N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme

Abstract

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

FIGURE 1.

N-Glycosylation pathway in P. tricornutum based on bioinformatic analysis of the genome database. Sequences of the N-glycosylation pathway identified in the P. tricornutum genome are numbered in bold type. Glc, glucose; PP-Dol, dolicholpyrophosphate; Man, mannose; GlcNAc, N-acetylglucosamine; ALG, asparagine linked glycosylation.

FIGURE 2.

Catalytic amino acids are very conserved in the putative GnT I protein from P. tricornutum. Protein sequences alignment between rabbit (1FOA) and P. tricornutum, as proposed by the Swiss-Pdb viewer program (56). Secondary structural elements are represented above the alignment for the P. tricornutum GnT I and below the alignment for the rabbit GnT I with a bold right arrow as the β strand and a looped line as the α helix. Essential residues for the binding of the donor substrate (UDP-GlcNAc) are indicated by arrowheads above the alignment: in black when identical and in white when not. Rabbit GnT I disulfide bridges are also numbered. The figure was created with the Espript program (57).

FIGURE 3.

P. tricornutum glycoproteins harbor N-linked oligosaccharides. A, affinodetection using concanavalin A (Con A) and immunodetection using antibodies raised against the core β(1,2)-xylose (anti-Xyl) and core α(1,3)-fucose (anti-Fuc) epitopes of proteins isolated from green onion used as a positive control (lanes 1) and from P. tricornutum (lanes 2). B, affinodetection by concanavalin A of proteins extracted from P. tricornutum treated (+) or not (−) with Endo H and PNGase F. C, affinodetection with RCA 120 of P. tricornutum proteins treated (+) or not (−) with bovine β(1,4)-galactosyltransferase. Plant-derived IgG was used as a positive control of the galactose transfer efficiency (34). The arrows indicate the migration of heavy (H) and light (L) chains.

FIGURE 4.

High mannose type N-glycans are the main oligosaccharides N-linked to P. tricornutum proteins. A, MALDI-TOF mass spectrum of N-linked glycans released by PNGase A from glycoproteins of P. tricornutum and labeled with 2-AB. B, MALDI-TOF mass spectrum of the pool of N-glycans after treatment with jack bean α-mannosidase. C, MALDI-TOF mass spectrum of 2-AB-labeled N-linked glycans released by PNGase F from glycoproteins of P. tricornutum. Man-3 to Man-9 are the paucimannose and high mannose type N-glycans Man₃GlcNAc₂ to Man₉GlcNAc₂. *, contaminants; ■, potassium adducts.

FIGURE 5.

Expression of the GnT I gene in P. tricornutum and in CHO Lec1 mutant. A, expression of the GnT I gene of P. tricornutum by real time quantitative PCR over a 1-month period. The relative gene expression was normalized to two reference genes encoding for histone H4 and ribosomal protein small subunit 30 S. B, Western blot analysis using anti-V5 antibodies of proteins isolated from two transformants (lanes 2 and 4) of CHO Lec1 expressing the P. tricornutum GnT I fused to a V5 tag. GnT I was detected at the expected molecular mass of 56 kDa.

FIGURE 6.

P. tricornutum GnT I complements N-glycan maturation deficiency in CHO Lec1 mutant. MALDI-TOF mass spectra of glycans N-linked to proteins extracted from CHO cells. A, CHO Lec1 mutant; B, CHO wild type; and C, transformant 4 of CHO Lec1 mutant complemented with P. tricornutum GnT I gene. Man-4 to Man-9 are the high mannose type N-glycans Man₄GlcNAc₂ to Man₉GlcNAc₂ 70). Black square, GlcNAc; gray circle, Man; white circle, Gal; gray triangle, fucose.

FIGURE 7.

GnT I are predicted in other microalgae. Phylogenetic tree of GnT I from algae, plants, and animals based on the maximum likelihood method. The scale bar (0.4) represents the number of amino acid residue substitutions per site. Microalgal GnT I sequences are indicated in bold type. Accession numbers for the different reference sequences are indicated under “Experimental Procedures.”