Malaysian Journal of Analytical Sciences Vol 21 No 6 (2017): 1210 - 1218

FLUORESCENCE AND EVAPORATIVE LIGHT SCATTERING HPLC PROFILING OF INTRACELLULAR ASPARAGINE (N)-LINKED OLIGOSACCHARIDES FROM Saccharomyces cerevisiae USING alg8 MUTANT

(Pemprofilan KCPT Pendarfluor dan Penyerakan Cahaya Sejatan Oligosakarida Terpaut-(N) Asparagina Intrasel daripada Saccharomyces cerevisiae Menggunakan Mutan alg8)

Iqbal Jalaludin¹, Amirul Husna Sudin¹, Ikram Mohd Said^1,2, Kamalrul Azlan Azizan², Syarul Nataqain Baharum², Abdul Munir Abdul Murad³, Farah Diba Abu Bakar³, Nor Muhammad Mahadi⁴, Mark R. Wormald⁵,

Dominic S. Alonzi⁵, Mukram Mohamed Mackeen^1,2*

¹School of Chemical Sciences and Food Technology

²Institute of Systems Biology (INBIOSIS)

³School of Biosciences and Biotechnology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

⁴Malaysia Genome Institute,

Jalan Bangi, 43000 Kajang, Selangor, Malaysia

⁵Oxford Glycobiology Institute,

University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

*Corresponding author: mukram.mackeen@ukm.edu.my

Received: 21 June 2017; Accepted: 30 September 2017

Abstract

N-glycans are biologically important oligosaccharides associated with the asparagine residue that may exist in protein-bound or unbound forms in all eukaryotes (including yeasts) and some bacteria. The- core structure of these oligosaccharides is based on the trimannosyl chitobiose structure resulting from cellular N-glycosylation. Preparative-scale amounts of these oligosaccharides are important for chemical, structural and functional studies due to their biological significance. Therefore, we explored a biochemical approach of oligosaccharide preparation using mutant-derived monoglucosylated lipid-linked oligosaccharides (LLOs) required for the assembly of N-linked glycoproteins and non-monoglucosylated free-oligosaccharides (fOSs) from misfolded N-linked glycoproteins using an N-glycosylation (alg) mutant of Saccharomyces cerevisiae. Oligosaccharide extracts of fOSs and LLOs from the alg8 S. cerevisiae mutant lacking the ALG8 gene were profiled using fluorescence- and evaporative light scattering-based HPLC. LLOs did not produce accumulated levels of the target mutant- related monoglucosylated (Glc₁Man₉GlcNAc₂) at 100 ml scale. However, it was possible to detect truncated oligomannose (paucimannose) structures in the fOSs of the alg8 mutant.

Keywords: N-linked glycosylation, N-glycans, Free oligosaccharides, Lipid-linked oligosaccharides, Saccharomyces cerevisiae

Abstrak

N-glikan merupakan oligosakarida yang penting dalam biologi yang bersekutu dengan residu asparagina yang hadir dalam keadaan terikat atau tidak terikat kepada protein dalam semua eukariot (termasuk yis) dan sesetengah bakteria. Struktur asas oligosakarida ini adalah berdasarkan struktur kitobiose trimanosa yang terhasil melalui pengglikosilan-N. Oligosakarida ini penting untuk kajian kimia, struktur dan fungsi disebabkan kepentingan biologinya. Oleh itu, kami telah mengkaji kaedah penyediaan oligosakarida dengan pendekatan biokimia menggunakan yis mutan (alg) Saccharomyces cerevisiae yang menghasilkan monoglukosa oligosakarida terpaut-lipid (LLO) yang diperlukan dalam pengumpulan glikoprotein terpaut-N dan bukan-monoglukosa oligosakarida bebas (fOS) daripada glikoprotein terpaut-N silap lipatan. Ekstrak oligosakarida fOS dan LLO daripada mutan alg8 S. cerevisiae tanpa gen ALG8 telah diprofil menggunakan KCPT-pendarfluor dan pengesan penyerakan cahaya sejatan. LLO didapati tidak menghasilkan sebatian sasaran monoglukosa (Glc₁Man₉GlcNAc₂) berkaitan-mutan pada skala 100 ml. Walau bagaimanapun, struktur fOS oligomanosa terpangkas (pausimanosa) boleh dikesan daripada mutan alg8.

Kata kunci: pengglikosilan terpaut-N, N-glikan, Oligosakarida bebas, Oligosakarida terpaut-lipid, Saccharomyces cerevisiae

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