N-Glycosylation Profiles of the Green Microalgae Chlorella Zofingiensis

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

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