N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Abstract

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

Human protein paucimannosylation: cues from the eukaryotic kingdoms

“…The N ‐acetyl‐β‐hexosaminidases (Hex) (EC 3.2.1.52), which form a family of glycoside hydrolases expressed widely across Plantae (Intra, Pavesi, & Horner, ), are hydrolytic enzymes directly responsible for generating plant PMPs from hybrid‐/complex‐type N ‐glycoprotein intermediates (Li & Li, ; Oikawa et al ., ; Gutternigg et al ., ). Three paralogous genes, HEXO1‐3 , encoding various Hex isoenzyme variants, have been reported in A. thaliana (Strasser et al ., ; Liebminger et al ., ), Nicotiana benthamiana (Castilho et al ., ; Shin et al ., ) and L. japonicus (Pedersen et al ., ). Two of these isoenzymes, the vacuolar HEXO1 and the plasma membrane‐located HEXO3 were demonstrated to contribute equally to PMP formation in A. thaliana roots whereas HEXO3 is responsible for PMP production in A. thaliana leaves.…”

“…LjHEXO1 and LjHEXO2 ), homologous to A. thaliana HEXO1 and HEXO3, respectively . L. japonicus seeds carrying HEXO deficiencies showed a dramatic reduction of PMPs thereby confirming the association between plant Hex and PMPs (Pedersen et al ., ). Due to their similar substrate preferences and enzymatic activities, the Hex isoenzymes may compensate for each other provided that they share subcellular location(s) (Strasser et al ., ; Gutternigg et al ., ).…”

The Golgi Localization of GnTI Requires a Polar Amino Acid Residue within Its Transmembrane Domain

“…Importantly, the central function of GnTI in complex N-glycan formation is conserved in higher eukaryotes and GnTI deficiency is embryo-lethal in mammals (Ioffe and Stanley, 1994). GnTI-deficient Lotus japonicus displays severe growth defects and rice (Oryza sativa) lacking GnTI shows developmental phenotypes leading to early lethality (Fanata et al, 2013;Pedersen et al, 2017). Arabidopsis (Arabidopsis thaliana) lacking GnTI is viable, but less tolerant to abiotic stress and display severe growth defects when combined with mutations that affect plant cell wall formation (von Schaewen et al, 1993;Kang et al, 2008).…”

Venoms of Neotropical wasps lack cross-reactive carbohydrate determinants enabling reliable protein-based specific IgE determination

“…Recent findings suggest developmental implications of the a1,3-core fucosylation in plants, supporting also the neural relevance of the epitope in insects. 9 Against this background not the absence of CCDs but their presence on HBV and YJV might represent the biological peculiarity.…”