Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.
Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular 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 specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool 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. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 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-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our 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 gene locus and is not necessary for in vitro depolymerase activity.
Gerardy-Schahn
R
R
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
Bethe
A
A
Brennecke
T
T
Mühlenhoff
M
M
Eckhardt
M
M
Ziesing
S
S
Lottspeich
F
F
Frosch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004790:Enzyme Induction; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics*; D006168:Guinea Pigs; D007106:Immune Sera; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics*; D009500:Neutralization Tests; D016415:Sequence Alignment; D012794:Sialic Acids / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics*
D007106:Immune Sera; D012794:Sialic Acids; D014764:Viral Proteins; C021319:polysialic acid; D006026:Glycoside Hydrolases; C035355:endo-N-acetylneuraminidase; C090534:endo-alpha-sialidase; D009439:Neuraminidase