Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii.

Nannan Xu,Lara Hoepfner,Marzieh Karimi,Simon Kelterborn,Lu-Ning Liu,Oliver Bäumchen,Peter Hegemann,Kaiyao Huang,Julia Beißel,Antoine Girot,Longsheng Zhao,Martin Scholz,Michael Hippler,Anne Oltmanns

doi:10.7554/elife.58805

Abstract

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

Highlights

N-glycosylation, as one of the major post-translational modifications, takes place along the ER/Golgi secretion route and most N-linked glycans are found on proteins facing the extracellular space
To test whether N-glycan maturation in Golgi is important for flagellar surface motility in C. reinhardtii, two insertional mutants (IM) such as IMMan1A, IMXylT1A and their double mutant IMMan1AxIMXylT1A were studied
Microbead binding was found diminished in IM strains, implying that the flagellar surface has an altered affinity toward microbeads

Summary

Introduction

N-glycosylation, as one of the major post-translational modifications, takes place along the ER/Golgi secretion route and most N-linked glycans are found on proteins facing the extracellular space. Initial steps of N-glycosylation in the ER are highly conserved among most eukaryotes and consist of the synthesis of a common prebuilt N-glycan precursor onto a dolichol phosphate. Following the transfer of the glycan precursor onto the asparagine of the consensus sequence N-X-S/T of a nascent protein (where X can be any amino acid except proline), the glycoprotein is folded by the glycan recognizing chaperones Calnexin and Calreticulin (Stanley and Taniguchi, 2017).

Methods

Results

Conclusion