The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase

Abstract

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Global view of domain-specific O-linked mannose glycosylation in glycoengineered cells

“…We first aimed to improve these three key steps in our established workflow (13,17) for identification and quantification of O-Man proteins and establish an optimized workflow for O-Man glycoproteomics. Previous glycoproteomics studies have demonstrated that the majority (72%) of proteins undergoing O-Man glycosylation consists of single-pass or multi-pass transmembrane proteins (13,(17)(18)(19)35) (Fig. S1A).…”

“…HCD/ETciD-based bottom-up analyses identified 3882 glyco-PSMs from 120 unique O-Man proteins (Fig. 2A), 33 of which have not been described as O-Man substrates before (13,(17)(18)(19)35).…”

“…More recently, we have shown that mammals have evolved multiple biosynthetic pathways for O-Man initiation based on the GT-CA type enzymes TMTC1, TMTC2, TMTC3, TMTC4 and TMEM260 (17)(18)(19)(20). The TMTC1-4 and TMEM260 enzymes, classified in the CAZy database as GT105 and GT117, respectively, serve distinct classes of extracellular Ig-like protein domains found on transmembrane adhesion molecules (cadherins) and receptors (plexins) (17,18). Unlike α-DG, O-Man on Ig-like domains is not elongated into complex structures but is rather present as single α-linked mannose monosaccharides on β-strands of the Ig-like domains.…”

“…The recently identified TMEM260 enzyme selectively serves extracellular immunoglobulin, plexin, transcription factor (IPT) domains found among a subset of structurally related plasma membrane receptors, including hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (MST1R or Recepteur d'Origine Nantais, RON) and members of the plexin family (17,18). The IPT domain is a distinct subclass of the Ig-like fold, and while the functions of their O-Man glycans, located on conserved Ser/Thr residues within β-strands of IPT domains, remains unknown, TMEM260-driven O-Man glycosylation appears to be critical for receptor maturation and epithelial morphogenesis (18). O-Man glycosylation fulfils important roles during development, and dysregulation of O-Man initiation is associated with severe developmental disorders in humans.…”

“…Mutations in POMT1/POMT2 genes is linked to a subclass of congenital muscular dystrophies known as αdystroglycanopathies, characterized by progressive muscular degeneration and developmental abnormalities in brain and eyes (23)(24)(25). Genetic defects in TMTC1-4 are primarily associated with neurological disorders, including brain malformation and hearing loss (26)(27)(28)(29)(30) while bi-allelic mutations in TMEM260 underlie the SHDRA syndrome, characterized by congenital heart defects, kidney phenotypes and neurodevelopmental disorders (18,31,32).…”