title: Semi-microbiological synthesis of an active lysinoalanine-bridged analog of glucagon-like-peptide-1.
abstract: Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog. Our data show: (i) GLP-1 fused to the nisin leader peptide is very well exported via the nisin transporter NisT, (ii) production of leader-GLP-1 via NisT is higher than via the SEC system, (iii) leader-GLP-1 exported via NisT was more efficiently dehydrated by the nisin dehydratase NisB than when exported via the SEC system, (iv) individual serines and threonines in GLP-1 are dehydrated by NisB to a significantly different extent, (v) an introduced Ser30 is well dehydrated and can be coupled to Lys34 to form a lysinoalanine-bridged GLP-1 analog, (vi) a lysinoalanine(30-34) variant's conformation shifts in the presence of 25% trifluoroethanol towards a higher alpha helix content than observed for wild type GLP-1 under identical condition, (vii) a lysinoalanine(30-34) GLP-1 variant has retained significant activity. Taken together the data extend knowledge on the substrate specificities of NisT and NisB and their combined activity relative to export via the Sec system, and demonstrate that introducing a lysinoalanine bridge is an option for modifying therapeutic peptides.
authors.affiliation: Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands.
authors.forename: Anneke
authors.initials: A
authors.lastname: Kuipers
authors.affiliation: Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands.
authors.forename: Louwe
authors.initials: L
authors.lastname: de Vries
authors.affiliation: Mass Spectrometry Core Facility, Department of Pharmacy, University of Groningen, 9713 AV Groningen, The Netherlands; Department of Pediatrics, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
authors.forename: Marcel P
authors.initials: MP
authors.lastname: de Vries
authors.affiliation: Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands.
authors.forename: Rick
authors.initials: R
authors.lastname: Rink
authors.affiliation: Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands.
authors.forename: Tjibbe
authors.initials: T
authors.lastname: Bosma
authors.affiliation: Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands; Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands. Electronic address: moll@lanthiopharma.com.
authors.forename: Gert N
authors.initials: GN
authors.lastname: Moll
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1 / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008241:Lysinoalanine / Q000737:chemistry*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D000069816:SEC Translocation Channels / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C079126:NisB protein, Lactococcus lactis; C094910:NisT protein, Lactococcus lactis; D000069816:SEC Translocation Channels; D008241:Lysinoalanine; D013912:Threonine; D012694:Serine; D052216:Glucagon-Like Peptide 1; C015102:dehydroalanine; D006836:Hydro-Lyases; D000409:Alanine
keywords: Dehydroalanine; Glucagon like peptide-1; Lanthipeptide; Lysine; Nisin