Tgm2 alleviates LPS-induced apoptosis by inhibiting JNK/BCL-2 signaling pathway through interacting with Aga in macrophages.

Sepsis is an unusual systemic infection caused by bacteria, which is a life-threatening organ dysfunction. The innate immune system plays an important role in this process; however, the specific mechanisms remain unclear. Using the LPS + treated mouse model, we found that the survival rate of Tgm2-/- mice was lower than that of the control group, while the inflammation was much higher. We further showed that Tgm2 suppressed apoptosis by inhibiting the JNK/BCL-2 signaling pathway. More importantly, Tgm2 interacted with Aga and regulated mitochondria-mediated apoptosis induced by LPS. Our findings elucidated a protective mechanism of Tgm2 during LPS stimulation and may provide a new reference target for the development of novel anti-infective drugs from the perspective of host immunity.

Knockout of the CMP-Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays.

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP-sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.

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

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single-chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5-Å-resolution structure of this new AGU model enzyme and built an enzyme-substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a "molecular clamp" capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Aspartylglycosaminuria: a review.

Aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease, is the most common disorder of glycoprotein degradation with a high prevalence in the Finnish population. It is a lifelong condition affecting on the patient's appearance, cognition, adaptive skills, physical growth, personality, body structure, and health. An infantile growth spurt and development of macrocephalia associated to hernias and respiratory infections are the key signs to an early identification of AGU. Progressive intellectual and physical disability is the main symptom leading to death usually before the age of 50 years.The disease is caused by the deficient activity of the lysosomal enzyme glycosylasparaginase (aspartylglucosaminidase, AGA), which leads to a disorder in the degradation of glycoasparagines - aspartylglucosamine or other glycoconjugates with an aspartylglucosamine moiety at their reducing end - and accumulation of these undegraded glycoasparagines in tissues and body fluids. A single nucleotide change in the AGA gene resulting in a cysteine to serine substitution (C163S) in the AGA enzyme protein causes the deficiency of the glycosylasparaginase activity in the Finnish population. Homozygosity for the single nucleotide change causing the C163S mutation is responsible for 98% of the AGU cases in Finland simplifying the carrier detection and prenatal diagnosis of the disorder in the Finnish population. A mouse strain, which completely lacks the Aga activity has been generated through targeted disruption of the Aga gene in embryonic stem cells. These Aga-deficient mice share most of the clinical, histopathologic and biochemical characteristics of human AGU disease. Treatment of AGU mice with recombinant AGA resulted in rapid correction of the pathophysiologic characteristics of AGU in non-neuronal tissues of the animals. The accumulation of aspartylglucosamine was reduced by up to 40% in the brain tissue of the animals depending on the age of the animals and the therapeutic protocol. Enzyme replacement trials on human AGU patients have not been reported so far. Allogenic stem cell transplantation has not proved effective in curing AGU.

Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1)--distinct characteristics in neurons.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCLs) are collectively the most common type of recessively inherited childhood encephalopathies. The most severe form of NCL, infantile neuronal ceroid lipofuscinosis (INCL), is caused by mutations in the CLN1 gene, resulting in a deficiency of the lysosomal enzyme, palmitoyl protein thioesterase 1 (PPT1). The deficiency of PPT1 causes a specific death of neocortical neurons by a mechanism, which is currently unclear. To understand the function of PPT1 in more detail, we have further analyzed the basic properties of the protein, especially focusing on possible differences in non-neuronal and neuronal cells. RESULTS: Our study shows that the N-glycosylation of N197 and N232, but not N212, is essential for PPT1's activity and intracellular transport. Deglycosylation of overexpressed PPT1 produced in neurons and fibroblasts demonstrates differentially modified PPT1 in different cell types. Furthermore, antibody internalization assays showed differences in PPT1 transport when compared with a thoroughly characterized lysosomal enzyme aspartylglucosaminidase (AGA), an important observation potentially influencing therapeutic strategies. PPT1 was also demonstrated to form oligomers by size-exclusion chromatography and co-immunoprecipitation assays. Finally, the consequences of disease mutations were analyzed in the perspective of our new results, suggesting that the mutations increase both the degree of glycosylation of PPT1 and its ability to form complexes. CONCLUSION: Our current study describes novel properties for PPT1. We observe differences in PPT1 processing and trafficking in neuronal and non-neuronal cells, and describe for the first time the ability of PPT1 to form complexes. Understanding the basic characteristics of PPT1 is fundamental in order to clarify the molecular pathogenesis behind neurodegeneration in INCL.

Autoproteolytic activation of human aspartylglucosaminidase.

Aspartylglucosaminidase (AGA) belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily characterized by an N-terminal nucleophile as the catalytic residue. Three-dimensional structures of the Ntn hydrolases reveal a common folding pattern and equivalent stereochemistry at the active site. The activation of the precursor polypeptide occurs autocatalytically, and for some amidohydrolases of prokaryotes, the precursor structure is known and activation mechanisms are suggested. In humans, the deficient AGA activity results in a lysosomal storage disease, aspartylglucosaminuria (AGU) resulting in progressive neurodegeneration. Most of the disease-causing mutations lead to defective molecular maturation of AGA, and, to understand the structure-function relationship better, in the present study, we have analysed the effects of targeted amino acid substitutions on the activation process of human AGA. We have evaluated the effect of the previously published mutations and, in addition, nine novel mutations were generated. We could identify one novel amino acid, Gly258, with an important structural role on the autocatalytic activation of human AGA, and present the molecular mechanism for the autoproteolytic activation of the eukaryotic enzyme. Based on the results of the present study, and by comparing the available information on the activation of the Ntn-hydrolases, the autocatalytic processes of the prokaryotic and eukaryotic enzymes share common features. First, the critical nucleophile functions both as the catalytic and autocatalytic residue; secondly, the side chain of this nucleophile is oriented towards the scissile peptide bond; thirdly, conformational strain exists in the precursor at the cleavage site; finally, water molecules are utilized in the activation process.

Aspartylglucosaminidase (AGA) is efficiently produced and endocytosed by glial cells: implication for the therapy of a lysosomal storage disorder.

BACKGROUND: Aspartylglucosaminuria (AGU) represents diseases affecting the central nervous system and is caused by a deficiency of a lysosomal enzyme, aspartylglucosaminidase (AGA). AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose-6-phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies. METHODS: Adenoviruses containing tissue-specific glial fibrillary acidic protein (GFAP) promoter and neuron-specific enolase (NSE) promoter were generated to target expression of AGA in Aga-deficient mouse primary glial and neuronal cell cultures. In addition an endogenous AGA promoter was used. The experimental design was planned to measure the enzymatic activities in the cells and media of neurons and glia infected with each specific virus. The endocytosis of AGA was analyzed by incubating neuronal and glial cells with media produced by each virus-cell combination. RESULTS: AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell-specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA. CONCLUSIONS: These data indicate the importance of glial cells in the expression and transport of AGA. Subsequently, new approaches can be developed for therapeutic intervention.

Two-step dimerization for autoproteolysis to activate glycosylasparaginase.

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.

Glycosylasparaginase inhibition studies: competitive inhibitors, transition state mimics, noncompetitive inhibitors.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

Human leukocyte glycosylasparaginase: cell-to-cell transfer and properties in correction of aspartylglycosaminuria.

Aspartylglycosaminuria (AGU), a severe lysosomal storage disease, is caused by the deficiency of the lysosomal enzyme, glycosylasparaginase (GA), and accumulation of aspartylglucosamine (GlcNAc-Asn) in tissues. Here we show that human leukocyte glycosylasparaginase can correct the metabolic defect in Epstein-Barr virus (EBV)-transformed AGU lymphocytes rapidly and effectively by mannose-6-phosphate receptor-mediated endocytosis or by contact-mediated cell-to-cell transfer from normal EBV-transformed lymphocytes, and that 2-7% of normal activity is sufficient to correct the GlcNAc-Asn metabolism in the cells. Cell-to-cell contact is obligatory for the transfer of GA since normal transformed lymphocytes do not excrete GA into extracellular medium. The combined evidence indicates that cell-to-cell transfer of GA plays a main role in enzyme replacement therapy of AGU by normal lymphocytes.

Bone marrow transplantation in aspartylglucosaminuria--histopathological and MRI study.

This study comprised two patients with aspartylglucosaminuria (AGU), who were followed up for 4 and 7 years. The patients underwent allogeneic bone marrow transplantation (BMT) at the ages of 2 and 2.6 years. Both patients had abnormal speech development and gross motor clumsiness. At the time of the BMT, they were mentally retarded. We report on follow-up data of these patients obtained by MRI, in addition to the histopathological, biochemical and clinical investigations. MR images of six non-transplanted patients and seven healthy children served as controls. In the non-transplanted patients, MRI revealed evident delay of myelination in contrast to the two transplanted patients showing fair or evident grey- vs. white matter differentiation on T2-weighted images. The aspartylglucosaminidase (AGA) activity in blood leukocytes reached a heterozygous level. Urinary excretion of aspartylglucosamine and glycoasparagines slowly decreased but remained about a third of the pre-BMT level 5 years after BMT. Storage lysosomes in electron microscopic investigations were not decreased 6 months after BMT, but after 1.5-2 years, rectal mucosa samples showed a decrease in the storage vacuoles of different cells. Three years after BMT, no cells with storage vacuoles were present. Allogeneic BMT slowly normalises the pathological, biochemical and MRI findings in patients with AGU.

Expression and endocytosis of lysosomal aspartylglucosaminidase in mouse primary neurons.

Aspartylglucosaminuria (AGU) is a neurodegenerative lysosomal storage disease that is caused by mutations in the gene encoding for a soluble hydrolase, aspartylglucosaminidase (AGA). In this study, we have used our recently developed mouse model for AGU and analyzed processing, intracellular localization, and endocytosis of recombinant AGA in telencephalic AGU mouse neurons in vitro. The processing steps of AGA were found to be similar to the peripheral cells, but both the accumulation of the inactive precursor molecule and delayed lysosomal processing of the enzyme were detected. AGA was distributed to the cell soma and neuronal processes but was not found in the nerve terminals. Endocytotic capability of cultured telencephalic neurons was comparable to that of fibroblasts, and endocytosis of AGA was blocked by free mannose-6-phosphate (M6P), indicating that uptake of the enzyme was mediated by M6P receptors (M6PRs). Uptake of extracellular AGA was also studied in the tumor-derived cell lines rat pheochromocytoma (PC12) and mouse neuroblastoma cells (N18), which both endocytosed AGA poorly as compared with cultured primary neurons. Expression of cation-independent M6PRs (CI-M6PRs) in different cell lines correlated well with the endocytotic capability of these cells. Although a punctate expression pattern of CI-M6PRs was found in fibroblasts and cultured primary neurons, the expression was beyond the detection limit in PC12 and N18 cells. This indicates that PC12 and N18 are not feasible cell lines to describe neuronal uptake of mannose-6-phosphate-tagged proteins. This in vitro data will form an important basis for the brain-targeted therapy of AGU.

Activation and oligomerization of aspartylglucosaminidase.

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 His124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His204-Asp205 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.

Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase.

Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.

Adenovirus-mediated gene transfer results in decreased lysosomal storage in brain and total correction in liver of aspartylglucosaminuria (AGU) mouse.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease leading to mental retardation, which is caused by deficiency of aspartylglucosaminidase (AGA). AGU is strongly enriched in the Finnish population in which one major mutation called AGU(Fin) has been identified. The molecular pathogenesis of AGU as well as the biology of the AGA enzyme have been extensively studied, thus giving a profound basis for therapeutic interventions. In this study we have performed adenovirus-mediated gene transfer to the recently produced mouse model of AGU, which exhibits similar pathophysiology as that in humans. Recombinant adenovirus vectors encoding for the human AGA and AGU(Fin) polypeptides were first applied in primary neurons of AGU mouse to demonstrate wild-type and mutant AGA expression in vitro. In vivo, both of the adenovirus vectors were injected into the tail vein of AGU mice and the expression of AGA was demonstrated in the liver. The adenovirus vectors were also injected intraventricularly into the brain of AGU mice resulting in AGA expression in the ependymal cells lining the ventricles and further, diffusion of AGA into the neighbouring neurons. Also, AGA enzyme injected intraventricularly was shown to transfer across the ependymal cell layer. One month after administration of the wild-type Ad-AGA, a total correction of lysosomal storage in the liver and a partial correction in brain tissue surrounding the ventricles was observed. After administration of the Ad-AGU virus the lysosomal storage vacuoles in liver or brain remained unchanged. These data demonstrate that the lysosomal storage in AGU can be biologically corrected and furthermore, in the brain a limited number of transduced cells can distribute AGA enzyme to the surrounding areas.

Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase.

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.

Primary folding of aspartylglucosaminidase. Significance of disulfide bridges and evidence of early multimerization.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme involved in the degradation of N-linked glycoproteins in lysosomes. AGA is synthesized as an inactive precursor molecule, which is rapidly activated in the endoplasmic reticulum by a proteolytic cleavage into alpha- and beta-subunits. We have recently determined the three-dimensional structure of AGA and shown that it is a globular molecule with a heterotetrameric (alphabeta)2 structure. On the basis of structural and functional analyses, AGA seems to be the first mammalian protein belonging to a newly described protein family, the N-terminal nucleophile hydrolases. Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA. To gain further information on the structural determinants influencing the early folding of AGA, we used site-specific mutagenesis of cysteine residues to define the role of intrachain disulfide bridges in the folding and activation of the enzyme. The N-terminal disulfide bridges in both the alpha- and beta-subunits seem to have only a stabilizing role, whereas the C-terminal disulfide bridge in both subunits evidently plays an important role in the early folding and activation of AGA.

Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation.

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three-dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-terminal residue of the beta subunit and most importantly its alpha-amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three-dimensional structure and whose catalytic mechanisms with an N-terminal nucleophile seem basically to be similar.