N-Glycosylation Profiles of the Green Microalgae Chlorella Zofingiensis

Year 2021, Issue: 2 - Special Issue for 2nd International Environmental Chemistry Congress, 147 - 155, 08.02.2021

Mehmet Emrah Yaman , Erdinç Aladağ , Hacı Mehmet Kayılı , Yücel Kadıoğlu , Bekir Salih

https://doi.org/10.15671/hjbc.717600

Cited By: 1

Abstract

Abstract
Nowadays, the use of microalgae species as raw materials in biopharmaceutical production is on the agenda. The reason behind this idea is that microalgae are cell factories that are able to efficiently utilize carbon dioxide for the production of numerous biologically active compounds. However, there are several problems that remain to be solved in the production of recombinant protein from microalgaes. One of the critical requirements is to produce a bio-compatible N-glycosylation profile from the secreted recombinant proteins. However, the knowledge about the glycosylation machinery and N-glycan profiles of microalgae spices are quite limited. In the study, it was aimed to characterize N-glycan profiles of a green microalgae, Chlorella zofingiensis. To achieve this, photoautotrophically grown Chlorella zofingiensis extracts including (glyco-)proteins were enzymatically deglycosylated and labelled with 2-aminobenzoic acid tag. Released N-glycans were purified with a HILIC-based approach and analyzed by MALDI-TOF(/TOF)-MS. The results showed that C. zofingiensis included oligomannosidic type N-glycan patterns. In addition, N-glycosylation profiles of C. zofingiensis by MALDI-MS revealed that most of the oligomannosidic N-glycans were phosphorylated.


Öz
Günümüzde, mikroalg türlerinin biyofarmasötik üretiminde hammadde olarak kullanımı konusu tartışılmaktadır. .Bu fikrin altında yatan temel neden mikro alglerin, biyolojik olarak aktif çok sayıda bileşenin üretimi için karbon dioksiti etkin bir şekilde kullanabilen hücre fabrikaları olmasıdır. Bununla birlikte, mikroalglerden rekombinant protein üretiminde kullanılması için çözülmesi gereken birkaç sorun mevcuttur. Kritik gereksinimlerden birisi salgılanan rekombinant proteinlerden biyo-uyumlu bir N-glikozilasyon profile üretmektir. Ancak mikroalg türlerinin glikozilasyon makineleri ve N-glikan profilleri hakkındaki bilgiler oldukça sınırlıdır. Bu çalışmada, yeşil bir mikroalg türü olan Chlorella zofingiensis ‘in N-glikan profilinin karakterize edilmesi amaçlandı. Bu amaçla (gliko-)proteinleri içeren fotoototrofik olarak yetiştirilmiş Chlorella zofingiensis ekstreleri enzimatik olarak deglikozile edildi ve 2-aminobenzoik asit etiketi ile etiketlendi. Serbest hale getirilen N-glikanlar HILIC bazlı bir yaklaşımla saflaştırıldı ve MALDI-TOF (/TOF)-MS ile analiz edildi. Sonuçlar C. zofingiensis'in oligomannozidik türde N-glikan modellerini içerdiğini göstermiştir. Ek olarak, MALDI-MS tarafından C. zofingiensis'in N-glikosilasyon profilleri, oligomannosidik N-glikanların neredeyse yarısının fosforilenmiş olduğunu ortaya koymuştur.

Keywords

Chlorella zofingiensis, microalgae N-glycosylation, MALDI-MS, mannose-6-phosphate

Supporting Institution

Türkiye Cumhuriyeti Kalkınma Bakanlığı

Project Number

2016 K121230

Thanks

Experimental stıdies were performed in SAREG (Salih Research Group) Laboratories, Department of Chemistry, Hacetteoe University (https://sareglab.org/). This work was supported by Ministry of Development-Republic of Turkey with the project Number: 2016 K121230. Bekir Salih gratefully acknowledges the Turkish Academy of Science (TUBA) for the partial financial support.

References

• 1. G. Walsh, Biopharmaceuticals: biochemistry and biotechnology, John Wiley & Sons2013.
• 2. N.K. Tripathi, A. Shrivastava, Scale up of biopharmaceuticals production. Nanoscale Fabrication, Optimization, Scale-Up and Biological Aspects of Pharmaceutical Nanotechnology, Elsevier2018, pp. 133-172.
• 3. A.F. Jozala, D.C. Geraldes, L.L. Tundisi, V.d.A. Feitosa, C.A. Breyer, S.L. Cardoso, P.G. Mazzola, L.d. Oliveira-Nascimento, C.d.O. Rangel-Yagui, P.d.O. Magalhães, Biopharmaceuticals from microorganisms: from production to purification, Braz. J. Microbiol. 47 (2016) 51-63.
• 4. D.C. Andersen, L. Krummen, Recombinant protein expression for therapeutic applications, Curr. Opin. Biotechnol. 13(2) (2002) 117-123.
• 5. R. Kothari, A. Pandey, S. Ahmad, A. Kumar, V.V. Pathak, V. Tyagi, Microalgal cultivation for value-added products: a critical enviro-economical assessment, 3 Biotech 7(4) (2017) 243.
• 6. O. Pignolet, S. Jubeau, C. Vaca-Garcia, P. Michaud, Highly valuable microalgae: biochemical and topological aspects, J. Ind. Microbiol. Biotechnol. 40(8) (2013) 781-796.
• 7. R. Mócsai, R. Figl, C. Troschl, R. Strasser, E. Svehla, M. Windwarder, A. Thader, F. Altmann, N-glycans of the microalga Chlorella vulgaris are of the oligomannosidic type but highly methylated, Sci. Rep. 9(1) (2019) 1-8.
• 8. E. Becker, Micro-algae as a source of protein, Biotechnol. Adv. 25(2) (2007) 207-210.
• 9. P. Jacobs, N. Callewaert, N-glycosylation engineering of biopharmaceutical expression systems, Curr. Mol. Med. 9(7) (2009) 774-800.
• 10. L. Zhang, S. Luo, B. Zhang, Glycan analysis of therapeutic glycoproteins, MAbs, Taylor & Francis, (2016), 205-215.
• 11. G.Y. Wiederschain, Essentials of glycobiology, Springer Science & Business Media, (2009).
• 12. P. Stanley, R.D. Cummings, Structures common to different glycans. Essentials of Glycobiology [Internet]. 3rd edition, Cold Spring Harbor Laboratory Press2017.
• 13. H. Lis, N. Sharon, Protein glycosylation: structural and functional aspects, Eur. J. Biochem. 218(1) (1993) 1-27.
• 14. P. Ahmad, F. Bano, Posttranslational Modifications in Algae: Role in Stress Response and Biopharmaceutical Production. Protein Modificomics, Elsevier2019, pp. 313-337.
• 15. M. Aebi, N-linked protein glycosylation in the ER, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1833(11) (2013) 2430-2437.
• 16. D. Ghaderi, R.E. Taylor, V. Padler-Karavani, S. Diaz, A. Varki, Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins, Nat. Biotechnol. 28(8) (2010) 863.
• 17. F. Li, J.X. Zhou, X. Yang, T. Tressel, B. Lee, Current therapeutic antibody production and process optimization, BioProcessing Journal 5(4) (2007) 16.
• 18. D. Ghaderi, M. Zhang, N. Hurtado-Ziola, A. Varki, Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, Biotechnol. Genet. Eng. Rev. 28(1) (2012) 147-176.
• 19. Z. Zhang, D. Sun, T. Wu, Y. Li, Y. Lee, J. Liu, F. Chen, The synergistic energy and carbon metabolism under mixotrophic cultivation reveals the coordination between photosynthesis and aerobic respiration in Chlorella zofingiensis, Algal research 25 (2017) 109-116.
• 20. P. Feng, Z. Deng, Z. Hu, L. Fan, Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors, Bioresour. Technol. 102(22) (2011) 10577-10584.
• 21. J. Liu, Z. Sun, H. Gerken, Z. Liu, Y. Jiang, F. Chen, Chlorella zofingiensis as an alternative microalgal producer of astaxanthin: biology and industrial potential, Mar. Drugs 12(6) (2014) 3487-3515.
• 22. B. Yang, J. Liu, Y. Jiang, F. Chen, Chlorella species as hosts for genetic engineering and expression of heterologous proteins: progress, challenge and perspective, Biotechnol. J. 11(10) (2016) 1244-1261.
• 23. J. Huang, J. Liu, Y. Li, F. Chen, ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFİNGİENSİS (CHLOROPHYTA) 1, J. Phycol. 44(3) (2008) 684-690.
• 24. J. Liu, Z. Sun, H. Gerken, J. Huang, Y. Jiang, F. Chen, Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker, Appl. Microbiol. Biotechnol. 98(11) (2014) 5069-5079.
• 25. T. Mamedov, V. Yusibov, Green algae Chlamydomonas reinhardtii possess endogenous sialylated N‐glycans, FEBS Open Bio 1(1) (2011) 15-22.
• 26. O. Levy-Ontman, M. Fisher, Y. Shotland, Y. Weinstein, Y. Tekoah, S. Arad, Genes involved in the endoplasmic reticulum N-glycosylation pathway of the red microalga Porphyridium sp.: a bioinformatic study, Int. J. Mol. Sci. 15(2) (2014) 2305-2326.
• 27. E. Mathieu-Rivet, M. Scholz, C. Arias, F. Dardelle, S. Schulze, F. Le Mauff, G. Teo, A.K. Hochmal, A. Blanco-Rivero, C. Loutelier-Bourhis, Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures, Mol. Cell. Proteomics 12(11) (2013) 3160-3183.
• 28. M.H. Selman, M. Hemayatkar, A.M. Deelder, M. Wuhrer, Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides, Anal. Chem. 83(7) (2011) 2492-2499.
• 29. A. Ceroni, K. Maass, H. Geyer, R. Geyer, A. Dell, S.M. Haslam, GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans, J. Proteome Res. 7(4) (2008) 1650-1659.
• 30. V. Gomord, A.C. Fitchette, L. Menu‐Bouaouiche, C. Saint‐Jore‐Dupas, C. Plasson, D. Michaud, L. Faye, Plant‐specific glycosylation patterns in the context of therapeutic protein production, Plant Biotechnol. J. 8(5) (2010) 564-587.
• 31. O. Levy-Ontman, S. Arad, D.J. Harvey, T.B. Parsons, A. Fairbanks, Y. Tekoah, Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp, J. Biol. Chem. 286(24) (2011) 21340-21352.
• 32. I.B. Wilson, F. Altmann, Structural analysis of N-glycans from allergenic grass, ragweed and tree pollens: core α1, 3-linked fucose and xylose present in all pollens examined, Glycoconj. J. 15(11) (1998) 1055-1070.