Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis.

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

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.

Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase.

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure-function relationships of glycans.

Biochemical and structural insights into an allelic variant causing the lysosomal storage disorder - aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by defects of the hydrolase glycosylasparaginase (GA). Previously, we showed that a Canadian AGU mutation disrupts an obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterizations of a model enzyme corresponding to a Finnish AGU allele, the T234I variant. Unlike the Canadian counterpart, the Finnish variant is capable of a slow autoprocessing to generate detectible hydrolyzation activity of the natural substrate of GA. We have determined a 1.6 Å-resolution structure of the Finnish AGU model and built an enzyme-substrate complex to provide a structural basis for analyzing the negative effects of the point mutation on KM and kcat of the mature enzyme. ENZYME: Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma.

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and ß-subunits also form an (αß)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and ß for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps' success.

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

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 ß-hydroxamate (ß-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-ß-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a ß-AHA molecule. A hydrolysis mechanism of ß-AHA by GA is proposed based on this complex model.

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.

Functional Analysis of the Ser149/Thr149 Variants of Human Aspartylglucosaminidase and Optimization of the Coding Sequence for Protein Production.

Aspartylglucosaminidase (AGA) is a lysosomal hydrolase that participates in the breakdown of glycoproteins. Defects in the AGA gene result in a lysosomal storage disorder, aspartylglucosaminuria (AGU), that manifests mainly as progressive mental retardation. A number of AGU missense mutations have been identified that result in reduced AGA activity. Human variants that contain either Ser or Thr in position 149 have been described, but it is unknown if this affects AGA processing or activity. Here, we have directly compared the Ser149/Thr149 variants of AGA and show that they do not differ in terms of relative specific activity or processing. Therefore, Thr149 AGA, which is the rare variant, can be considered as a neutral or benign variant. Furthermore, we have here produced codon-optimized versions of these two variants and show that they are expressed at significantly higher levels than AGA with the natural codon-usage. Since optimal AGA expression is of vital importance for both gene therapy and enzyme replacement, our data suggest that use of codon-optimized AGA may be beneficial for these therapy options.

Aspartylglucosaminuria caused by a novel homozygous mutation in the AGA gene was identified by an exome-first approach in a patient from Japan.

BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme, aspartylglucosaminidase (AGA). This disorder is rare in the general population except in Finland. Since the most characteristic feature of this disorder is a progressive developmental regression, patients often show no specific symptoms in the initial stages, and thus early diagnosis is often challenging. CASE REPORT: We encountered a 16-year-old boy who began to show difficulties in his speech at the age of 6years. Due to a mild regression in his development, he gradually lost common daily abilities. His diagnosis was first obtained through exome sequencing that identified a novel homozygous mutation in the AGA gene. This result was reasonable because of parental consanguinity. Reduced enzymatic activity of AGA was then confirmed. His urine was retrospectively screened by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and a specific pattern of abnormal metabolites was identified. CONCLUSIONS: Because both exome sequencing and MALDI-TOF-MS screening are adaptable and comprehensive, future combinatory use of these methods would be useful for diagnosis of rare inborn errors of metabolism such as AGU.

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.

[A family with two children diagnosed with aspartylglucosaminuria-case report and literature review].

OBJECTIVE: The authors sought to investigate the clinical features and characteristics of genetic mutation in patients with aspartylglucosaminuria. METHOD: Clinical data of two pediatric siblings in a family were analyzed retrospectively and relative literature was reviewed in order to study the clinical features, imaging and enzymatic characteristics and genetic mutations. RESULT: Case 1, the proband, male, he was hospitalized at 20 months of age because of fever and hepatosplenomegaly for nine days. This child was of moderate nutritional status and normal development. Blood tests showed hemoglobin 78.0 g/L, RBC3.18 × 10¹²/L, WBC 4.06 × 109/L, neutrophils 0.236, lymphocytes 0.631, platelets 34 × 109/L, C-reactive protein 17 mg/L. Blood biochemistry showed alanine aminotransferase 67.1 U/L, aspartate aminotransferase 74.1 U/L, serum albumin 32.8 g/L, direct bilirubin 10.5 µmol/L, lactate dehydrogenase 301.7 U/L. Bone marrow cytology showed reactive morphological changes in bone marrow cells. Atypical lymphocytes could be seen in both peripheral blood and bone marrow smears. Cranial MRI showed poor myelination. Aspartylglucosaminidase activity in peripheral leucocytes of the proband 5.7 nmol/(g × min) vs. normal control>26.6 nmol/(g × min). On his AGA gene and that of his parents, a heterozygous mutation site located in exon 3, c.392C>T (p.S131L), was identified as a novel mutation inherited from his father. The mutation from his mother has not been detected. The proband was not responsive to the anti-infectious medication, nutritional intervention and symptomatic treatment.He died one month after diagnosis.His elder brother, Case 2, showed fever, recurrent respiratory tract infection and progressive psychomotor regression with hepatosplenomegaly from the age of four years. Cranial MRI revealed extensive symmetrical leukodystrophy in bilateral cerebra, cerebellum and brainstem.He died at the age of six years.Related literature was summarized, and no Chinese AGU cases had been reported; 221 foreign cases were collected. The clinical and imaging characteristics were summarized. Delay in language development was one of the clinical symptoms that the majority of parents of AGU children first noted. CONCLUSION: Patients with aspartylglucosaminuria lack of specific symptoms.For children with unexplained delayed speech and progressive mental retardation, the possibility of AGU should be considered, and efforts be made for enzymatic and genetic diagnosis. c.392C> T (p.S131L) was identified as a novel mutation of AGA gene.

Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology.

In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein-carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed.

Molecular and biochemical analysis of an aspartylglucosaminidase from the venom of the parasitoid wasp Asobara tabida (Hymenoptera: Braconidae).

The most abundant venom protein of the parasitoid wasp Asobara tabida was identified to be an aspartylglucosaminidase (hereafter named AtAGA). The aim of the present work is the identification of: 1) its cDNA and deduced amino acid sequences, 2) its subunits organization and 3) its activity. The cDNA of AtAGA coded for a proalphabeta precursor molecule preceded by a signal peptide of 19 amino acids. The gene products were detected specifically in the wasp venom gland (in which it could be found) under two forms: an (active) heterotetramer composed of two alpha and two beta subunits of 30 and 18 kDa respectively and a homodimer of 44 kDa precursor. The activity of AtAGA enzyme showed a limited tolerance toward variations of pH and temperatures. Since the enzyme failed to exhibit any glycopeptide N-glycosidase activity toward entire glycoproteins, its activity seemed to be restricted to the deglycosylation of free glycosylasparagines like human AGA, indicating AtAGA did not evolve a broader function in the course of evolution. The study of this enzyme may allow a better understanding of the functional evolution of venom enzymes in hymenopteran parasitoids.

Components of Asobara venoms and their effects on hosts.

Hymenoptera of the Asobara genus are endophagous parasitoids of Drosophila larvae. In these apocrita insects whose venom gland is associated with the female reproductive tract, the wasp venom is injected into the host along with the parasitoid egg during oviposition. We conducted a comparative study of the venom apparatuses from three Asobara spp.: the European Asobara tabida, the Asiatic A. japonica and the African A. citri. Light and electron microscopy of venom glands, together with the biochemical analysis of their contents, revealed important differences between Asobara spp. In addition, the physiological effects of female wasp's venom injected into Drosophila larvae differed greatly between the tested Asobara spp.

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.

Massive accumulation of Man2GlcNAc2-Asn in nonneuronal tissues of glycosylasparaginase-deficient mice and its removal by enzyme replacement therapy.

Aspartylglycosaminuria (AGU) is caused by deficient enzymatic activity of glycosylasparaginase (GA). The disease is characterized by accumulation of aspartylglucosamine (GlcNAc-Asn) and other glycoasparagines in tissues and body fluids of AGU patients and in an AGU mouse model. In the current study, we characterized a glycoasparagine carrying the tetrasaccharide moiety of alpha-D-Man-(1-->6)-beta-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-beta-D-GlcNAc-(1-->N)-Asn (Man2GlcNAc2-Asn) in urine of an AGU patient and also in the tissues of the AGU mouse model. Quantitative analysis demonstrated a massive accumulation of the compound especially in nonneuronal tissues of the AGU mice, in which the levels of Man2GlcNAc2-Asn were typically 30-87% of those of GlcNAc-Asn. The highest level of Man2GlcNAc2-Asn was found in the liver, spleen, and heart tissues of the AGU mice, the respective amounts being 87%, 76%, and 57% of the GlcNAc-Asn levels. In the brain tissue of AGU mice the Man2GlcNAc2-Asn storage was only 9% of that of GlcNAc-Asn. In contrast to GlcNAc-Asn, the storage of Man2GlcNAc2-Asn markedly increased in the liver and spleen tissues of AGU mice as they grew older. Enzyme replacement therapy with glycosylasparaginase for 3.5 weeks reduced the amount of Man2GlcNAc2-Asn by 66-97% in nonneuronal tissues, but only by 13% in the brain tissue of the AGU mice. In conclusion, there is evidence for a role for storage of glycoasparagines other than aspartylglucosamine in the pathogenesis of AGU, and this possibility should be taken into consideration in the treatment of the disease.

Elevation of plasma aspartylglucosaminidase is a useful marker for the congenital disorders of glycosylation type I (CDG I).

Elevated plasma aspartylglucosaminidase activity was found in 21/25 cases of CDG Ia, in single cases of CDG Ib, Ic and If, and in 15/16 cases of CDG Ix. The CDG I patients in whom the activity was not raised were either atypical clinically (CDG Ia) or very young (CDG Ih).

Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (LlA) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg.L(-1) of culture, respectively. The enzymes are heat-stable up to 60 degrees C and show both isoaspartyl dipeptidase and l-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.