Biology subjects

Halim, A.

Publications and source records attributed to Halim, A..

5 recordsLinked to original sources

bioRxiv2026

Structure and mechanism of the human TMEM260 O-mannosyltransferase

Protein O-linked mannose (O-Man) glycosylation is essential for mammalian development, and mutations in its biosynthetic glycosyltransferases cause severe muscular, neurological and cardiac disorders. Despite its biological and clinical importance, the structural basis of mammalian O-Man biosynthesis has remained unknown. Here we report cryo-electron microscopy (cryo-EM) structures of human TMEM260, an endoplasmic reticulum glycosyltransferase that selectively catalyzes O-mannosylation of semaphorin plexin receptors and receptor tyrosine kinases cMET and RON, key regulators of cell guidance and migration. Structures of TMEM260 in a ternary complex with its natural donor dolichyl-phosphate-{beta}-mannose (Dol-P-Man) and an acceptor peptide derived from plexin-B2, together with binary complexes with Dol-P-Man or a synthetic donor analogue, capture physiologically relevant, substrate-loaded states and reveal the structural basis of O-Man transfer. We identify a conserved O-mannosylation sequon that underlies acceptor specificity and show that TMEM260 modifies extended polypeptide substrates, consistent with a co-translational glycosylation mechanism. These findings establish the molecular mechanism of a mammalian O-mannosyltransferase required for the maturation of physiologically critical receptors and provide a structural framework for interpreting TMEM260-associated congenital malformations.

biophysics↗

bioRxiv2025

O-mannose glycosylations influence E-cadherin functional interactions

Cadherins are plasma membrane proteins that play critical roles in maintaining cell-cell adhesion and modulating cell signaling during development. Their functions are mediated by extracellular cadherin (EC) domains, which facilitate adhesive interactions and enable the formation of cis- and trans assemblies at adherens junctions and desmosomes. EC domains adopt a characteristic immunoglobulin-like fold composed of seven {beta}-strands (A-G) and are modified by N -linked and O -linked glycosylations, including O -linked mannose monosaccharides (O -Man) on conserved serine and threonine residues of B- and G-strands. O -Man glycosylations on EC domains are catalyzed by the TMTC1-4 enzymes, with different TMTC enzymes modifying B- or G-strands. Given the site-specific deposition of O -Man glycans by dedicated enzymes and the central role of EC domains in cadherins functions, we hypothesized that these PTMs may fine-tune cellular adhesion and otherwise contribute to diverse physical interactions that involve cadherins. To test these hypotheses, we assayed for changes in protein-protein interactions formed with epithelial (E)-cadherin in model cells where O -Man were genetically ablated. Herein, we report O -Man-dependent E-cadherin (CDH1) protein interactions, revealed by affinity proteomics, and we orthogonally validate an altered association between CDH1 and CDH3 (P-cadherin). We show different interactomic changes associated with O -Man ablation on B- vs. G-strands, highlighting the importance of these PTMs in CDH1-associated interaction. These findings provide new insights into how O -Man regulates CDH1-dependent protein complexes.

cell biology↗

bioRxiv2024

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

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification (PTM) that fulfills important biological roles during embryonic development. Three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including -dystroglycan, cadherins and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knock-out (KO) of O-Man glycosyltransferase genes. We established a human cell library for analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man glycosylated adhesion molecules and receptors.

molecular biology↗

bioRxiv2021

Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo

The eIF2 initiation complex is central to maintaining a functional translation machinery. Extreme stress such as life-threatening sepsis exposes vulnerabilities in this tightly regulated system, resulting in an imbalance between the opposing actions of kinases and phosphatases on the main regulatory subunit eIF2. Here, we report that translation shutdown is a hallmark of established sepsis-induced kidney injury brought about by excessive eIF2 phosphorylation and sustained by blunted expression of the counterregulatory phosphatase subunit Ppp1r15a. We determined that the blunted Ppp1r15a expression persists because of the presence of an upstream open reading frame (uORF). Overcoming this barrier with genetic approaches enabled the derepression of Ppp1r15a, salvaged translation and improved kidney function in an endotoxemia model. We also found that the loss of this uORF has broad effects on the composition and phosphorylation status of the immunopeptidome that extended beyond the eIF2 axis. Collectively, our findings define the breath and potency of the highly conserved Ppp1r15a uORF and provide a paradigm for the design of uORF-based translation rheostat strategies. The ability to accurately control the dynamics of translation during sepsis will open new paths for the development of therapies at codon level precision.

physiology↗

bioRxiv2019

ER 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 TMTC1-4 genes 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. Using genetically engineered cells lacking all four TMTC genes, overexpression of TMTC3 rescued O-linked glycosylation of E-cadherin and cell adherence. The knockdown of the Tmtcs in Xenopus laevis embryos caused a delay in gastrulation that was rescued by the addition of human TMTC3. Mutations in TMTC3 have been linked to neuronal cell migration diseases including Cobblestone lissencephaly. Analysis of TMTC3 mutations associated with Cobblestone lissencephaly found that three of the variants exhibit reduced stability and missence mutations were unable to complement TMTC3 rescue of gastrulation in Xenopus embryo development. Our study demonstrates that TMTC3 regulates O-linked glycosylation and cadherin-mediated adherence, providing insight into its effect on cellular adherence and migration, as well the basis of TMTC3-associated Cobblestone lissencephaly.

cell biology↗