αA 66-80 Peptide Accumulates in Aged Lenses Due to Enzymatic Hydrolysis of αA-Crystallin

Abstract

Purpose: : Peptide αA 66-80 and its truncated forms viz., 66-75, 67-80 and 67-75 are found to accumulate in human aged and cataract lenses. The objective of this study was to find out the role of lens enzymes in generation of αA 66-80 peptide and its truncated forms in aged lenses.

Methods: : Bovine lens water soluble extract was fractionated on a Superdex 200 gel filtration column. The fractions were incubated with internally quenched peptide MCA-SEVRSDRD[Lys(Dnp)]RR (αA 62-70) at 37^oC for 30 min and the hydrolysis of the substrate was analyzed by measuring fluorescent intensity at 395nm in micro plate reader. Two synthetic peptides, 100µg [succinyl-ISEVRSDRDK-Biotin (αA 61-70) and succinyl-DVKHFSPEDK-Biotin (αA 76-85)], cleavage of which would indicate the mechanism of αA 66-80 generation in vivo, were incubated with the enzyme rich fractions of bovine lens extract (5mg protein) for 8h at 37^oC. The hydrolyzed peptides were subjected to RP-HPLC and LC-MS analysis to identify the fragments. To determine the mechanistic class of protease(s) involved in the hydrolysis, αA 62-70 peptide was used. Different inhibitors belonging to serine, cysteine and metalloprotease were tested.

Results: : Enzyme(s) hydrolyzing the above synthetic peptides co-eluted with β_L crystallin. The RP-HPLC analysis of lens enzyme hydrolyzed αA 61-70 peptide, showed 16% hydrolysis after 8h of incubation at 37^oC. The LC-MS results of the hydrolyzed peptide, showed the peaks corresponding to succinyl-61-65 and succinyl-61-66 with nearly equal intensity. The αA 76-85 peptide was 6 times more susceptible to hydrolysis than 61-70 peptide. Peaks corresponding to succinyl-76-78, succinyl-76-80 and succinyl-76-82 were identified in LC-MS analysis. Succinyl-76-82 fragment was most abundant followed by succinyl-76-78 and succinyl-76-80. Metalloprotease inhibitor, 1,10 Phenanthroline (1mM), completely inhibited the hydrolysis of internally quenched substrate αA 62-70 peptide.

Conclusions: : The present investigation reveals that the αA 66-80 and its truncated forms accumulate in aged lens because of enzymatic hydrolysis of αA-crystallin. A metalloprotease might be responsible for this hydrolysis.

Keywords: crystallins • cataract • aging