material of the human lens is comprised of proteins that have very low rates of turnover in vivo over many decades, it is natural to assume that these polypeptides undergo extensive post-translational modifications during the lifetime of the individual. This is especially true in the central, nuclear region that contains the oldest cells. In this regard, oxidation of cysteine residues to cystine disulfide groups in this region of the lens has already been shown to occur during cataract formation . Therefore. it is possible that other post-translational modifications may also occur during the opacification process of the human nuclear cataract.
Although deamidation is probably the most common post-translational modification in lens proteins, it has been difficult to identify and quantitate deamidation of individual asparagine and glutamine residues present in proteins from the human cataractous lens. Much of the difficulty arises from the large number of asparagine and glutamine residues found in lens proteins. In addition, deamidation results in a molecular weight change of only one atomic mass unit, making it difficult to identify and quantitate the modification by many techniques based upon molecular weight changes. To circumvent these problems, we have developed methodology that uses reverse phase chromatography and synthetic peptide standards to quantitate tryptic peptides that contain the amidated and deamidated forms of a particular asparagine or glutamine residue.
Using this experimental approach, it was possible to show that deamidation of specific asparagine and glutamine residues of a-A crystallin did not change in proteins from human cataracts .
In the present report, quantitation of asparagine-143 from gS-crystallin was stimulated by an earlier study that suggested increased deamidation of this residue during cataract formation . This earlier study used a solid-phase radioimmunoassay to show increased amounts of the deamidated sequences EL(I)PDYR and L(I)FYEL(I)PDYR present in digests from cataractous lenses as compared with normal lenses. It was impossible, however, to definitively assign these sequences to a specific lens protein. Since the amidated form of the peptide was not detected by radioimmunoassay, it was also impossible to quantitate the degree of deamidation of this sequence. In the present study, a direct approach involving resolution and quantitation of both the amidated and deamidated forms of the tryptic peptide was used. In addition, since the sequences of all major lens proteins are now known, it was possible to assign the site of deamidation to asparagine-143 of gS-crystallin.
The results of the analyses demonstrate that, relative to normal lenses of the same age range, asparagine-143 of gS-crystallin from cataractous lenses has undergone increased deamidation. This is the first report to show that, in the aged human lens, deamidation is simply not an age-related phenomenon, but rather may be accelerated during the lens opacification process. Analyses of protein deamidation, both in vivo and in vitro, have demonstrated that deamidation is not a random process occurring equally among all asparagine residues of a protein [1,2]. Both primary sequence , as well as secondary structure , influence the rate of this process. Age-dependent deamidation is, therefore, a selective process that may be accelerated in only certain asparagine residues. Consistent with this observation is the previous report that specific asparagine and glutamine residues of aA-crystallin did not undergo increased deamidation during human senile cataractogenesis . The increased deamidation of asparagine-143 of gS-crystallin present in human cataracts may, therefore, reflect specific changes in protein structure or environment that accelerate this post-translational modification.
Although the exact role of deamidation during human cataractogenesis is presently not known, the introduction of a negative charge during this process may have significant effects upon the structural properties of gS-crystallin. Based upon sequence homology and known structure of g- and b-crystallins from other species, Zarina et al.  have modeled a structure for human gS-crystallin. Based upon this structure, asparagine-143 would be exposed on the surface of the molecule. Since it has been hypothesized that short-range interactions of lens crystallins are necessary to maintain lens transparency , possible changes in the surface properties of this major lens crystallin resulting from deamidation may perturb protein-protein interactions, resulting in changes in the transparent properties of the lens. In addition, introduction of a negative charge may induce alterations in the overall structural properties of gS-crystallin, changing its ability to associate with other crystallins, resulting in the formation of high molecular weight aggregates that are known to increase during cataract formation. These aggregates may by themselves scatter enough light to cause opacification . Future studies, now in progress, will be directed towards the quantitation of asparagine-143 deamidation found in these aggregates. In addition, the current methodology can be used to identify and quantitate possible deamidation of other asparagine and glutamine residues of gS-crystallin, to obtain a compete picture of the deamidation process that has occurred in this molecule from the cataractous and normal human lens.
The authors wish to acknowledge the help of P. Buhr for technical assistance and K. Wyatt and E. Sparks for the preparation of the figure and tables. Cataractous lenses were provided by Dr. V. Reddy, supported from a grant by the NEI.
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