Kuipers Anneke, de Vries Louwe, de Vries Marcel P, Rink Rick, Bosma Tjibbe, Moll Gert N
Lanthio Pharma, a MorphoSys AG company, 9727 DL Groningen, The Netherlands.
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.
Peptides. 2017 May;91:33-39. doi: 10.1016/j.peptides.2017.03.004. Epub 2017 Mar 12.
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.
一些修饰的胰高血糖素样肽-1（GLP-1）类似物对治疗2型糖尿病非常重要。在此，我们研究了乳酸乳球菌中表达的GLP-1类似物是否可能是乳链菌肽脱水酶和转运酶修饰和输出的底物。随后，我们通过将形成的脱氢丙氨酸与赖氨酸偶联引入了赖氨酰丙氨酸，并研究了形成的赖氨酰丙氨酸桥接的GLP-1类似物的结构和活性。我们的数据表明：（i）与乳链菌肽前导肽融合的GLP-1通过乳链菌肽转运蛋白NisT能很好地输出，（ii）通过NisT产生前导肽-GLP-1比通过SEC系统更高，（iii）通过NisT输出的前导肽-GLP-1比通过SEC系统输出时更有效地被乳链菌肽脱水酶NisB脱水，（iv）GLP-1中的单个丝氨酸和苏氨酸被NisB脱水的程度有显著差异，（v）引入的Ser30能很好地被脱水，并可与Lys34偶联形成赖氨酰丙氨酸桥接的GLP-1类似物，（vi）在25%三氟乙醇存在下，赖氨酰丙氨酸（30-34）变体的构象向更高的α-螺旋含量转变，比在相同条件下野生型GLP-1观察到的更高，（vii）赖氨酰丙氨酸（30-34）GLP-1变体保留了显著的活性。综合这些数据扩展了关于NisT和NisB的底物特异性及其相对于通过Sec系统输出的联合活性的知识，并证明引入赖氨酰丙氨酸桥是修饰治疗性肽的一种选择。
