Uncoupling Intramolecular Processing and Substrate Hydrolysis in the N-Terminal Nucleophile Hydrolase hASRGL1 by Circular Permutation

Abstract

The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50 %), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like, hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side-chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.

Loss of ASRGL1 expression is an independent biomarker for disease-specific survival in endometrioid endometrial carcinoma

“…The human ASRGL1 protein contains 308 amino acids and is activated by autocleavage at amino acid 168 to form an alpha-chain and a betachain (Fig. 3A), which dimerize into a heterodimer [16]. ASRGL1 is relatively stable in its inactive full-length form [35], and can be detected on the Western blot as a band at 40 kDa.…”

“…Here we have used an antibody against the isoaspartyl peptidase/ L-asparaginase protein ASRGL1, an enzyme involved in the production of L-aspartate [15,16] to assess its prognostic value in EC on two large tissue microarray (TMA) cohorts, together consisting of over 480 EC tumor samples. In a systematic search for novel EC biomarkers using The Human Protein Atlas [17][18][19], ASRGL1 was identified as a candidate biomarker based on differential expression in EC.…”

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

“…This engineering design physically links the original N- and C-termini, and the protein is now expressed such that the active site nucleophile Thr168 is the new N-terminus once the initiator Met residue is cleaved during translation. 6 The cp-hASRGL1 construct therefore uncouples the intramolecular processing event needed for subsequent hydrolysis of substrate. The utility of this approach is evidenced by high expression yields and enzymatic activity that is comparable to that of fully activated wt-hASRGL1.…”

“…This strategy allows the characterization of active site variants such as hASRGL1-Thr168Ser that are incapable of intramolecular processing (even in the presence of high glycine concentrations). 3,6 …”

“…6 In this study, we exploited this property of the enzyme and developed a differential scanning fluorimetry (DSF) assay that can quantify individual hASRGL1 species formed during activation. This advance allowed observation for the first time of three distinct molecular complexes during maturation, namely, the unprocessed αβ – βα homodimer, a half-processed α / β – βα heterotrimer, and the fully processed α / β – β / α heterotetramer.…”