RESUMO
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.
Assuntos
Diatomáceas/enzimologia , Retículo Endoplasmático/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Biologia Computacional/métodos , Cricetinae , Cricetulus , Diatomáceas/genética , Retículo Endoplasmático/genética , Teste de Complementação Genética/métodos , Estudo de Associação Genômica Ampla/métodos , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , N-Acetilglucosaminiltransferases/genética , Polissacarídeos/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Extraction of natural substances and chemical synthesis are the main sources of pharmaceutical molecules. When possible, one may transfer the gene of the molecule in living cells creating individual factories producing on demand and in a safe way the requested molecule. Today, bacteria, yeast, mammalian cells and plants constitute the main plate-forms for various commercial products. Microalgae present numerous advantages and could offer a powerful tool for the production of commercial molecules in a near future.
