Site-Specific N-Glycosylation of Caprine Lysostaphin Restricts its Bacteriolytic Activity Toward Staphylococcus Aureus.

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoidStaphylococcus aureusmastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goatβ-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn125but not Asn232is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn232to Gln232substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn125to Gln125substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability toS. aureus. This reveals that N-glycosylation at Asn125of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability toS. aureus. [ABSTRACT FROM PUBLISHER]