Site-Specific N-Glycosylation of Caprine Lysostaphin Restricts its Bacteriolytic Activity Toward Staphylococcus Aureus
Journal
Animal Biotechnology
Journal Volume
24
Journal Issue
2
Pages
129
Date Issued
2013-04-01
Author(s)
Abstract
Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis 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 goat β-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 Asn125 but not Asn232 is 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 (Asn232 to Gln232 substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn125 to Gln125 substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn125 of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus. © 2013 Copyright Taylor and Francis Group, LLC.
Subjects
Glycosylation | Goat | Lysostaphin | Mastitis
Publisher
TAYLOR & FRANCIS INC
Type
journal article