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 (SHDRA), but the function of the encoded protein remains unknown. We report that TMEM260 is an ER-located protein O-mannosyltransferase that selectively glycosylates defined extracellular immunoglobulin, plexin, transcription factor (IPT) domains of the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knock out in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies a new, receptor-specific O-mannosylation pathway that serves critical functions during epithelial morphogenesis.

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

“…3E ). Taken together, these results, which agree with previous studies (13,17,18), validated the approach and showed that our O-Man deconstruction cell library is suitable for analyses of pathway-specific substrate specificities.…”

“…2C ). As recently described, IPT domains are TMEM260 substrates (18), and here, we identified 7 members (PLXNA1-4, PLXNB2-3, PLXND1) of the plexin family, as well as cMET and RON receptors, which account to 9 proteins (of n=12 predicted) with O-Man on IPT domains. Interestingly, we also identified 26 proteins with O-Man on distinct Ig-like domains, including Ig-like C2-type domains (n=5), Fibronectin type-III domains (n=4) and Ig-like V-type domains (n=2), indicating that O-Man glycosyltransferases selectivity is not limited to the Ig-like fold subclasses defined by EC- and IPT-domains.…”

“…Remarkably, our platform allowed detection of O-Man glycosylation on several other proteins, both in regions with no assigned domain/unstructured regions or annotated domains (UniProt). Few of these candidates were also identified previously, such as integrins and, interestingly, a site on β-DG where O-Man glycosylation was abolished by KO of TMEM260 (18). Here, we also identified members of α-integrins which where O-Man glycosylated in the HEK293 TMTC1-4 cell line, while ITGB1 showed site-specific glycosylation in both HEK293 TMTC1-4 and HEK293 TMEM260 .…”

“…Furthermore, we assessed the approach for differential labelling of peptides by stable isotopes. In our most recent study (18), we employed diethyl labeling (DEL) for two-plex experiments, which is a cost-effective, flexible, and readily available method that utilizes 13 C isotopologues of acetaldehyde, as an alternative to dimethyl labelling (DML) (39). In agreement with previous studies (39), the use of diethyl labelling improved quantification accuracy and retention of short hydrophilic O-Man glycopeptides on C18 columns ( Fig.…”

“…The TMTC1-4 enzymes are dedicated to the cadherin superfamily of adhesion molecules and selectively glycosylate highly conserved Ser/Thr residues with O-Man in two β-strands (B and G) of extracellular cadherin (EC) domains (17), which are functional units that direct homo/heterophilic cis- and trans -binding important for cell-cell interactions (21,22). 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).…”