Pantethine inhibits the formation of high-T_c protein aggregates in γB crystallin solutions

Abstract

Purpose. Solutions of the bovine lens protein γB (or γII) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, γII_H. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (≈32% of total protein by weight) and loosely associated dimers (≈66%). γII_H has a phase separation temperature (T_ph), at least 40d`C higher than that of native γII crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of γII_H.

Methods. γII crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The T_ph of the solutions was monitored as a function of incubation time. Corresponding to each T_ph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of γII_H formed.

Results. Incubation of γII crystallin with increasing amounts of pantethine lowers T_ph and suppresses the formation of γII_H. With pantethine to protein mole ratios of 0.66, 1 and 2, the T_ph of γII crystallin is lowered from 8d`C in the native protein, to 2d`C, -3d`C and -4.3d`C respectively, at a protein concentration of ≈200 mg/ml. The amount of γII_H accumulated decreases from ∼ 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-trcatcd protein solutions. For complete suppression of the rise in T_ph and inhibition of γII_H formation, a 2:1 mole ratio of pantethine to protein is required.

Conclusions. We suggest that pantethine reacts with two cys-leine residues of γII crystallin by forming a mixed disulfide, and effectively suppresses protein aggregation and lowers T_ph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.

Key Words:

• gamma crystallin
• aggregation
• phase separation
• pantethine
• inhibition
• lens
• cataract
• bovine (cattle)