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

Mutations in the 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 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains.

In-Depth Profiling of -Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated -glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of -glycans in biological material.

Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells.

Endoplasmic reticulum transmembrane protein TMTC3 contributes to O-mannosylation of E-cadherin, cellular adherence, and embryonic gastrulation.

Protein glycosylation plays essential roles in protein structure, stability, and activity such as cell adhesion. The cadherin superfamily of adhesion molecules carry O-linked mannose glycans at conserved sites and it was recently demonstrated that the ransembrane and tetratricopeptide repeat-ontaining proteins 1-4 (TMTC1-4) gene products contribute to the addition of these O-linked mannoses. Here, biochemical, cell biological, and organismal analysis was used to determine that TMTC3 supports the O-mannosylation of E-cadherin, cellular adhesion, and embryonic gastrulation.

A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation.

Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented.

Multiple distinct O-Mannosylation pathways in eukaryotes.

Protein O-mannosylation (O-Man), originally discovered in yeast five decades ago, is an important post-translational modification (PTM) conserved from bacteria to humans, but not found in plants or nematodes. Until recently, the homologous family of ER-located protein O-mannosyl transferases (PMT1-7 in yeast; POMT1/POMT2 in humans), were the only known enzymes involved in directing O-Man biosynthesis in eukaryotes. However, recent studies demonstrate the existence of multiple distinct O-Man glycosylation pathways indicating that the genetic and biosynthetic regulation of O-Man in eukaryotes is more complex than previously envisioned.

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion.

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the genes, which were found to be imperative for cdh and pcdh O-Man glycosylation.

Mammalian -mannosylation of cadherins and plexins is independent of protein -mannosyltransferases 1 and 2.

Protein mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries linked mannose (-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the Man glycans. Deficiencies in enzymes catalyzing Man biosynthesis, including the two human protein mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae.

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammalsO-mannosylation is the only type ofO-glycosylation. In an essential step toward the full understanding of proteinO-mannosylation we mapped theO-mannose glycoproteome in baker's yeast.

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast.

Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system.