Studies on Enzymes Acting on Glycopeptides*

Makino, Mayumi; Kojima, Tsugio; Ohgushi, Tadayasu; Yamashina, Ikuo

doi:10.1093/oxfordjournals.jbchem.a128760

Cited by 84 publications

(43 citation statements)

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“…The only mammalian Ntn hydrolase whose crystallographic structure is known, aspartylglucosaminidase (glycosylasparaginase, N 4 -(␤-N-acetyl-D-glucosaminyl)-L-asparaginase; EC 3.5.1.26), is a lysosomal amidohydrolase that cleaves asparagine from the oligosaccharide as one of the final steps in the breakdown of glycoproteins (12). AGA is activated in the lumen of the endoplasmic reticulum (ER) from a single chain precursor by a cleavage that creates the 27-kDa pro-␣-and 17-kDa ␤-subunits and exposes the active site N-terminal threonine in the beginning of the ␤-subunit (13).…”

Section: N-terminal Nucleophile Hydrolases (Ntn Hydrolases)mentioning

confidence: 99%

Activation and Oligomerization of Aspartylglucosaminidase

Saarela¹,

Lainé²,

Tikkanen³

et al. 1998

Journal of Biological Chemistry

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Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His 124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr 206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His 204 -Asp 205 seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.

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Section: N-terminal Nucleophile Hydrolases (Ntn Hydrolases)mentioning

confidence: 99%

Activation and Oligomerization of Aspartylglucosaminidase

Saarela¹,

Lainé²,

Tikkanen³

et al. 1998

Journal of Biological Chemistry

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“…Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a lysosomal amidase that catalyzes one of the final steps in the ordered breakdown of glycoproteins (Makino et al, 1968;Tarentino et al, 1975). The enzyme cleaves the bond between Asn and N-acetylglucosamine and has a substrate specificity that requires both a free a-carboxyl and a-amino group on the Asn (Aronson and Kuranda, 1989).…”

Section: Introductionmentioning

confidence: 99%

Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

et al. 1993

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Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations have been identified in AGU patients, elucidation of the molecular pathogenesis of the disease has been hampered by the missing information on the cellular events resulting in the maturation and activation of the enzyme. Here we used the expression of in vitro mutagenized constructs of the AGA cDNA to define three specific proteolytic trimming steps resulting in mature AGA. Removal of the signal peptide is immediately followed by proteolytic cleavage of the precursor into two subunits and results in biologically active enzyme already in the endoplasmic reticulum. This early activation has not previously been described for lysosomal enzymes. The subsequent lysosomal trimming does not influence the enzymatic activity of AGA. It consists only of a single proteolytic cleavage which removes 10 amino acids from the C‐terminal end of the larger subunit, in contrast to the multistep lysosomal processing observed in several other hydrolases.

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“…Glycosylasparaginase is a member of the recently described structural superfamily of enzymes termed as N-terminal nucleophile (Ntn) hydrolases (7). The hydrolysis of ␤-aspartylglycosylamines catalyzed by glycosylasparaginase is initiated by the binding of the ␤-aspartyl moiety into the active site of the enzyme through its free ␣-amino and ␣-carboxyl groups (8,9). The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10,11).…”

mentioning

confidence: 99%

Glycosylasparaginase-catalyzed Synthesis and Hydrolysis of β-Aspartyl Peptides

Noronkoski¹,

Stoineva²,

Ivanov³

et al. 1998

Journal of Biological Chemistry

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␤-Aspartyl di-and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of ␤-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of ␤-aspartyl peptides by transferring the ␤-aspartyl moiety from other ␤-aspartyl peptides or ␤-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the ␤-aspartyl donor in the formation of ␤-aspartyl peptides. The data show that synthesis and degradation of ␤-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of ␤-aspartyl peptides.␤-Aspartyl and ␥-glutamyl peptides are normal constituents of mammalian urine (1, 2) and tissues (3). Although ␥-glutamyltransferase (EC 2.3.2.2, GGT) (1) 1 is the key enzyme in the synthesis and hydrolysis of ␥-glutamyl compounds such as glutathione in the ␥-glutamyl cycle (4), little is known about the metabolism of ␤-aspartyl peptides.Glycosylasparaginase (GA; aspartylglucosaminidase;asparagine; ␤-aspartylglucosamine; GlcNAc-Asn) during degradation of glycoproteins. Genetic deficiency of glycosylasparaginase causes a lysosomal storage disease aspartylglycosaminuria (McKusick 208400) that is the most common disorder of glycoprotein degradation in humans and is clinically characterized by severe mental and motor retardation (5, 6).Glycosylasparaginase is a member of the recently described structural superfamily of enzymes termed as N-terminal nucleophile (Ntn) hydrolases (7). The hydrolysis of ␤-aspartylglycosylamines catalyzed by glycosylasparaginase is initiated by the binding of the ␤-aspartyl moiety into the active site of the enzyme through its free ␣-amino and ␣-carboxyl groups (8, 9). The enzyme uses the ␥-hydroxyl and ␣-amino group of its ␤-chain N-terminal threonine as an active site nucleophile and general base in the formation of ␤-aspartyl enzyme, which is subsequently deacylated by water to L-aspartic acid (10, 11). The GA-catalyzed hydrolysis of L-asparagine occurs in a similar manner, resulting in the formation of ␤-aspartyl enzyme and ammonia (12). The mechanism of action of glycosylasparaginase and the structural properties of its substrate-binding site (13) led us to consider that the enzyme might have a role in the metabolism of ␤-aspartyl peptides. In the present study, we demonstrate that synthesis and degradation of ␤-aspartyl peptides are new significant functions of glycosylasparaginase. This suggests that glycosylasparaginase could have an important role in the metabolism of ␤-aspartyl peptides present in body fluids and tissues. EXPERIMENTAL PROCEDURESMaterials-GA was purified to homogeneity from an NIH-3T3 cell line overexpre...

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Studies on Enzymes Acting on Glycopeptides*

Cited by 84 publications

References 0 publications

Activation and Oligomerization of Aspartylglucosaminidase

Activation and Oligomerization of Aspartylglucosaminidase

Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

Glycosylasparaginase-catalyzed Synthesis and Hydrolysis of β-Aspartyl Peptides

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