bims-meglyc 2024-04-21 papers

Issue of 2024‒04‒21
three papers selected by
Silvia Radenkovic, UMC Utrecht

bioRxiv. 2024 Apr 03. pii: 2024.04.03.587922. [Epub ahead of print]

Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N.

Eri Hirata, Ken-Taro Sakata, Grace I Dearden, Indu Menon, George N Chiduza, Anant K Menon.

The oligosaccharide needed for protein N -glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including CDG 1N which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what role Rft1 plays in N -glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N -glycosylated. The majority of CDG 1N mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

DOI: https://doi.org/10.1101/2024.04.03.587922

Mol Genet Genomic Med. 2024 Apr;12(4): e2422

Congenital disorders of glycosylation with multiorgan disruption and immune dysregulation caused by compound heterozygous variants in MAN2B2.

Shiqi Fan, Huanhuan Wu, Rongrong Wang, Qian Chen, Xue Zhang.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a type of inborn error of metabolism (IEM) resulting from defects in glycan synthesis or failed attachment of glycans to proteins or lipids. One rare type of CDG is caused by homozygous or compound heterozygous loss-of-function variants in mannosidase alpha class 2B member 2 (MAN2B2). To date, only two cases of MAN2B2-CDG have been reported worldwide.METHODS: Trio whole-exome sequencing (Trio-WES) was conducted to screen for candidate variants. N-glycan profiles were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MAN2B2 expression was evaluated by western blotting. MX dynamin like GTPase 1 (MX1) function was estimated via Thogoto virus (THOV) minireplicon assay.
RESULTS: Trio-WES identified compound heterozygous MAN2B2 (hg19, NM_015274.1) variants (c.384G>T; c.926T>A) in a CDG patient. This patient exhibited metabolic abnormalities, symptoms of digestive tract dysfunction, infection, dehydration, and seizures. Novel immune dysregulation characterized by abnormal lymphocytes and immunoglobulin was observed. The MAN2B2 protein level was not affected, while LC-MS/MS showed obvious disruption of N-glycans and N-linked glycoproteins.
CONCLUSION: We described a CDG patient with novel phenotypes and disruptive N-glycan profiling caused by compound heterozygous MAN2B2 variants (c.384G>T; c.926T>A). Our findings broadened both the genetic and clinical spectra of CDG.

Keywords: MAN2B2 ; N‐glycan; congenital disorders of glycosylation; inborn error of metabolism

DOI: https://doi.org/10.1002/mgg3.2422

Heliyon. 2024 Apr 30. 10(8): e28787

Functional prediction of the potential NGLY1 mutations associated with rare disease CDG.

Shuying Yuan, Yanwen Chen, Lin Zou, Xinrong Lu, Ruijie Liu, Shaoxing Zhang, Yuxin Zhang, Cuiying Chen, Dongqing Cheng, Li Chen, Guiqin Sun.

Genetic diseases are currently diagnosed by functional mutations. However, only some mutations are associated with disease. It is necessary to establish a quick prediction model for clinical screening. Pathogenic mutations in NGLY1 cause a rare autosomal recessive disease known as congenital disorder of deglycosylation (NGLY1-CDDG). Although NGLY1-CDDG can be diagnosed through gene sequencing, clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, taken NGLY1-CDDG as an example, a comprehensive and practical predictive model for pathogenic mutations on NGLY1 through an NGLY1/Glycopeptide complex model was constructed, the binding sites of NGLY1 and glycopeptides were simulated, and an in vitro enzymatic assay system was established to facilitate quick clinical decisions for NGLY1-CDDG patients. The docking model covers 42 % of reported NGLY1-CDDG missense mutations (5/12). All reported mutations were subjected to in vitro enzymatic assay in which 18 mutations were dysfunctional (18/30). In addition, a full spectrum of functional R328 mutations was assayed and 11 mutations were dysfunctional (11/19). In this study, a model of NGLY1 and glycopeptides was built for potential functional mutations in NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-l-cysteine and dithiothreitol, on NGLY1 was examined. The established in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations in NGLY1-CDDG. This method could also be applied as a comprehensive and practical predictive model for the other rare genetic diseases.

Keywords: Deglycosylation; Enzymatic activity; Gain of function mutation; PNGase; Rare disease

DOI: https://doi.org/10.1016/j.heliyon.2024.e28787