Structural and functional roles of deamidation of N146 and/or truncation of NH2- or COOH-termini in human αB-crystallin

Abstract

Purpose: The purpose of the study was to determine the relative effects of deamidation and/or truncation on the structural and functional properties of αB-crystallin.

Methods: Using wild-type (WT) αB-crystallin and the αB deamidated mutant (i.e., αB N146D), we generated NH(2)-terminal domain deleted (residues no. 1-66; αB-NT), deamidated plus NH(2)-terminal domain deleted (αB N146D-NT), COOH-terminal extension deleted (residues no. 151-175; αB-CT), and deamidated plus COOH-terminal extension deleted (αB N146D-CT) mutants. All of the proteins were purified and their structural and functional (chaperone activity with insulin as target protein) properties were determined and compared to WT αB-crystallin.

Results: The desired deletions in the αB-crystallin mutants were confirmed by DNA sequencing and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. The homomers of αB-CT and its deamidated form (αB N146D-CT) became water insoluble, whereas the αB N146D, αB-NT, and αB N146D-NT species remained water-soluble. CD spectroscopic studies revealed that the mutants with deletion of NH(2)- or COOH-termini or deamidation showed increased β-sheet and decreased α-helical contents with the exception of αB N146D-CT, which showed a substantial increase in α-helix and decrease in β-sheet content. Results of intrinsic Trp fluorescence suggested little change in Trp microenvironment of αB N146D relative to WT αB, but substantial alterations on deletion of COOH-terminal extension or a combination of this deletion plus deamidation. Hydrophobic binding studies using the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) showed that, relative to WT αB structure, the N146 deamidation, COOH-terminal extension deletion or a combination of this deamidation and deletion resulted in a relatively compact structure whereas the NH(2)-terminal domain deletion and a combination of this deletion plus deamidation resulted in a relaxed structure. All the αB mutants showed higher molecular mass ranging between 1.2×10(6) to 5.4×10(6) Da, relative to WT αB which had a molecular mass of 5.8×10(5) Da. Chaperone activity across all αB species decreased in the following order: WTαB > αB N146D-CT > αB N146D-NT > αB-NT > αB-CT > αB N146D. Specifically, substantial losses in chaperone activity (only 10% to 20% protection) were seen in αB N146D, αB-NT, and αB-CT. However, in the species with the combination of deamidation plus NH(2)- or COOH-terminal deletion, the percent protection was about 24% in αB N146D-NT and about 40% in αB N146D-CT.

Conclusions: Although all mutants formed oligomers even after deamidation, on deletion of either NH(2)-terminal domain or COOH-terminal extension or a combination of these deletions and deamidation, their structural properties were substantially altered. The results suggested that the NH(2)-terminal domain is relatively more important than the COOH-terminal extension for the chaperone function of αB. The non-deamidated N146 residue, NH(2)-terminal domain and COOH-terminal extension are also of critical importance to the maintenance of αB-crystallin chaperone activity.

Figure 1

Schematic diagram showing the regions and residue numbers of the NH₂-terminal domain, α-crystallin domain, COOH-terminal extension and deamidation sites in WT αB-crystallin and its mutants. WT αB-crystallin is a full-length protein containing all residues from 1 to 175. Residue N146 was deamidated (i.e., N to D) in the deamidated mutants. NH₂-terminal domain deleted mutants (αB-NT and αB N146D-NT) are missing residues no. 1–66, while COOH-terminal extension deleted mutants (αB-CT and αB N146D-CT) are missing residues no. 151–175.

Figure 2

SDS–PAGE analysis of purified, His-tagged WT αB-crystallin and its deamidated, NH₂- or COOH-terminally deleted mutants, and deamidated plus deleted mutants, following Ni²⁺-affinity column purification (see Methods). Lane 1 – molecular weight marker; Lane 2 – WT αB-crystallin; Lane 3 – αB N146D; Lane 4 – αB-NT; Lane 5 – αB N146D-NT; Lane 6 – αB-CT; αB N146D-CT.

Figure 3

Far-UV CD spectra of WT αB-crystallin and its mutant proteins. Spectra were recorded using protein preparations of 0.2 mg/ml, dissolved in 50 mM sodium phosphate buffer (pH 7.8), and a cell path length of 0.5 mm. The reported spectra are the average of 5 scans corrected for the buffer blank and smoothed. A: WT αB-crystallin and deamidated mutant. B: WT αB-crystallin and NH₂-terminal domain deleted mutants. C: WT αB-crystallin and COOH-terminal extension deleted mutants.

Figure 4

Intrinsic Trp and total fluorescence spectra of WT αB-crystallin and its mutant proteins. A: Total fluorescence spectra (Ex 290 nm, Em 300–400 nm) were recorded for the NH₂-terminal domain deleted mutants because Trp residues 9 and 60 were deleted along with the deletion of this domain. B: Intrinsic Trp fluorescence spectra (Ex 295 nm, Em 300–400 nm) were recorded for mutants containing the NH₂-terminal domain. The dotted lines indicate the wavelength of maximum peak fluorescence (λ_max) observed in WT αB-crystallin, used to determine whether a blue or red shift in wavelength occurred.

Figure 5

Fluorescence spectra of WT αB-crystallin and its mutants following ANS binding. Spectra were recorded by excitation at 390 nm and emission from 400 to 600 nm using 0.2 mg/ml protein preparations mixed with 15 μl of 0.8 mM ANS (dissolved in methanol) and incubated at 37 °C for 15 min. A: WT αB-crystallin and its deamidated and NH₂-terminal domain deleted mutants. B: WT αB-crystallin and its deamidated and COOH-terminal extension deleted mutants. The dotted lines indicated the wavelength of maximum peak fluorescence (λ_max) observed in WT αB-crystallin, used to determine whether a blue or a red shift in wavelength occurred.

Figure 6

Comparison of chaperone activity of WT αB-crystallin and its mutant proteins. The chaperone activity, calculated as % protection, was assayed by measuring DTT-induced insulin (100 μg) aggregation in the presence of a chaperone/insulin ration (1:1) at 25 °C. Error bars=Percent Error (±1%).