Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits.

A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.

The first demonstration of a procaryotic glycosylasparaginase.

Glycosylasparaginase was purified to near homogeneity from intracellular lysates of Flavobacterium meningosepticum. The enzyme is a heterodimer with an estimated molecular weight of 38 kDa and consists of one alpha-subunit (18 kDa) and one beta-subunit (16 kDa). The beta-subunit of the Flavobacterium enzyme has a direct evolutionary relationship to the beta-subunit of mammalian glycosylasparaginases as evidenced by: (1) strong cross-reactivity with antibodies made to the denatured rat beta-subunit, (2) a high degree of homology with the amino-terminus of the corresponding eukaryotic enzymes, and (3) irreversible inactivation with 5-diazo-4-oxo-L-norvaline, a reagent known to react with the catalytic amino-terminal threonine residue on the beta-subunit of a mammalian glycosylasparaginase.

Human aspartylglucosaminidase. A biochemical and immunocytochemical characterization of the enzyme in normal and aspartylglucosaminuria fibroblasts.

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is an essential enzyme in the degradation of asparagine-linked glycoproteins. In man, deficient activity of this enzyme leads to aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease. Here we used affinity-purified polyclonal antibodies against the native AGA and its denatured subunits to establish the molecular structure and intracellular location of the enzyme in normal and AGU fibroblasts. Inactivation of the enzyme was found to coincide with the dissociation of the heterodimeric enzyme complex into subunits. Although the subunits were not linked by covalent forces, the intrapolypeptide disulphide bridges were found to be essential for the normal function of AGA. AGA was localized into lysosomes in control fibroblasts by both immunofluorescence microscopy and immuno-electron microscopy, whereas in AGU cells the location of antigen was different, suggesting that, owing to the mutation, a missing disulphide bridge, most of the enzyme molecules get retarded in the cis-Golgi region and most probably face intracellular degradation.