Sabry, S. | bioRxiv Science

bioRxiv2026

Dolichol biosynthesis in yeast traces the expanded reaction pathway of higher eukaryotes

The recent reassignment of Saccharomyces cerevisiae Dfg10 as a polyprenal reductase left two unresolved steps in yeast dolichol biosynthesis: the conversion of polyprenol to polyprenal and dolichal to dolichol. In humans, both of these steps are carried out by DHRSX, an enzyme with a unique dual specificity. We found that yeast Env9 catalyzes an NADPH-dependent reduction of dolichal to dolichol. However, in contrast to DHRSX, Env9 does not catalyze the conversion of polyprenol to polyprenal. Instead, our data indicates that this reaction is catalyzed by another yeast short chain oxidoreductase, Tda5. Thus, we provide evidence that the dual role of DHRSX in human dolichol synthesis is fulfilled by two dedicated yeast enzymes, Env9 and Tda5. Accordingly, deletion of ENV9 and TDA5 led to the accumulation of polyisoprenoid intermediates, transfer of immature lipid-linked oligosaccharides onto nascent proteins, and defective N-glycosylation. This is similar to what had been observed in DHRSX-deficient mammalian cells and yeast cells with DFG10 deleted. Furthermore, we discovered that loss of Dfg10 results in a deficiency of cell wall -mannan, revealing a critical sensitivity of yeast mannan biosynthesis to the quality of nascent N-linked glycans.

biochemistry↗

bioRxiv2025

ALG13 loss-of-function alters glycosylation, impairs neuronal maturation, and drives network hypoactivity in a cortical organoid model of CDG

BackgroundCongenital disorders of glycosylation (CDGs) are a group of rare metabolic diseases recognized for their neurological presentations, including developmental delay and seizures. However, the link between glycosylation defects and cortical brain network pathology remains elusive. MethodsTo address this unmet need, we generated iPSC derived human cortical organoids (hCOs) for ALG13-CDG, which is the second most common CDG that is also X-linked. To comprehensively understand the impact of glycosylation defects on cortical pathology in CDG, we combined electrophysiological recordings using multi-electrode arrays (MEA) with comprehensive molecular profiling via multiomics, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was also evaluated in both iPSCs and organoids. ResultsALG13-CDG hCOs revealed reduced glycosylation of proteins critical for extracellular matrix (ECM), neuronal migration, lipid metabolism, calcium ion homeostasis, and neuronal excitability. Dysregulation in related pathways was corroborated by proteomics and scRNA-seq, which also showed altered communication patterns in these pathways. Trajectory analysis revealed an inversion in neuronal development, with early inhibitory and delayed excitatory development, indicating an excitatory and inhibitory (E/I) imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst dynamics, and shorter axonal extensions. Despite this, transcriptomic and proteomic data revealed upregulation of excitatory receptors suggesting latent hyperexcitability. Altered lipid and sugar (GlcNAc) metabolism and skewed X-inactivation were also observed. ConclusionsOur study provides the first evidence of glycosylation defects in an ALG13-CDG human cortical organoid (hCO) model and links these defects to disrupted neuronal developmental trajectories and dysregulation of key pathways essential for brain function. We identify mistimed neuronal maturation and an excitatory/inhibitory (E/I) imbalance as early drivers of network hypoactivity and immature burst dynamics, with downstream compensatory hyperexcitability that may contribute to seizure susceptibility. While specific to ALG13-CDG, these mechanisms likely extend to other glycosylation disorders with overlapping neurological features. This work offers new mechanistic insight into cortical dysfunction associated with impaired protein glycosylation and highlights potential targets for therapeutic intervention.

neuroscience↗