The human asparaginase-like protein 1 hASRGL1 is an Ntn hydrolase with beta-aspartyl peptidase activity

Abstract

Herein we report the bacterial expression, purification, and enzymatic characterization of the human asparaginase-like protein 1 (hASRGL1). We present evidence that hASRGL1 exhibits beta-aspartyl peptidase activity consistent with enzymes designated as plant-type asparaginases, which had thus far been found in only plants and bacteria. Similar to nonmammalian plant-type asparaginases, hASRGL1 is shown to be an Ntn hydrolase for which Thr168 serves as the essential N-terminal nucleophile for intramolecular processing and catalysis, corroborated in part by abolishment of both activities through the Thr168Ala point mutation. In light of the activity profile reported here, ASRGL1s may act synergistically with protein l-isoaspartyl methyl transferase to relieve accumulation of potentially toxic isoaspartyl peptides in mammalian brain and other tissues.

Fig 1

Western blot analysis of BL21(DE3) cells expressing human ASRGL1 (hASRGL1) or hASRGL1-T168A. Samples corresponding to an equal number of cells were loaded in each lane. S, soluble whole cell lysate fraction; I, insoluble whole cell lysate fraction.

Fig 2

SDS-PAGE of human ASRGL1 (hASRGL1) and its Thr168Ala point mutant (hASRGL1-T168A) following in vitro incubation at 37°C over time. Samples corresponding to an equivalent mass of total enzyme were loaded in each lane.

Fig 3

Sequence alignment of human asparaginase-like protein 1 (hASRGL1) with plant-type asparaginases generated using ClustalW2 () and JalView () . The asterisk indicates the conserved autoproteolytic cleavage site and corresponds to residue Thr168 for the hASRGL1 sequence. Residue conservation across the alignment is denoted by the degree of shading. The sequences of the plant-type asparaginases correspond to the following UniProtKB numbers: E. coli (P37595); L. Luteus (Q9ZSD6); Ana.7120 (Q8YQB1); Syn.6803 (P74383).

Fig 4

Progression of intramolecular processing of human ASRGL1 (hASRGL1) following in vitro incubation at 37°C as determined by relative AspAMC hydrolysis rate over time. At various time points, equivalent aliquots of enzyme were withdrawn and analyzed using the fluorometric AspAMC activity assay as described under “Experimental Procedures” with the final time point serving as the maximum rate observed. The intramolecular processing reaction as evaluated through this approach exhibited t_1/2 = 20 ± 3h to maximum level of observed in vitro processing.

Fig 5

Predicted human ASRGL1 (hASRGL1) active site overlaid against the human aspartylglucosaminidase (AGA) active site. A model of hASRGL1 structure based on E. coli isoaspartyl aminopeptidase/L-asparaginase (EcAIII) was obtained from Phyre () [Job code: 45ae21e95bf1f71f, SCOP code c2zakA, E-value 7.5e-41, Identity 35%, Estimated precision 100%] and aligned to human AGA (PDB: 1APZ; with bound l-aspartate) using PyMol (). Bound l-aspartate is shown as spheres. Amino acids conserved between the two structures are colored by CPK and are numbered by hASRGL1 sequence (asterisk indicates the nucleophilic Thr). Amino acids shaded green are specific to human AGA and those shaded orange are specific to hASRGL1. For additional detail, please refer to the “Discussion” section.