MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF BACTERIOPHAGE-PK1E-ENCODED ENDONEURAMINIDASE ENDO NE

Homopolymeric beta-2,8-linked sialic acid (PSA) has been found as a ca psular component of sepsis- and meningitis-causing bacterial pathogens , and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifical ly recognized and degraded by a phage-encoded enzyme, the endo-N-acety lneuraminidase E (Endo NE). Endo NE therefore has become a valuable to ol in the study of bacterial pathogenesis and eukaryotic morphogenesis . In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expresse d in Escherichia coil, the enzyme migrates as a single band of approxi mately 74 kDa in SDS-PAGE, A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF, Both phage-in duced lysis of bacteria and the catalysis of PSA degradation by the re combinant enzyme are efficiently inhibited by a polyclonal antiserum r aised against the intact phage particle, The C-terminal region seems t o he essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Ou r data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gen e locus and is not necessary for in vitro depolymerase activity.