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Autoproteolytic activation of human aspartylglucosaminidase.

ABSTRACT: 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.

SUBMITTER: Saarela J

PROVIDER: S-EPMC1223969 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a 'native' molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.

Project description:To study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection.Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFN?), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively.Corneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1? and MIP-1?, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation.Human macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.

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Autoproteolytic activation of human aspartylglucosaminidase.

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