MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF FLAVOBACTERIUM-MENINGOSEPTICUM GLYCOSYLASPARAGINASE - A SINGLE-GENE ENCODES THE ALPHA-SUBUNITS AND BETA-SUBUNITS

A full-length insert for the Flavobacterium meningosepticum N-4-(N-ace tyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500 -bp HindIII fragment and cloned into the plasmid vector pBluescript. D NA sequencing revealed an open reading frame of 1020 nucleotides encod ing a putative 45-amino-acid leader sequence and a deduced precursor p olypeptide of 295 amino acids, In F, meningosepticum this precursor po lypeptide undergoes proteolytic processing by an as yet unknown mechan ism 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-polyac rylamide gel electrophoresis to consist of the typical alpha- and beta -subunits, The recombinant beta-subunit cross-reacted to antibody spec ific for the rat liver beta-subunit, and Edman analysis demonstrated t hat its amino-terminus corresponded exactly to that of the mature nati ve glycosylasparagine beta-subunit, A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 3 0% structural identity and 60% overall similarity between the prokaryo tic and eukaryotic forms of the enzyme. Even more striking was the con servation of the amino acid sequence in both proteins where the post-t ranslational cleavage to generate the active enzyme occurs, Our data d emonstrate that deglycosylation of asparagine-linked glycans via hydro lysis of the AspNHGlcNAc linkage is an important reaction which has be en preserved during evolution. (C) 1995 Academic Press. Inc.