To test if Gg laforin exists in a dynamic monomer dimer state, we collected the fractions from the monomer peak, concentrated the fractions, and re loaded these fractions over the same column. Gg laforin eluted as a 36 kDa protein, and no dimer shoulder was present dur ing this second purification, suggesting that monomeric Gg laforin does not convert Calcitriol to a dimer. The protein content and purity of the Superdex 200 mono meric fraction was assessed by collecting fractions and analyzing them by SDS PAGE. Gg laforin purified via this multi step protocol migrated as a highly pure 36 kDa pro tein. Previous studies have shown that Hs laforin dimers are resistant to SDS denaturation to a small extent, but there was no indication from the gel that a Gg laforin dimer species was present.

To further define the size and oligomeric state of Gg laforin, the Superdex 200 purified Gg laforin protein was analyzed using dynamic light scattering. The hydrodynamic radius of the detected species corresponded to a 31. 6 14. 5 kDa protein, the approximate size of the monomeric Gg laforin. Cumulatively, these data demon strate that Gg laforin can be cleaved from the His6 SUMO fusion tag, monomeric Gg laforin can be resolved by size exclusion chromatography, and the monomers remain to serine within the DSP of Hs laforin inactivates the enzyme. We cloned and purified a corresponding Gg laforin C253S mutant, and as expected this mutant displayed no activity and was used as a negative control. Hs laforin is the only human phosphatase known to bind and dephosphorylate glycogen and amylopectin monomeric during subsequent chromatography steps.

Thus, Gg laforin behaves in a similar manner as previously reported for Hs laforin. Gg laforin monomer binds glucans The CBM of Hs laforin distinguishes this phosphatase from other protein tyrosine phosphatase superfamily members in that the CBM enables Hs laforin to bind carbohydrates. Gg laforin is predicted to possess a CBM due to the high similarity between Hs laforin and Gg laforin in this region. The CBM of Gg laforin is highly similar to the Hs laforin CBM and was previously shown to bind glycogen in vitro. Using agarose beads conjugated to the carbohydrate amylose, we in vestigated the glucan binding properties of Gg laforin. The Vaccinia H1 related phosphatase is a human phos phatase from the same DSP superfamily as laforin, but VHR lacks a CBM and is therefore unable to bind carbohy drates.

Hs laforin, Batimastat Gg laforin and VHR were each incu bated with amylose beads for 30 min at 4 C, the beads were then pelleted by centrifugation, the supernatant was re moved, and the beads were treated with SDS PAGE buffer to release the proteins bound to the beads. Subsequently, proteins in the supernatant were precipitated and resus pended in SDS PAGE buffer. Proteins in the supernatant and pellet fractions were separated by SDS PAGE and an alyzed by Western blotting.