Abstract:
:Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant γS-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asn143 deamidated (2N-HGS) and the other with two additional Gln residues (92 and 120) deamidated (2N2Q-HGS). Isoelectric focusing measurements showed the expected lowering of the pI from 6.9 in HGS to ∼6.5 in 2N-HGS and to ∼6.1 in 2N2Q-HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q-HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein-protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2Q-HGS of 8.90 × 10(-4), 7.10 × 10(-4), and 6.65 × 10(-4) mL mol g(-2), respectively). Further substantiation is provided by estimates of the excess binding energy of protein-protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid-liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein-protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Pande A,Mokhor N,Pande Jdoi
10.1021/acs.biochem.5b00185subject
Has Abstractpub_date
2015-08-11 00:00:00pages
4890-9issue
31eissn
0006-2960issn
1520-4995journal_volume
54pub_type
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