Biosynthesis of animal-free recombinant chondroitin sulfate E using a functional chondroitin sulfotransferase in E. coli.
Chondroitin
Recombinant glycosaminoglycan
Sulfotransferase
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
15 Aug 2024
15 Aug 2024
Historique:
received:
10
06
2024
accepted:
02
08
2024
revised:
22
07
2024
medline:
15
8
2024
pubmed:
15
8
2024
entrez:
15
8
2024
Statut:
epublish
Résumé
Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli. Moreover, there is no experimentally derived three-dimensional structure available for this specific sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks. KEY POINTS: • Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished. • First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.
Identifiants
pubmed: 39145804
doi: 10.1007/s00253-024-13275-3
pii: 10.1007/s00253-024-13275-3
doi:
Substances chimiques
Chondroitin Sulfates
9007-28-7
Sulfotransferases
EC 2.8.2.-
Recombinant Proteins
0
N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase
EC 2.8.2.-
chondroitin 4-sulfotransferase
EC 2.8.2.5
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
440Subventions
Organisme : Rensselaer Polytechnic Institute
ID : Biocatalysis Constellation Fund
Informations de copyright
© 2024. The Author(s).
Références
Abdallah MM, Fernández N, Matias AA, Bronze MR (2020) Hyaluronic acid and Chondroitin sulfate from marine and terrestrial sources: extraction and purification methods. Carbohydr Polym 243:116441
pubmed: 32532391
doi: 10.1016/j.carbpol.2020.116441
Alexandrov A, Dutta K, Pascal SM (2001) MBP fusion protein with a viral protease cleavage site: one-step cleavage/purification of insoluble proteins. Biotechniques 30(6):1194–1198
pubmed: 11414203
doi: 10.2144/01306bm01
Awofiranye AE, Hudson J, Tithi AD, Linhardt RJ, Vongsangnak W, Koffas MAG (2022) Chondroitin sulfate and its derivatives: a review of microbial and other production methods. Fermentation 8:323
doi: 10.3390/fermentation8070323
Badri A, Williams A, Linhardt RJ, Koffas MA (2018) The road to animal-free glycosaminoglycan production: current efforts and bottlenecks. Curr Opin Biotechnol 53:85–92
pubmed: 29291494
doi: 10.1016/j.copbio.2017.12.018
Badri A, Williams A, Awofiranye A, Datta P, Xia K, He W, Fraser K, Dordick JS, Linhardt RJ, Koffas MAG (2021) Complete biosynthesis of a sulfated chondroitin in Escherichia coli. Nat Commun 12(1):1–10
doi: 10.1038/s41467-021-21692-5
Baik JY, Gasimli L, Yang B, Datta P, Zhang F, Glass CA, Esko JD, Linhardt RJ, Sharfstein ST (2012) Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin. Metab Eng 14(2):81–90
pubmed: 22326251
pmcid: 3299301
doi: 10.1016/j.ymben.2012.01.008
Bergefall K, Trybala E, Johansson M, Uyama T, Naito S, Yamada S, Kitagawa H, Sugahara K, Bergström T (2005) Chondroitin sulfate characterized by the E-disaccharide unit is a potent inhibitor of herpes simplex virus infectivity and provides the virus binding sites on gro2C cells. J Biol Chem 280(37):32193–32199
pubmed: 16027159
doi: 10.1074/jbc.M503645200
Bishnoi M, Jain A, Hurkat P, Jain SK (2016) Chondroitin sulphate: a focus on osteoarthritis. Glycoconj J 33(5):693–705
pubmed: 27194526
doi: 10.1007/s10719-016-9665-3
Bourin MC, Lindahl U (1993) Glycosaminoglycans and the regulation of blood coagulation. The Biochem J 289(Pt 2):313–330
pubmed: 8380990
doi: 10.1042/bj2890313
Cai C, Solakyildirim K, Yang B, Beaudet JM, Weyers A, Linhardt RJ, Zhang F (2012) Semi-synthesis of chondroitin sulfate-E from chondroitin sulfate-A. Carbohydr Polym 87(1):822–829
pubmed: 22140285
pmcid: 3225962
doi: 10.1016/j.carbpol.2011.08.075
De Marco A (2009) Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Factories 8(1):26
doi: 10.1186/1475-2859-8-26
Dickendesher TL, Baldwin KT, Mironova YA, Koriyama Y, Raiker SJ, Askew KL, Wood A, Geoffroy CG, Zheng B, Liepmann CD, Katagiri Y, Benowitz LI, Geller HM, Giger RJ (2012) NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans. Nat Neurosci 15(5):703–712
pubmed: 22406547
pmcid: 3337880
doi: 10.1038/nn.3070
Fuentes EP, Diaz VB (1998) Oligosaccharide mapping of chondroitin sulfate obtained from different animal sources. ACTA Farm Bonaer 17:135–142
Goldenzweig A, Fleishman SJ (2018) Principles of protein stability and their application in computational design. Annu Rev Biochem 87:105–129
pubmed: 29401000
doi: 10.1146/annurev-biochem-062917-012102
Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y, Dym O, Unger T, Albeck S, Prilusky J, Lieberman RL, Aharoni A, Silman I, Sussman JL, Tawfik DS, Fleishman SJ (2016) Automated structure- and sequence-based design of proteins for high bacterial expression and stability. Mol Cell 63(2):337–346
pubmed: 27425410
pmcid: 4961223
doi: 10.1016/j.molcel.2016.06.012
Guerrini M, Beccati D, Shriver Z, Naggi A, Viswanathan K, Bisio A, Capila I, Lansing JC, Guglieri S, Fraser B, Al-Hakim A, Gunay NS, Zhang Z, Robinson L, Buhse L, Nasr M, Woodcock J, Langer R, Venkataraman G, Linhardt RJ, Casu B, Torri G, Sasisekharan R (2008) Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 26(6):669–675
pubmed: 18437154
pmcid: 3491566
doi: 10.1038/nbt1407
Habuchi O (2022) Functions of chondroitin/dermatan sulfate containing GalNAc4,6-disulfate. Glycobiology 32(8):664–678
pubmed: 35552694
doi: 10.1093/glycob/cwac030
Habuchi O, Yamagata T, Suzuki S (1971) Biosynthesis of the acetylgalactosamine 4,6-disulfate unit of squid chondroitin sulfate by transsulfation from 3’-phosphoadenosine 5’-phosphosulfate. J Biol Chem 246(23):7357–7365
pubmed: 4256953
doi: 10.1016/S0021-9258(19)45894-8
Habuchi O, Moroi R, Ohtake S (2002) Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage. Anal Biochem 310(2):129–136
pubmed: 12423630
doi: 10.1016/S0003-2697(02)00277-4
He W, Zhu Y, Shirke A, Sun X, Liu J (2017) Expression of chondroitin-4- O -sulfotransferase in Escherichia coli and Pichia pastoris. Appl Microbiol Biotechnol 101(18):6919–6928
pubmed: 28761999
doi: 10.1007/s00253-017-8411-5
Ishii M, Maeda N (2008) Oversulfated chondroitin sulfate plays critical roles in the neuronal migration in the cerebral cortex. J Biol Chem 283(47):32610–32620
pubmed: 18819920
doi: 10.1074/jbc.M806331200
Ito Y, Habuchi O (2000) Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage. J Biol Chem 275(44):34728–34736
pubmed: 10871629
doi: 10.1074/jbc.M909633199
Izumikawa T, Sato B, Kitagawa H (2014) Chondroitin sulfate is indispensable for pluripotency and differentiation of mouse embryonic stem cells. Sci Rep 4:3701
pubmed: 24424429
pmcid: 3892716
doi: 10.1038/srep03701
Kawai Y, Seno N, Anno K (1966) Chondroitin polysulfate of squid cartilage. Jpn Biochem Soc 60(3):317–321
Koike T, Izumikawa T, Tamura JI, Kitagawa H (2012) Chondroitin sulfate-E fine-tunes osteoblast differentiation via ERK1/2, Smad3 and Smad1/5/8 signaling by binding to N-cadherin and cadherin-11. Biochem Biophys Res Commun Academic Press 420(3):523–529
doi: 10.1016/j.bbrc.2012.03.024
Lénon M, Ke N, Szady C, Sakhtah H, Ren G, Manta B, Causey B, Berkmen M (2020) Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions. Appl Microbiol Biotechnol 104(22):9693–9706
pubmed: 32997203
pmcid: 7595990
doi: 10.1007/s00253-020-10920-5
Li J, Sparkenbaugh EM, Su G, Zhang F, Xu Y, Xia K, He P, Baytas S, Pechauer S, Padmanabhan A, Linhardt RJ, Pawlinski R, Liu J (2020) Enzymatic synthesis of chondroitin sulfate E to attenuate bacteria lipopolysaccharide-induced organ damage. ACS Cent Sci 6(7):1199–1207
pubmed: 32724854
pmcid: 7379384
doi: 10.1021/acscentsci.0c00712
Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Factories 11(1):753
doi: 10.1186/1475-2859-11-56
Miyazaki T, Miyauchi S, Tawada A, Anada T, Suzuki O (2010) Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells. Dent Mater J 29(4):403–410
pubmed: 20610874
doi: 10.4012/dmj.2009-132
Mizumoto S, Watanabe M, Yamada S, Sugahara K (2013) Expression of n-acetylgalactosamine 4-sulfate 6-o-sulfotransferase involved in chondroitin sulfate synthesis is responsible for pulmonary metastasis. Biomed Res Int 2013:656319
pubmed: 23555092
pmcid: 3595098
doi: 10.1155/2013/656319
Nagai N, Habuchi H, Esko JD, Kimata K (2004) Stem domains of heparan sulfate 6-O-sulfotransferase are required for Golgi localization, oligomer formation and enzyme activity. J Cell Sci 117(Pt 15):3331–3341
pubmed: 15226404
doi: 10.1242/jcs.01191
Ohtake S, Ito Y, Fukuta M, Habuchi O (2001) Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene. J Biol Chem 276(47):43894–43900
pubmed: 11572857
doi: 10.1074/jbc.M104922200
Park C (2022) (2022) Visual interpretation of the meaning of k
doi: 10.1021/acs.jchemed.1c01268
Plaas AH, West LA, Wong-Palms S, Nelson FR (1998) Glycosaminoglycan sulfation in human osteoarthritis. Disease-related alterations at the non-reducing termini of chondroitin and dermatan sulfate. J Biol Chem 273(20):12642–12649
pubmed: 9575226
doi: 10.1074/jbc.273.20.12642
Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C, Huang HC, Chang YJ, Chen PS, Wu HL (2005) Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation 111(13):1627–1636
pubmed: 15795324
doi: 10.1161/01.CIR.0000160364.05405.B5
Sim JS, Im AR, Cho SM, Jang HJ, Jo JH, Kim YS (2007) Evaluation of chondroitin sulfate in shark cartilage powder as a dietary supplement: raw materials and finished products. Food Chem 101(2):532–539
doi: 10.1016/j.foodchem.2006.02.011
Sterner E, Li L, Paul P, Beaudet JM, Liu J, Linhardt RJ et al (2014Jan) Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity. Anal Bioanal Chem 406(2):525–536
pubmed: 24271188
doi: 10.1007/s00216-013-7470-4
Suzuki S, Saito H, Yamagata T, Anno K, Seno N, Kawai Y, Furuhashi T (1968) Formation of three types of disulfated disaccharides from chondroitin sulfates by chondroitinase digestion. J Biol Chem 243(7):1543–1550
pubmed: 5647269
doi: 10.1016/S0021-9258(18)93576-3
Tamura J, Nakada Y, Taniguchi K, Yamane M (2008) Synthesis of chondroitin sulfate E octasaccharide in a repeating region involving an acetamide auxiliary. Carbohydr Res 343(1):39–47
pubmed: 17950715
doi: 10.1016/j.carres.2007.09.009
Tully SE, Mabon R, Gama CI, Tsai SM, Liu X, Hsieh-Wilson LC (2004) A Chondroitin sulfate small molecule that stimulates neuronal growth. J Am Chem Soc 126(25):7736–7737
pubmed: 15212495
doi: 10.1021/ja0484045
Verkoczy LK, Guinn BA, Berinstein NL (2000) Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells. J Biol Chem 275(28):20967–20979
pubmed: 10749872
doi: 10.1074/jbc.M001866200
Volpi N (2019) Chondroitin sulfate safety and quality. Molecules 24(8):1447
pubmed: 31013685
pmcid: 6515237
doi: 10.3390/molecules24081447
Zou P, Zou K, Muramatsu H, Ichihara-Tanaka K, Habuchi O, Ohtake S, Ikematsu S, Sakuma S, Muramatsu T (2003) Glycosaminoglycan structures required for strong binding to midkine, a heparin-binding growth factor. Glycobiology 13(1):35–42
pubmed: 12634322
doi: 10.1093/glycob/cwg001