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

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.

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells.

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.