Purification and Characterisation of Ovine Lp82 and Its Role in Cataractogenesis

Abstract

Purpose:: To purify and characterise the lens specific calpain, Lp82 and determine its ability to modify ovine crystallins and the role Lp82 plays in cataract formation in the heritable ovine cataract.

Methods:: Lenses were obtained post-mortem from lambs up to the age of 7 weeks old. Lenses were homogenised, then centrifuged to remove insoluble protein. Total soluble protein was loaded on to a DEAE ion exchange column. Lp82 was eluted with an increasing NaCl concentration. The fractions containing Lp82 were pooled and concentrated by ultrafiltration. The Lp82 was then run through a Superose 6 Gel Filtration column and Lp82 containing fractions were pooled and concentrated by ultrafiltration. The final step was purification on a Reactive Red Sepharose Column. The activity of the Lp82 was assessed using BODIPY-casein assay. BODIPY-casein assay was used to determine Ca²⁺ concentration for ½ maximal activity and optimal pH for Lp82. The purified Lp82 was incubated with purified ovine α and ß crystallins in the presence of Ca²⁺ at 37ºC. Lp82 induced changes in crystallins were visualised using two dimensional electrophoresis (2DE). The Lp82 induced changes were compared to the soluble fraction of cataract lenses using 2DE. Ovine Lp82 mRNA was transcribed to cDNA, amplified and sequenced.

Results:: This procedure resulted in a purified Lp82 that could be used for subsequent studies. The Ca²⁺ concentration required for ½ maximal activity of Lp82 was 200µM which is similar to that of the ubiquitous ovine calpain II. Incubation of both α and ß crystallins with purified Lp82 and Ca²⁺ at 37ºC resulted in truncation of both α and ß crystallins. The truncations produced buy incubation with Lp82 were similar to some of those found in the soluble fraction of a cataractous ovine lens when compared using 2DE. The sequence of ovine Lp82 was similar to Lp82 in other species.

Conclusions:: These experiments are the first to purify and characterise Lp82 from the ovine lens. They show that Lp82 in the ovine lens may play a role in the truncation of crystallins found in ovine cataractous lenses.

Keywords: crystallins • proteolysis • cataract