title: Comparison of alternative nucleophiles for Sortase A-mediated bioconjugation and application in neuronal cell labelling.
abstract: The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.
authors.affiliation: CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052, Australia. Charlotte.Williams@csiro.au.
authors.forename: Samuel
authors.initials: S
authors.lastname: Baer
authors.forename: Julie
authors.initials: J
authors.lastname: Nigro
authors.forename: Mariusz P
authors.initials: MP
authors.lastname: Madej
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Nisbet
authors.forename: Randy
authors.initials: R
authors.lastname: Suryadinata
authors.forename: Gregory
authors.initials: G
authors.lastname: Coia
authors.forename: Lisa P T
authors.initials: LP
authors.lastname: Hong
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Adams
authors.forename: Charlotte C
authors.initials: CC
authors.lastname: Williams
authors.forename: Stewart D
authors.initials: SD
authors.lastname: Nuttall
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005029:Ethylenediamines / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D016229:Amyloid beta-Peptides; D001426:Bacterial Proteins; D005029:Ethylenediamines; D007140:Immunoglobulin Fab Fragments; D034521:Nuclear Matrix-Associated Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C031234:ethylenediamine; C035647:glycyl-glycyl-glycine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases