bims-meglyc 2024-10-20 papers

Issue of 2024–10–20
three papers selected by
Silvia Radenkovic, UMC Utrecht

J Thromb Haemost. 2024 Oct 16. pii: S1538-7836(24)00611-1. [Epub ahead of print]

Novel insights into Antithrombin Deficiency enabled by Mass Spectrometry-based Precision Diagnostics.

Mirjam Kruijt, Maria Eugenia de la Morena-Barrio, Javier Corral, Christa M Cobbaert, L Renee Ruhaak.

BACKGROUND: Although P5 (preventive, personalized, predictive, participatory, psychocognitive) medicine and patient focused healthcare are gaining ground in various healthcare areas, the diagnosis of antithrombin deficiency (ATD) is still based on crude diagnostic tests clustering patients into clinically heterogeneous subgroups whereby relevant thrombophilia phenotypes may go unnoticed. Clinical pathways and the majority of evidence are based on these tests, therefore generic treatment is still the norm.
OBJECTIVES: To unravel the heterogeneity of ATD, a mass spectrometry (LC-MRM-MS)-based test for antithrombin was developed allowing molecular characterization of the antithrombin proteoforms in patient plasma. This study provides the first insight into the tests' clinical performance.
METHODS: Plasma from 91 unrelated ATD patients and 41 patients with a congenital disorder of glycosylation affecting antithrombin glycosylation were characterized functionally, genetically, and analyzed by LC-MRM-MS. An established data analysis strategy was applied for quantitation and molecular characterization of antithrombin proteoforms.
RESULTS: The test recognized patients with a quantitative defect, discriminated between type I and type II ATD, and identified variant proteoforms. Overall, the diagnostic sensitivity for ATD was 100% for LC-MRM-MS compared to 81.1% by the functional test. Type II ATD, a subtype prone to misdiagnosis, revealed an even larger difference of 100% identification by LC-MRM-MS versus 56.8% by functional test.
CONCLUSIONS: The qualitative and quantitative MS-based AT-test can serve as a platform for investigating the molecular basis of the clinical heterogeneity of ATD. This Precision Diagnostics approach for ATD can lower diagnostic uncertainty and modernize the ATD diagnostic and clinical pathways.

Keywords: Hereditary Antithrombin Deficiency; Mass Spectrometry; Molecular Testing; Protein Isoforms

DOI: https://doi.org/10.1016/j.jtha.2024.10.005

Anal Bioanal Chem. 2024 Oct 17.

GlycoMaple: recent updates and applications in visualization and analysis of glycosylation pathways.

Wei-Ze Kong, Morihisa Fujita.

Post-translational modifications including glycosylation, phosphorylation, and lipidation expand the functionality and diversity of proteins. Protein glycosylation is one of the most abundant post-translational modifications in mammalian cells. The glycosylation process is regulated at multiple steps, including transcription, translation, protein folding, intracellular transport, and localization, and activity of glycosyltransferases and glycoside hydrolases. The glycosylation process is also affected by the concentration of sugar nucleotides in the lumen of the Golgi apparatus. Unlike the synthesis of other biological macromolecules, such as nucleic acids and proteins, glycan biosynthesis is not a template-driven process. In addition, the chemical complexity of glycan structures makes the glycosylation network extraordinarily intricate. We previously developed a web-based tool specially focused on glycan metabolic pathways known as GlycoMaple, which is able to easily visualize and estimate glycosylation pathways based on gene expression data. We recently updated GlycoMaple to incorporate the new genes and glycosylation pathways. Here, we introduce and discuss the uses and upgrades of GlycoMaple.

Keywords: Glycan biosynthesis; GlycoMaple; Glycogene; Glycosylation

DOI: https://doi.org/10.1007/s00216-024-05594-1

J Biol Chem. 2024 Oct 10. pii: S0021-9258(24)02377-9. [Epub ahead of print] 107875

Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells.

Takfarinas Kentache, Charlotte R Althoff, Francesco Caligiore, Erika Souche, Céline Schulz, Julie Graff, Eline Pieters, Pamela Stanley, Joseph N Contessa, Emile Van Schaftingen, Gert Matthijs, François Foulquier, Guido T Bommer, Matthew P Wilson.

Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.

Keywords: CHO glycosylation mutants; Glycosylation; N-linked glycosylation; dolichol; glycobiology; glycoconjugate; glycoprotein biosynthesis; isoprenoid; lipid synthesis; polyprenal; polyprenol

DOI: https://doi.org/10.1016/j.jbc.2024.107875