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GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients
Maria E de la Morena-Barrio, Trinidad Hernández-Caselles, Javier Corral1
1Centro Regional de Hemodonación Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Universidad de Murcia, Ronda de Garay S/N, 30003 Murcia, Spain. javier.corral@carm.es.
Background:
Mutations in PMM2 impair phosphomannomutase-2 activity and cause the most frequent congenital disorder of glycosylation, PMM2-CDG. Mannose-1-phosphate, that is deficient in this disorder, is also implicated in the biosynthesis of glycosylphosphatidyl inositol (GPI) anchors.
Objective:
To evaluate whether GPI-anchor and GPI-anchored proteins are defective in PMM2-CDG patients.
Methods:
The expression of GPI-anchor and seven GPI-anchored proteins was evaluated by flow cytometry in different cell types from twelve PMM2-CDG patients. Additionally, neutrophil CD16 and plasma hepatic proteins were studied by Western blot. Transferrin glycoforms were evaluated by HPLC.
Results:
Patients and controls had similar surface expression of GPI-anchor and most GPI-anchored proteins. Nevertheless, patients displayed a significantly diminished binding of two anti-CD16 antibodies (3G8 and KD1) to neutrophils and also of anti-CD14 (61D3) to monocytes. Interestingly, CD16 immunostaining and asialotransferrin levels significantly correlated with patients' age. Analysis by flow cytometry of CD14 with MΦP9, and CD16 expression in neutrophils by Western blot using H-80 ruled out deficiencies of these antigens.
Conclusions:
PMM2 mutations do not impair GPI-anchor or GPI-anchored protein expression. However, the glycosylation anomalies caused by PMM2 mutations might affect the immunoreactivity of monoclonal antibodies and lead to incorrect conclusions about the expression of different proteins, including GPI-anchored proteins. Neutrophils and monocytes are sensitive to PMM2 mutations, leading to abnormal glycosylation in immune receptors, which might potentially affect their affinity to their ligands, and contribute to infection. This study also confirms less severe hypoglycosylation defects in older PMM2-CDG patients.
Insights
Mutations in phosphomannomutase-2 (PMM2) do not affect glycosylphosphatidyl inositol (GPI) anchor expression in PMM2-CDG patients. However, altered glycosylation may impact immune receptor function and antibody binding.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Phosphomannomutase-2 (PMM2) mutations cause PMM2-congenital disorder of glycosylation (CDG), leading to mannose-1-phosphate deficiency.
- Mannose-1-phosphate is crucial for glycosylphosphatidyl inositol (GPI) anchor biosynthesis.
Purpose of the Study:
- To investigate potential defects in GPI anchors and GPI-anchored proteins in patients with PMM2-CDG.
- To assess the impact of PMM2 mutations on immune cell surface protein expression and immunoreactivity.
Main Methods:
- Flow cytometry was used to evaluate GPI anchor and GPI-anchored protein expression on various cell types from PMM2-CDG patients.
- Western blot analysis was performed on neutrophil CD16 and plasma hepatic proteins.
- High-performance liquid chromatography (HPLC) analyzed transferrin glycoforms.
Main Results:
- Surface expression of GPI anchors and most GPI-anchored proteins remained similar between patients and controls.
- PMM2-CDG patients showed significantly reduced binding of specific antibodies (3G8, KD1, 61D3) to neutrophils and monocytes.
- CD16 immunostaining and asialotransferrin levels correlated with patient age, while flow cytometry and Western blot ruled out antigen deficiencies.
Conclusions:
- PMM2 mutations do not inherently impair GPI anchor or GPI-anchored protein expression.
- Glycosylation anomalies in PMM2-CDG can affect monoclonal antibody immunoreactivity, potentially leading to misinterpretation of protein expression.
- Neutrophils and monocytes exhibit abnormal glycosylation of immune receptors, possibly affecting ligand affinity and contributing to infection susceptibility in PMM2-CDG.
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