Elucidating How the Loss of Protein Glycans Leads to Disease: Investigating the Causes of Disease Through Gene Variants in Undiagnosed Patients (Prof. Shoko Nishihara, Director of GaLSIC)

[Key Points]

• Although information on genetic variants is accumulating rapidly, the relationship between many of these variants and disease remains unclear.
• We identified cases in which genetic variants may lead to disease by causing the loss of protein glycans and impairing protein function.

• The study suggests that the loss of glycans from the phosphatase ACP3 may impair enzymatic activity, protein stability, and secretion outside the cell, potentially resulting in neurodevelopmental abnormalities.

[Overview]

A research group led by Lecturer Noriyoshi Manabe and Director and Professor Yoshiki Yamaguchi of the Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, in collaboration with Director Shoko Nishihara of Glycan and Life Systems Integration Center (GaLSIC), Soka University, Director Tadashi Kaname of the Department of Genome Medicine, National Center for Child Health and Development, Professor Jun-ichi Furukawa of the Institute for Glyco-Core Research (iGCORE), Nagoya University, Professor Yoshihiro Akimoto of Kyorin University, and other researchers, has been investigating the causes of disease by analyzing genetic data from patients with undiagnosed diseases.
In this study, the researchers identified genetic variants affecting glycans, which serve as molecular markers attached to proteins, among more than six million genomic variant¹ records from patients with undiagnosed diseases collected at the National Center for Child Health and Development. They then focused on ACP3, an enzyme that removes phosphate groups in the body. Detailed analyses revealed that the loss of glycans caused by a particular variant may impair the enzyme's activity, protein stability, and secretion outside the cell, potentially contributing to disease.
These findings were published in iScience, a scientific journal published by Cell Press in the United States, on October 16, 2026. This research was supported by the Research Program on Practical Application of Innovative Pharmaceutical and Medical Technologies for Rare and Intractable Diseases of the Japan Agency for Medical Research and Development (AMED; project management numbers 22ek0109446, 24ek0109760, 25ek0109815, and 26ek0109886) and Grants-in-Aid for Scientific Research (26K02160).

[Background]

In recent years, advances in genetic analysis technologies have led to the accumulation of an enormous amount of information on genetic variants (Figure 1). However, fewer than half of these variants have been clearly classified as either disease-causing or having no known impact. More than half remain variants of uncertain significance.
Some genetic variants, known as missense variants², change the amino acids that make up a protein. Such changes are known to affect the three-dimensional structure of proteins and may also affect glycans, which are molecular markers attached to proteins. Determining whether variants of uncertain significance are responsible for disease could lead to the development of new treatments.

Genetic variants include those found in healthy individuals that have no harmful effects, as well as those that cause disease. However, many reported variants have not been classified into either category and remain variants of uncertain significance. Evaluating the relationship between each of these variants and disease is essential for accurate diagnosis, a better understanding of diseases, and the development of treatments.

[Research Findings]

First, we narrowed down missense variants² that were considered likely to cause abnormalities in glycosylation from more than six million variant records from patients with undiagnosed diseases accumulated at the National Center for Child Health and Development. Among these, we focused on three patients carrying the T335M variant in ACP3, an enzyme that removes phosphate groups. This variant replaces the amino acid threonine at position 335 with methionine. As a result, the glycan that would normally be attached to asparagine at position 333 of ACP3 is lost. Since all three patients exhibited neurodevelopmental abnormalities, we hypothesized that the loss of this glycan might be responsible for their conditions and conducted further analyses.

We prepared recombinant proteins³ produced in cells, including normal (wild-type) ACP3 and a modified form of ACP3 in which the glycan was absent. We compared these proteins from various perspectives, including enzymatic activity, intracellular localization, protein resistance to degradation (protease resistance assay⁴), and glycan structure analyzed using mass spectrometer⁵.

The results confirmed that the T335M variant shared by the patients resulted in the loss of the glycan modification found in wild-type ACP3. ACP3 carrying this variant was barely secreted outside the cells into the culture medium and instead remained inside the cells. Further investigation revealed that the protein failed to reach the Golgi apparatus⁶, the cellular pathway through which proteins are processed and transported for secretion, and instead remained in the endoplasmic reticulum⁷, located upstream of the Golgi apparatus. Furthermore, the protein retained inside the cells had almost completely lost its normal enzymatic activity. Experiments examining its resistance to degradation also showed that it was more susceptible to degradation than wild-type ACP3, indicating that its three-dimensional structure had become unstable.

Mass spectrometry⁵ further revealed that the glycan lost as a result of this variant was exclusively a type known as a high-mannose-type glycan⁸. In contrast, at other glycosylation sites on ACP3, another type of glycan known as a complex-type glycan⁹ accounted for the majority of the glycans present.

The researchers also used molecular dynamics simulations¹⁰, a computational method, to analyze how glycans affect the three-dimensional structure of ACP3. The results showed that the high-mannose-type glycan attached to asparagine at position 333 forms a stable network of interactions, including hydrogen bonds, with surrounding amino acids. It remains relatively immobile and appears to function as a structural support. These findings indicate that the glycan attached to Asn333 acts as a structural clamp that helps maintain the structure of ACP3.

Taken together, these results demonstrate that the T335M variant of ACP3, found in all three patients, reduces structural stability through abnormal glycosylation, leading to a loss of enzymatic activity and impaired secretion outside the cell. In particular, the findings suggest that high-mannose-type glycans play an important role in maintaining the structural stability of ACP3. These molecular changes are thought to contribute to the neurodevelopmental symptoms observed in the patients (Figure 2).

 

The T335M variant eliminates glycosylation at asparagine 333. As a result, ACP3 may remain in the endoplasmic reticulum and aggregate, leading to an unstable three-dimensional structure and a loss of its function as a phosphatase.

[Future Perspectives]

This study focused on patients carrying the T335M variant of ACP3. However, more than 1,000 other variants have been identified that may cause disease through abnormalities in glycan modification. By systematically clarifying the relationships between these variants and disease, the researchers believe that it may be possible to propose new treatments in the future. Further analyses will therefore continue.

[Article Information]

• Title: Loss of the Asn333 high-mannose N-glycan impairs ACP3 folding and drives disease-associated dysfunction
• Journal: iScience
• Authors: Noriyoshi Manabe¹, Kazuyoshi Itoh², Shiho Ohno¹, Ayumi Miura¹, Aika Miyata¹, Sora Yashima¹, Sachiko Komatsu³, Tsutomu Fujimura³, Hisatoshi Hanamatsu⁴, Jun-ichi Furukawa⁴, Kiyohiko Angata², Akira Togayachi², Kiyoko F. Aoki-Kinoshita²˒⁴, Yoshihiro Akimoto⁵, Tadashi Kaname⁶, Shoko Nishihara², and Yoshiki Yamaguchi¹

• Affiliations:
  1. Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University
  2. Glycan and Life Systems Integration Center (GaLSIC), Soka University
  3. Division of Bioanalytical Chemistry, Tohoku Medical and Pharmaceutical University
  4. Institute for Glyco-core Research (iGCORE), Nagoya University
  5. Kyorin University School of Medicine
  6. National Center for Child Health and Development

[Glossary]

1. Variant: An individual difference or change in a DNA sequence. Some variants cause disease, while others have no effect on the body.
2. Missense variant: A genetic change that causes one amino acid in a protein to be replaced by another amino acid.
3. Recombinant protein: A protein artificially produced by manipulating genetic material to make cells produce a specific protein.
4. Protease resistance assay: An experiment that evaluates the stability of a protein's three-dimensional structure by examining how resistant it is to degradation by proteases, enzymes that break down proteins.
5. Mass spectrometer: An instrument that precisely measures the mass of molecules. It is used to investigate the structures of proteins and glycans.
6. Golgi apparatus: A cellular organelle that processes glycans attached to proteins and prepares proteins for transport outside the cell.​​​​​​
7. Endoplasmic reticulum: A cellular organelle where proteins are synthesized and folded into their proper three-dimensional structures. Proteins that fail to fold correctly may remain there.​​​​​​​
8. High-mannose-type glycan: A relatively simple glycan structure containing multiple mannose sugar units. It is found during the early stages of protein production.​​​​​​​
9. Complex-type glycan: A glycan with a more complex structure that is formed when high-mannose-type glycans are processed in the Golgi apparatus.​​​​​​​
10. ​​​​​​​Molecular dynamics simulation: A computational method that uses the laws of physics to simulate the movements of atoms and molecules, allowing researchers to investigate changes in the three-dimensional structures of proteins and other molecules.