Marie-Estelle Losfeld

Last Name

Losfeld

First Name

Marie-Estelle

ORCID

0000-0002-8029-7822

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Publications 1 - 7 of 7

Microfluidic mimicry of the Golgi-linked N-glycosylation machinery
Isenrich, Florin N.; Losfeld, Marie-Estelle; Aebi, Markus; et al. (2025)
Lab on a Chip
The complexity of the eukaryotic glycosylation machinery hinders the development of cell-free protein glycosylation since in vitro methods struggle to simulate the natural environment of the glycosylation machinery. Microfluidic technologies have the potential to address this limitation due to their ability to control glycosylation parameters, such as enzyme/substrate concentrations and fluxes, in a rapid and precise manner. However, due to the complexity and sensitivity of the numerous components of the glycosylation machinery, very few "glycobiology-on-a-chip" systems have been proposed or reported in the literature. Herein, we describe the design, fabrication and proof-of-concept of a droplet-based microfluidic platform able to mimic N-linked glycan processing along the secretory pathway. Within a single microfluidic device, glycoproteins and glycosylation enzymes are encapsulated and incubated in water-in-oil droplets. Additional glycosylation enzymes are subsequently supplied to these droplets via picoinjection, allowing further glycoprotein processing in a user-defined manner. After system validation, the platform is used to perform two spatiotemporally separated consecutive enzymatic N-glycan modifications, mirroring the transition between the endoplasmic reticulum and early Golgi.
Glycosylation network mapping and site-specific glycan maturation in vivo
Losfeld, Marie-Estelle; Scibona, Ernesto; Lin, Chia-wei; et al. (2022)
iScience
Glycoprotein processing along a complex highly compartmentalized pathway is a hallmark of eukaryotic cells. We followed the kinetics of intracellular, site-specific glycan processing of a model protein with five distinct N-glycosylation sites and deduced a mathematical model of the secretory pathway that describes a complex set of processing reactions localized in defined intracellular compartments such as the endoplasmic reticulum the Golgi, or the lysosome. The model was able to accommodate site-specific N-glycan processing and we identified phosphorylated glycan structures of the mannose-6-phosphate pathway responsible for the lysosomal sorting of the glycoprotein. Importantly, our model protein can take different routes of the cellular secretory pathway, resulting in an increased glycan complexity of the secreted protein.
N-glycoprotein: role of protein structure on glycan microheterogeneity
Losfeld, Marie-Estelle; Lin, Chia-wei; Grant, Oliver C.; et al. (2015)
Glycobiology
A hierarchical structure in the N-glycosylation process governs the N-glycosylation output: prolonged cultivation induces glycoenzymes expression variations that are reflected in the cellular N-glycome but not in the protein and site-specific glycoprofile of CHO cells
Arigoni-Affolter, Ilaria; Losfeld, Marie-Estelle; Hennig, René; et al. (2024)
Glycobiology
N-glycosylation is a central component in the modification of secretory proteins. One characteristic of this process is a heterogeneous output. The heterogeneity is the result of both structural constraints of the glycoprotein as well as the composition of the cellular glycosylation machinery. Empirical data addressing correlations between glycosylation output and glycosylation machinery composition are seldom due to the low abundance of glycoenzymes. We assessed how differences in the glycoenzyme expression affected the N-glycosylation output at a cellular as well as at a protein-specific level. Our results showed that cellular N-glycome changes could be correlated with the variation of glycoenzyme expression, whereas at the protein level differential responses to glycoenzymes alterations were observed. We therefore identified a hierarchical structure in the N-glycosylation process: the enzyme levels in this complex pathway determine its capacity (reflected in the N-glycome), while protein-specific parameters determine the glycosite-specificity. What emerges is a highly variable and adaptable protein modification system that represents a hallmark of eukaryotic cells.
Glycan–protein interactions determine kinetics of N-glycan remodeling
Mathew, Corina; Weiß, R. Gregor; Giese, Christoph; et al. (2021)
RSC Chemical Biology
A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways.
Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae
Poljak, Kristina; Selevsek, Nathalie; Ngwa, Elsy; et al. (2018)
Molecular & Cellular Proteomics
Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in Saccharomyces cerevisiae by coupling stable isotope labeling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly.
N-Glycosylation Enhances Conformational Flexibility of Protein Disulfide Isomerase Revealed by Microsecond Molecular Dynamics and Markov State Modeling
Weiss, R. Gregor; Losfeld, Marie-Estelle; Aebi, Markus; et al. (2021)
The Journal of Physical Chemistry B
Secreted proteins of eukaryotes are decorated with branched carbohydrate oligomers called glycans. This fact is only starting to be considered for in silico investigations of protein dynamics. Using all-atom molecular dynamics (MD) simulations and Markov state modeling (MSM), we unveil the influence of glycans on the conformational flexibility of the multidomain protein disulfide isomerase (PDI), which is a ubiquitous chaperone in the endoplasmic reticulum (ER). Yeast PDI (yPDI) from Saccharomyces cerevisiae is glycosylated at asparagine side chains and the knowledge of its five modified sites enables a realistic computational modeling. We compare simulations of glycosylated and unglycosylated yPDI and find that the presence of glycan-glycan and glycan-protein interactions influences the flexibility of PDI in different ways. For example, glycosylation reduces interdomain interactions, shifting the conformational ensemble toward more open, extended structures. In addition, we compare our results on yPDI with structural information of homologous proteins such as human PDI (hPDI), which is natively unglycosylated. Interestingly, hPDI lacks a surface recess that is present in yPDI. We find that glycosylation of yPDI facilitates its catalytic site to reach close to this surface recess. Hence, this might point to a possible functional relevance of glycosylation in yeast to act on substrates, while glycosylation seems redundant for the human homologous protein. We conclude that glycosylation is fundamental for protein dynamics, making it a necessity for a truthful representation of the flexibility and function in in silico studies of glycoproteins.

Publications 1 - 7 of 7

Marie-Estelle Losfeld

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