Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices

• Published in: Cell Vol. 187; no. 8; pp. 1907 - 1921.e16
• Main Authors: Dai, Jin, Ma, Meisheng, Niu, Qingwei, Eisert, Robyn J., Wang, Xiangli, Das, Poulomi, Lechtreck, Karl F., Dutcher, Susan K., Zhang, Rui, Brown, Alan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.04.2024
• Subjects: cilia; Cilia - metabolism; cryo-EM; Glycoproteins - chemistry; Glycosylation; hydroxyproline; Hydroxyproline - chemistry; Hydroxyproline - metabolism; ion channels; mass spectrometry; mastigoneme; O-linked glycosylation; Plants - metabolism; polycystic kidney disease; post-translational modifications; mastigoneme; polycystic kidney disease; O-linked glycosylation; mass spectrometry; cilia; post-translational modifications; cryo-EM; ion channels; hydroxyproline
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2024.03.005

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes. [Display omitted] •Cryo-EM reveals the composition and structure of an algal mastigoneme filament•MST3 anchors mastigonemes to ciliary membranes via a PKD-like domain aided by SIP•O-linked hydroxyproline glycosylation sheaths PPII helices in a carbohydrate casing•Serine galactosylation hydrogen bonds with the poly(hydroxyproline) backbone Structure of the native C. reinhardtii mastigoneme allows visualization of the glycan patterns associated with different types of proline repetition in two different hydroxyproline-rich glycoproteins, providing insight into the hydroxyproline glycosylation code. The structure also provides an explanation for how mastigonemes are tethered to ciliary membranes.