Crystal structure of a mutant glycosylasparaginase shedding light on aspartylglycosaminuria-causing mechanism as well as on hydrolysis of non-chitobiose substrate

Abstract

Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in lysosomes of cells, resulting in a genetic condition called aspartylglycosaminuria (AGU). To better understand the mechanism of a disease-causing mutation with a single residue change from a glycine to an aspartic acid, we generated a model mutant enzyme at the corresponding position (named G172D mutant). Here we report a 1.8Å resolution crystal structure of mature G172D mutant and analyzed the reason behind its low hydrolase activity. Comparison of mature G172D and wildtype GA models reveals that the presence of Asp 172 near the catalytic site affects substrate catabolism in mature G172D, making it less efficient in substrate processing. Also recent studies suggest that GA is capable of processing substrates that lack a chitobiose (Glycan, N-acetylchiobios, NAcGlc) moiety, by its exo-hydrolase activity. The mechanism for this type of catalysis is not yet clear. L-aspartic acid β-hydroxamate (β-AHA) is a non-chitobiose substrate that is known to interact with GA. To study the underlying mechanism of non-chitobiose substrate processing, we built a GA-β-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a β-AHA molecule. A hydrolysis mechanism of β-AHA by GA is proposed based on this complex model.

The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria

“…In contrast, a Flavobacterium system has become an excellent model system for these studies. The justifications to use the bacterial model include the following: (a) the Favobacterial GA shares the same αββα fold with the human enzyme; (b) Favobacterial and human GAs have a high sequence homology with an expect value of 1e‐48 in BLAST; (c) both enzymes utilize the same autoproteolytic process to cleave single‐chain precursors into mature amidases that allow glycoasparagine digestion; (d) although different in glycosylations, these two enzymes have an essentially identical 3D structure; (e) all the amino acids around the catalytic center, those within 4 Å of the bound substrate NAcGlc‐Asn, are 100% identical between these two homologs; (f) the bacterial homolog allows production of crystallographic amount and purity of AGU model enzymes to determine three‐dimensional structures and study their detailed kinetic parameters . Overall, these facts advocate suitability of using the Flavobacterium homolog to study the effects of AGU mutations on GA catalytic activities.…”

“…However, this essential dimerization is not sufficient to trigger the autoproteolytic activation of GA because subtle structural changes can alter the dimerization status and thus prevent autoproteolytic activation. Data suggest that many AGU mutations remain as dimers but cannot undergo autoproteolysis and thus lack amidase activity for digesting glycoasparagines . These results suggest that the precise monomer–monomer interactions of GA play essential roles in GA autoprocessing and autoactivation.…”

“…Although AGU and other GA mutations have long been known to impair autoprocessing of GA precursor and/or impair its amidase activity in lysosomes, the molecular details of AGU variants have only begun to be studied carefully in vivo and in vitro . These studies indicate that different AGU substitutions result in different impacts on GA autoactivation, hydrolysis of glycoasparagines, and/or thermal stability of the enzyme.…”

“…Model structures of two AGU variants have recently been reported. The first one corresponds to the Canadian G203D variant . The second model structure corresponds to the Finnish T234I variant .…”

“…The second model structure corresponds to the Finnish T234I variant . These structural studies revealed that the Canadian variant appears to cause local conformational change outside of the amidase substrate site, which in turn disrupts the requisite autoprocessing step, thus persisting as a metabolically nonfunctional precursor protein . On the other hand, the T234I Finnish variant appears to have a detectible level of autoprocessing capability to generate a mature amidase .…”