title: Site-specific N-glycosylation of caprine lysostaphin restricts its bacteriolytic activity toward Staphylococcus aureus.
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 Asn(125) but not Asn(232) 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 (Asn(232) to Gln(232) substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn(125) to Gln(125) 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 Asn(125) of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.
authors.affiliation: Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan, ROC.
authors.forename: Ching-Ying
authors.initials: CY
authors.lastname: Huang
authors.forename: Jih-Tay
authors.initials: JT
authors.lastname: Hsu
authors.forename: Pei-Hsuan
authors.initials: PH
authors.lastname: Chung
authors.forename: Winston Teng-Kuei
authors.initials: WT
authors.lastname: Cheng
authors.forename: Yan-Nian
authors.initials: YN
authors.lastname: Jiang
authors.forename: Yu-Ten
authors.initials: YT
authors.lastname: Ju
mesh_terms: D000818:Animals; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D015169:Colony Count, Microbial / Q000662:veterinary; D005260:Female; D006031:Glycosylation; D015511:Goat Diseases / Q000188:drug therapy / Q000382:microbiology*; D006041:Goats; D007150:Immunohistochemistry / Q000662:veterinary; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism; D008248:Lysostaphin / Q000378:metabolism / Q000494:pharmacology*; D008413:Mastitis / Q000188:drug therapy / Q000382:microbiology / Q000662:veterinary*; D016297:Mutagenesis, Site-Directed / Q000662:veterinary; D011994:Recombinant Proteins / Q000494:pharmacology*; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development
chemical_list: D002364:Caseins; D011994:Recombinant Proteins; D007781:Lactoferrin; D008248:Lysostaphin