Cloning, Sequencing and Expression of the Sialidase Gene fromActinomyces viscosusDSM 43798

Abstract

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac.The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A.viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa.The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species. Klonierung, Sequenzanalyse und Expression des Sialidase-G ens aus Actinomyces viscosus DSM 43798Zusammenfassung: Chromosomale DNA von Actinomyces viscosus wurde mit Restriktionsendonucleasen verdaut und die Fragmente in pUC-Vektoren ligiert, welche zur Transformation von E. co/z-Zellen verwendet wurden. Die gesuchten Klone, die das Sialidase-Gen enthielten, wurden anhand der Enzymaktivität mit dem fluorogenen Sialidasesubstrat MUNeuSAcdetektiert.Die Identität der klonierten Sialidase wurde nach 5 700-facher Anreicherung durch Vergleich mit dem angereicherten Enzym von Actinomyces viscosus bestätigt. Beide Sialidasen waren identisch bezüglich der Molekularmasse, der Substratspezifität getestet

Evidence for recombination between a sialidase (nanH) ofActinomyces naeslundiiandActinomyces oris, previously named ‘Actinomyces naeslundiigenospecies 1 and 2’

“…All sialidase enzymes have a highly conserved array of residues, which in the tertiary structure of the enzyme, form the active and binding sites of the molecule (Crennell et al , 1993). In addition to these residues all sialidases also have a number of BNRs structural entities for which a function cannot be definitely ascribed because Asp boxes are found in many protein families (Henningsen et al , 1991; Copley et al , 2001). In determining the sequence diversity of nanH in these Actinomyces species, we selected a portion of the gene which contained the 12 residues involved in substrate interactions with, and stabilization of, the active site and the BNRs originally described in ‘ A. viscosus ’ strain DSM 43798, now identified as A. oris .…”

“…In this report, we have compared the nucleotide and amino acid sequences of the region of the nanH gene containing the active site (Crennell et al , 1993) and the five Asp boxes or bacterial neuraminidase repeats (BNRs, Henningsen et al , 1991) of these Actinomyces species. In the mouth A. naeslundii and A. oris occupy the same sites but A. oris is the predominant species (Bowden et al , 1999) while A. johnsonii was isolated from the gingival crevice (Johnson et al , 1990).…”

Molecular and Genetic Analyses of Actinomyces SPP

“…Whereas the sialidase from strain T14V is most active toward a-(2---3)-and at-(2-->6)-linked sialic acid and does Hoyer et al, 1992). Molecular cloning of the A. viscosus DSM 43798 sialidase gene also was facilitated when the previously described fluorogenic sialidase substrate was used as the screening tool (Henningsen et al, 1991 sequence homology, one of which included the five "Asp block" sequences (Yeung, 1993). Results of Southern blot analysis indicated the presence of the A. viscosus T14V nanH homologue in the genomes of 18 strains of five Actinomyces species that express various levels of sialidase activity (Yeung, 1993).…”

Improving Protein Pharmacokinetics by Genetic Fusion to Simple Amino Acid Sequences

“…Four sequences matching these criteria were identified and cloned at the TS C terminus to be tested in our pharmacokinetic assays. These sequences span the degenerate repetitive units present at the C termini of the sialidases from A. viscosus (32,42) and S. pneumoniae (33), the hydrophilic C-terminal extension of the closely related TS from the fish parasite T. carassii (34), and the proline-rich region of the EspF protein from enteropathogenic E. coli (35,36). The complete sequence of these domains together with the predicted molecular properties of the ensuing chimeric TS proteins is indicated in Fig.…”