Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Abstract

The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes L-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ–βα sandwich fold homodimer that cleaves its own peptide backbone at the G167–T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167–T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure–activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity

“…Sarcosine, for instance, accelerated the cleavage, while its isostere -alanine did not, indicating that the position of the glycine amino group provides the interactions necessary for the cleavage reaction. Ammonium, -alanine, betaine, O-phosphorylethanolamine, glycinamide, bicarbonate, taurine and l-asparagine (the substrate) do not accelerate the autoproteolysis reaction (Li et al, 2016).…”

“…Several studies have indicated that different Class 2 asparaginases have different autoproteolysis rates and that in some cases the process can be accelerated by small-molecule activators, at least in the case of human and guinea pig asparaginases . The first such activator to be reported was glycine (Su et al, 2013), which accelerates autoprocessing in vitro and in vivo (Li et al, 2016 (a)-(c) Schematic representation of the mechanism of maturation of Class 2 asparaginases. The process is started by the nucleophilic attack of (101) Thr on the scissile bond (100) Gly- (101) Thr (black).…”

“…Amino acids similar to glycine, such as alanine or serine, seem to be too large to fit the binding site near the scissile (100) Gly- (101) Thr bond and do not accelerate autoproteolysis (Su et al, 2013). It has been suggested that the glycine activator is first bound to (104) Arg and only later near the scissile bond, as the (104) Arg/Gln mutant (R196Q in HsAIII) requires a very high glycine concentration to rescue the autoprocessing, while without glycine no autoprocessing of the HsAIII R196Q variant takes place (Li et al, 2016). Tests of other compounds revealed that sarcosine, carbamate and phosphate also promoted the cleavage, albeit much less efficiently.…”

New Structural Insights into the Function of the Catalytically Active Human Taspase1

“…Despite its proximity to the catalytic site, this portion of the protein had not been thought to be important for catalytic activity, perhaps because its mobility had prevented it from being observed in the crystal structures previously reported (10-14, 28-36). While it has been demonstrated that mutations within the catalytic site of type 2 asparaginases, including Taspase1 (14), hASRGL1 (37), glycosylasparaginase (38), and hASNase3 (39), and others, abolish or significantly diminish the catalytic activity of the proteins, an essential role for a segment like the Pro183-Asp233 helix of Taspase1 had not previously been considered. In addition to cp-Taspase1 α41-233/β , we have generated two shorter protein variants, the cp-Taspase1 α41-206/β containing half the helix (Pro183-Asp206), and the cp-Taspase1 α41-183/β , which lacks the entire fragment.…”

RNAi‑mediated downregulation of asparaginase‑like protein 1 inhibits growth and promotes apoptosis of human cervical cancer line SiHa

“…Therefore, identifying the novel biomarker for cervical cancer intervention is essential. The gene encoding ASRGL1, one part of the N-terminal nucleophile (Ntn) hydrolase group ( 24 ), occurs in duplicate, and the corresponding transcriptional activation was assessed in endometrial and breast cancers ( 16 , 18 ). As the final step in the degradation of cell surface glycoproteins, ASRGL1 can remove the carbohydrate side chains from asparagine ( 25 ), thereby controlling the signaling function from the cell surface, metabolism of tumor cells, and cell growth ( 11 ).…”