Molecular cloning and functional expression of bacteriophage PK1E‐encoded endoneuraminidase Endo NE

Abstract

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.

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

“…2). In all samples, the specificity of the PSA signal was shown by EndoNE digestion, which degrades PSA and abolishes binding of mAb 735 (36). Expression levels of the recombinant epitope-tagged proteins were monitored by Western blot analysis using the anti-FLAG mAb M5 and the anti-HA mAb 12CA5.…”

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

“…Gradient gels were prepared with ProSieve 50 gel solution (BioWhittaker) and run in Tris/Tricine electrode buffer according to the manufacturer's instructions for 12% resolving and 3% stacking gels. For Western blot analysis proteins were blotted onto nitrocellulose membranes (Schleicher & Schuell), and blots were developed as described (18). For detection of His 6 -tagged proteins Penta-His antibody (Qiagen) was used at a concentration of 1 g/ml.…”

“…Cloning of endoNE revealed that the gene encodes a 90-kDa protein, whereas for the purified phage-borne and the recombinantly expressed endoNE an apparent molecular mass between 74 and 76 kDa was observed (8,18,19), indicating post-translational processing. In contrast, no discrepancy be-* This work was supported by Deutsche Forschungsgemeinschaft Grant GE 801/3-3 and by the Fonds der Chemischen Industrie.…”

“…SDS-resistant trimers, composed of three identical subunits of 105 kDa each, have been described for endoNF of coliphage K1F (9). For endoNE of K1E, hetero-oligomeric structures of 208 and 325 kDa were described, composed of endoNE and a not as yet identified 38-kDa protein (8,18). So far, genes encoding endosialidases have been cloned from four different phages: K1F (11), K1E (18,19), 63D (GenBank accession number AB015437), and the dual specificity phage K1-5 that can infect E. coli K1 and K5 (20).…”

“…For endoNE of K1E, hetero-oligomeric structures of 208 and 325 kDa were described, composed of endoNE and a not as yet identified 38-kDa protein (8,18). So far, genes encoding endosialidases have been cloned from four different phages: K1F (11), K1E (18,19), 63D (GenBank accession number AB015437), and the dual specificity phage K1-5 that can infect E. coli K1 and K5 (20). However, recombinant expression of active enzyme has been reported only in the case of endoNE (18,19).…”