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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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Glycoforms of antithrombin and α1-antitrypsin in plasma of one control, three adults, and three children with PMM2-CDG. Normal glycoforms are indicated by solid arrows and hypoglycosylated glycoforms by dashed arrows.
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Figure 6: Glycoforms of antithrombin and α1-antitrypsin in plasma of one control, three adults, and three children with PMM2-CDG. Normal glycoforms are indicated by solid arrows and hypoglycosylated glycoforms by dashed arrows.

Mentions: All patients exhibited a characteristic HPLC pattern for PMM2-CDG, with increased asialo- and disialotransferrin fractions (Table 2). Similarly, all patients had significant antithrombin deficiency (Table 2) and increased levels of abnormal glycoforms of antithrombin and α-antitrypsin (Figure 6).


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)

Glycoforms of antithrombin and α1-antitrypsin in plasma of one control, three adults, and three children with PMM2-CDG. Normal glycoforms are indicated by solid arrows and hypoglycosylated glycoforms by dashed arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Glycoforms of antithrombin and α1-antitrypsin in plasma of one control, three adults, and three children with PMM2-CDG. Normal glycoforms are indicated by solid arrows and hypoglycosylated glycoforms by dashed arrows.
Mentions: All patients exhibited a characteristic HPLC pattern for PMM2-CDG, with increased asialo- and disialotransferrin fractions (Table 2). Similarly, all patients had significant antithrombin deficiency (Table 2) and increased levels of abnormal glycoforms of antithrombin and α-antitrypsin (Figure 6).

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