Abstract
Purpose: :
The objectives of the study were to determine: (1) an association of an Arg–bond hydrolyzing proteinase activity with recombinant human ßA3/A1–crystallin, and (2) localization of the active site in a specific region of the crystallin.
Methods: :
PCR–based truncations were performed to delete: (i) N–terminal 21 amino acids (named ßA3/A1 [21] mutant), (ii) N–terminal 22 amino acids (ßA3/A1 [22] mutant), (iii) N–terminal extension (ßA3/A1 [N] mutant), (iv) N–terminal extension plus motif I (ßA3/A1 [N+1] mutant), (v) N–terminal extension plus motif I and II (ßA3/A1 [N+I+II] mutant), (vi) N–terminal extension plus motif I, II and connecting peptide (ßA3/A1 [N+I+II+CP] mutant), (vii) Motif III and IV (ßA3/A1 [III+IV] mutant), (viii) Motif IV (ßA3/A1 [IV] mutant). The recombinant wild type (WT) ßA3/A1–crystallin and individual mutant proteins were purified by Ni2+ affinity column chromatographic method. Individual proteins, following treatment with 0.1% sodium deoxycholate, were fractionated by HPLC using a size–exclusion TSK G–2500 PWXL column, and the column–eluted fractions were analyzed for ßA3/A1–crystallin and the proteinase activity using CBZ L–Arg p–nitroanilde (BAPNA) as a substrate.
Results: :
The affinity–purified recombinant WT ßA3/A1–crystallin, on incubation with BAPNA, showed almost no activity but exhibited about 5–10X higher activity following 0.1% sodium deoxycholate treatment and size–exclusion HPLC. Additionally, the proteinase activity peak and the crystallin peak (determined by absorbance at 280 nm and by SDS–PAGE analysis of the column fractions) showed co–elution. Similar co–elution analyses of proteinase activity and crystallin peak of different purified mutant proteins showed proteinase activity in the mutant proteins even after deletion of 21 or 22 N–terminal residues, motifs I, II and connecting peptide. However the enzyme activity was absent in mutant proteins with the deletion of motifs III and IV. The results suggested that the proteinase active site might be localized in the region forming motifs III and IV of ßA3/A1–crystallin.
Conclusions: :
An Arg–bond hydrolyzing activity was found to be associated with WT ßA3/A1–crystallin. Results of proteinase activity association with different deletion mutant proteins suggested that the enzyme active site might be localized in the region forming motifs III and IV.
Keywords: crystalline lens • protein modifications-post translational • cataract