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GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients.

de la Morena-Barrio ME, Hernández-Caselles T, Corral J, García-López R, Martínez-Martínez I, Pérez-Dueñas B, Altisent C, Sevivas T, Kristensen SR, Guillén-Navarro E, Miñano A, Vicente V, Jaeken J, Lozano ML - Orphanet J Rare Dis (2013)

Bottom Line: 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.This study also confirms less severe hypoglycosylation defects in older PMM2-CDG patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro 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.

ABSTRACT

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.

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Related in: MedlinePlus

Level of expression of glycosylphosphatidyl inositol (GPI) anchored-proteins on different blood cells in PMM2-CDG patients and control subjects. The study was done by flow cytometry using proaerolysin variant (FLAER). Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. PMN: Polymorphonuclear cells.
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Figure 2: Level of expression of glycosylphosphatidyl inositol (GPI) anchored-proteins on different blood cells in PMM2-CDG patients and control subjects. The study was done by flow cytometry using proaerolysin variant (FLAER). Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. PMN: Polymorphonuclear cells.

Mentions: We found that the level of FLAER binding to white blood cells from patients was similar to that of healthy individuals (Figure 2). In agreement with this result, a similar surface expression of most specific GPI-anchored proteins was observed in PMM2-CDG patients and in controls on lymphocytes (CD48), erythrocytes (CD55, and CD59), monocytes (CD55) and neutrophils (CD55, CD24 and CD66) (Figure 3A). A similar expression of non-GPI anchored proteins was also observed in patients and in controls on lymphocytes (CD3, CD19, and CD16), monocytes (CD33) and neutrophils (CD54) (Figure 3B).


GPI-anchor and GPI-anchored protein expression in PMM2-CDG patients.

de la Morena-Barrio ME, Hernández-Caselles T, Corral J, García-López R, Martínez-Martínez I, Pérez-Dueñas B, Altisent C, Sevivas T, Kristensen SR, Guillén-Navarro E, Miñano A, Vicente V, Jaeken J, Lozano ML - Orphanet J Rare Dis (2013)

Level of expression of glycosylphosphatidyl inositol (GPI) anchored-proteins on different blood cells in PMM2-CDG patients and control subjects. The study was done by flow cytometry using proaerolysin variant (FLAER). Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. PMN: Polymorphonuclear cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016514&req=5

Figure 2: Level of expression of glycosylphosphatidyl inositol (GPI) anchored-proteins on different blood cells in PMM2-CDG patients and control subjects. The study was done by flow cytometry using proaerolysin variant (FLAER). Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. PMN: Polymorphonuclear cells.
Mentions: We found that the level of FLAER binding to white blood cells from patients was similar to that of healthy individuals (Figure 2). In agreement with this result, a similar surface expression of most specific GPI-anchored proteins was observed in PMM2-CDG patients and in controls on lymphocytes (CD48), erythrocytes (CD55, and CD59), monocytes (CD55) and neutrophils (CD55, CD24 and CD66) (Figure 3A). A similar expression of non-GPI anchored proteins was also observed in patients and in controls on lymphocytes (CD3, CD19, and CD16), monocytes (CD33) and neutrophils (CD54) (Figure 3B).

Bottom Line: 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.This study also confirms less severe hypoglycosylation defects in older PMM2-CDG patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro 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.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus