Abstract
An osteoblastic protein, osteocalcin (OC), exists in vivo in two forms: carboxylated OC, and uncarboxylated or low-carboxylated OC (ucOC). ucOC acts as a hormone to regulate carbon and energy metabolism. Recent studies demonstrated that ucOC exerts insulinotropic effects, mainly through the glucagon-like peptide 1 (GLP-1) pathway. GLP-1 is an insulinotropic hormone secreted by enteroendocrine L cells in the small intestine. Thus, efficient delivery of ucOC to the small intestine may be a new therapeutic option for metabolic diseases such as diabetes and obesity. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse ucOC. Western blotting showed that the engineered strain (designated NZ-OC) produces and secretes the designed peptide (rOC) in the presence of nisin, an inducer of the recombinant gene. Highly-purified rOC was obtained from the culture supernatants of NZ-OC using immobilized metal affinity chromatography. An in vitro assay showed that purified rOC promotes GLP-1 secretion in a mouse intestinal neuroendocrine cell line, STC-1, in a dose-dependent manner. These results clearly demonstrate that NZ-OC secretes rOC, and that rOC can promote GLP-1 secretion by STC-1 cells. Genetically modified lactic acid bacteria (gmLAB) have been proposed over the last two decades as an effective and low-cost mucosal delivery vehicle for biomedical proteins. NZ-OC may be an attractive tool for the delivery of rOC to trigger GLP-1 secretion in the small intestine to treat diabetes and obesity.
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Cano-Garrido O, Seras-Franzoso J, Garcia-Fruitos E (2015) Lactic acid bacteria: reviewing the potential of a promising delivery live vector for biomedical purposes. Microb Cell Fact 14:137. doi:10.1186/s12934-015-0313-6
Ferron M, Hinoi E, Karsenty G, Ducy P (2008) Osteocalcin differentially regulates β cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci USA 105(13):5266–5270. doi:10.1073/pnas.0711119105
Ferron M, McKee MD, Levine RL, Ducy P, Karsenty G (2012) Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice. Bone 50(2):568–575. doi:10.1016/j.bone.2011.04.017
Kakonen SM, Hellman J, Pettersson K, Lovgren T, Karp M (1996) Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli. Protein Expr Purif 8(2):137–144. doi:10.1006/prep.1996.0085
Kanazawa I (2015) Osteocalcin as a hormone regulating glucose metabolism. World J Diabetes 6(18):1345–1354. doi:10.4239/wjd.v6.i18.1345
Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, Zhang Z, Kim JK, Mauvais-Jarvis F, Ducy P, Karsenty G (2007) Endocrine regulation of energy metabolism by the skeleton. Cell 130(3):456–469. doi:10.1016/j.cell.2007.05.047
Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6):705–717. doi:10.1007/s00253-005-0107-6
Mizokami A, Yasutake Y, Gao J, Matsuda M, Takahashi I, Takeuchi H, Hirata M (2013) Osteocalcin induces release of glucagon-like peptide-1 and thereby stimulates insulin secretion in mice. PLoS ONE 8(2):e57375. doi:10.1371/journal.pone.0057375
Mizokami A, Yasutake Y, Higashi S, Kawakubo-Yasukochi T, Chishaki S, Takahashi I, Takeuchi H, Hirata M (2014) Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion. Bone 69:68–79. doi:10.1016/j.bone.2014.09.006
Morello E, Bermudez-Humaran LG, Llull D, Sole V, Miraglio N, Langella P, Poquet I (2008) Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. J Mol Microbiol Biotechnol 14(1–3):48–58
Pi M, Wu Y, Quarles LD (2011) GPRC6A mediates responses to osteocalcin in β-cells in vitro and pancreas in vivo. J Bone Miner Res 26(7):1680–1683. doi:10.1002/jbmr.390
Pi M, Kapoor K, Ye R, Nishimoto SK, Smith JC, Baudry J, Quarles LD (2016) Evidence for osteocalcin binding and activation of GPRC6A in β-cells. Endocrinology 157(5):1866–1880. doi:10.1210/en.2015-2010
Rishavy MA, Berkner KL (2012) Vitamin K oxygenation, glutamate carboxylation, and processivity: defining the three critical facets of catalysis by the vitamin K-dependent carboxylase. Adv Nutr 3(2):135–148. doi:10.3945/an.111.001719
Rishavy MA, Hallgren KW, Yakubenko AV, Zuerner RL, Runge KW, Berkner KL (2005) The vitamin K-dependent carboxylase has been acquired by Leptospira pathogens and shows altered activity that suggests a role other than protein carboxylation. J Biol Chem 280(41):34870–34877. doi:10.1074/jbc.M504345200
Samazan F, Rokbi B, Seguin D, Telles F, Gautier V, Richarme G, Chevret D, Varela PF, Velours C, Poquet I (2015) Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis. Microb Cell Fact 14:104. doi:10.1186/s12934-015-0271-z
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Shigemori S, Shimosato T (2017) Applications of genetically modified immunobiotics with high immunoregulatory capacity for treatment of inflammatory bowel diseases. Front Immunol 8:22. doi:10.3389/fimmu.2017.00022
Shigemori S, Oshiro K, Wang P, Yamamoto Y, Wang Y, Sato T, Uyeno Y, Shimosato T (2014) Generation of dipeptidyl peptidase-IV-inhibiting peptides from β-lactoglobulin secreted by Lactococcus lactis. Biomed Res Int 2014:393598. doi:10.1155/2014/393598
Shigemori S, Watanabe T, Kudoh K, Ihara M, Nigar S, Yamamoto Y, Suda Y, Sato T, Kitazawa H, Shimosato T (2015) Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice. Microb Cell Fact 14:189. doi:10.1186/s12934-015-0378-2
Shigemori S, Ihara M, Sato T, Yamamoto Y, Nigar S, Ogita T, Shimosato T (2017) Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis. Appl Microbiol Biotechnol 101(1):341–349. doi:10.1007/s00253-016-7907-8
van Asseldonk M, de Vos WM, Simons G (1993) Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase. Mol Gen Genet 240(3):428–434
Wells JM, Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6(5):349–362. doi:10.1038/nrmicro1840
Wyszynska A, Kobierecka P, Bardowski J, Jagusztyn-Krynicka EK (2015) Lactic acid bacteria—20 years exploring their potential as live vectors for mucosal vaccination. Appl Microbiol Biotechnol 99(7):2967–2977. doi:10.1007/s00253-015-6498-0
Funding
This study was funded by a Grant-in-Aid from the Japan Society for the Promotion of Science Fellows (No. 14J06317) to SS and a grant from the Faculty of Agriculture, Shinshu University, to TSh.
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Namai, F., Shigemori, S., Sudo, K. et al. Recombinant Mouse Osteocalcin Secreted by Lactococcus lactis Promotes Glucagon-Like Peptide-1 Induction in STC-1 Cells. Curr Microbiol 75, 92–98 (2018). https://doi.org/10.1007/s00284-017-1354-3
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DOI: https://doi.org/10.1007/s00284-017-1354-3