Green algae Chlamydomonas reinhardtii possess endogenous sialylated N-glycans

Abstract

Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii, a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins.

Keywords: 2-AB, 2-aminobenzamide; CST, CMP-sialic acid transporter; Chlamydomonas reinhardtii; ER, endoplasmic reticulum; HPLC-FLD, high-performance liquid chromatography using fluorescence detection; HRP, horseradish peroxidase; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; MS, mass spectrometry; Mass spectrometry analysis; N-Glycan structure; PBS, phosphate buffered saline; PVDF, polyvinylidene fluoride; RCA, Ricinus communis agglutinin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Sialic acid; TBS, Tris-buffered saline; hEPO, human erythropoietin.

Fig. 1

Glycan profiles of extracts from C. reinhardtii. Cleavage of N-linked carbohydrates from glycoproteins of the total soluble fraction of C. reinhardtii was performed using N-glycosidase A (PNGase A, Roche). The released glycan pool was extracted and brought to dryness by centrifugal concentration. Analysis of N-linked oligosaccharides by HPLC-FLD was performed as described in Section 2. (A) Profile of glycans released from C. reinhardtii total soluble glycoproteins by PNGase A. (B) Profile of glycans released from C. reinhardtii total membrane glycoproteins by PNGase A. (C) Selected MS spectrum of ion at mass/charge (m/z) 2036.8 corresponding to Neu5Ac. The peak fraction N6 (from total membrane fraction) was analyzed using MALDI-TOF-MS. Number of sugars [4,4,1,1,0] in the structure: 4 (N-acetylhexosamines), 4 (hexose, mannose or galactose), 1 (fucose), 1 (Neu5Ac), 0 (Neu5Gc).

Fig. 2

Lectin blotting analysis of C. reinhardtii proteins using RCA₁₂₀. Eight microgram of C. reinhardtii total soluble protein (CS), 8 μg of C. reinhardtii total membrane protein (CM), 8 μg of total soluble proteins from N. benthamiana (PC), 8 μg of total soluble proteins from N. benthamiana infiltrated with human β1,4-galactosyltransferase (PT), and 250 ng of fibrinogen (F) were loaded onto the SDS–PAGE gel followed by a Western blot analysis. Galactosylated proteins were detected using the RCA₁₂₀ lectin at 10 μg/ml in PBS with 0.05% Tween. Fibrinogen (Cat. No. F8630, Sigma, St. Louis, MO) was used as a positive control. (A) After blocking with Carbo-free blocking buffer, membranes were incubated with the 1,4-galactose residue-specific lectin followed by avidin plus biotinylated HRP using ABC kit (Vector Laboratories); (B) after blocking, the membrane was incubated with avidin plus biotinylated HRP using ABC kit. M – protein marker. Arrows show non-specific signals due to algae-derived biotin staining.

Fig. 3

Lectin blotting analysis of C. reinhardtii proteins using MAA and SNA-I. Twenty microgram of C. reinhardtii total soluble protein (CS) and 20 μg of C. reinhardtii total membrane protein (CM) along with 2 μg fibrinogen (F), treated with sialidase (A, as indicated) and non-treated (B and C), were loaded onto the SDS–PAGE gel followed by a Western blot analysis. Sialic acid was detected using the MAA and SNA-I lectins at 50 μg/ml and 10 μg/ml, respectively, in the reaction buffer (RB) (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂). Fibrinogen was used as a positive control. (A and B) After blocking with Carbo-free blocking buffer, the membrane was incubated with the lectins followed by avidin plus biotinylated HRP using ABC kit; (C) after blocking, the membrane was incubated with avidin plus biotinylated HRP using ABC kit. M – protein marker. Arrows show non-specific signals due to algae-derived biotin staining.