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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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Expression of CD16 in neutrophils of PMM2-CDG patients and control subjects. A) Flow cytometry analysis using 3G8 monoclonal antibody in the whole cohort of patients and according to age. Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. B) Western blot analysis using the H-80 polyclonal antibody. As loading controls, we stained the membrane with Ponceau Red and evaluated the expression of tubulin.
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Figure 4: Expression of CD16 in neutrophils of PMM2-CDG patients and control subjects. A) Flow cytometry analysis using 3G8 monoclonal antibody in the whole cohort of patients and according to age. Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. B) Western blot analysis using the H-80 polyclonal antibody. As loading controls, we stained the membrane with Ponceau Red and evaluated the expression of tubulin.

Mentions: However, analysis of CD16 on granulocytes, performed in parallel by two different laboratories (Centro Regional de Hemodonación and Departamento de Bioquímica, Biología Molecular B de Inmunología), revealed that PMM2-CDG patients displayed a significantly diminished CD16 immunostaining using 3G8 mAb compared to controls (Figure 4A). In contrast, the staining of CD16 in lymphocytes, where this molecule exists as a transmembrane non GPI-linked form, was similar in PMM2-CDG patients and controls (Figure 3B). Two patients, P4 and P8, showed negligible binding of anti-CD16 using two different mAb: 3G8 and KD1 (Table 3). Interestingly, these two mAb seem to recognize different but close epitopes since preincubation with KD1 clone strongly reduced 3G8 staining of PMN cells (Additional file 2: Figure S2A). The reduction of CD16 expression on neutrophils seemed to be restricted to children with PMM2-CDG, since the three available adult PMM2-CDG patients displayed normal values (Figure 4A). To analyze whether the decreased CD16 immunostaining was a real deficiency of the molecule, or a defect in recognition by the antibody, we performed Western blot analysis using a polyclonal antibody (H-80) against neutrophils. This assay revealed similar levels of CD16 in neutrophils in PMM2-CDG and in controls (Figure 4B).


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)

Expression of CD16 in neutrophils of PMM2-CDG patients and control subjects. A) Flow cytometry analysis using 3G8 monoclonal antibody in the whole cohort of patients and according to age. Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. B) Western blot analysis using the H-80 polyclonal antibody. As loading controls, we stained the membrane with Ponceau Red and evaluated the expression of tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Expression of CD16 in neutrophils of PMM2-CDG patients and control subjects. A) Flow cytometry analysis using 3G8 monoclonal antibody in the whole cohort of patients and according to age. Values are expressed as % mean fluorescence intensity (MFI) vs that observed in controls. B) Western blot analysis using the H-80 polyclonal antibody. As loading controls, we stained the membrane with Ponceau Red and evaluated the expression of tubulin.
Mentions: However, analysis of CD16 on granulocytes, performed in parallel by two different laboratories (Centro Regional de Hemodonación and Departamento de Bioquímica, Biología Molecular B de Inmunología), revealed that PMM2-CDG patients displayed a significantly diminished CD16 immunostaining using 3G8 mAb compared to controls (Figure 4A). In contrast, the staining of CD16 in lymphocytes, where this molecule exists as a transmembrane non GPI-linked form, was similar in PMM2-CDG patients and controls (Figure 3B). Two patients, P4 and P8, showed negligible binding of anti-CD16 using two different mAb: 3G8 and KD1 (Table 3). Interestingly, these two mAb seem to recognize different but close epitopes since preincubation with KD1 clone strongly reduced 3G8 staining of PMN cells (Additional file 2: Figure S2A). The reduction of CD16 expression on neutrophils seemed to be restricted to children with PMM2-CDG, since the three available adult PMM2-CDG patients displayed normal values (Figure 4A). To analyze whether the decreased CD16 immunostaining was a real deficiency of the molecule, or a defect in recognition by the antibody, we performed Western blot analysis using a polyclonal antibody (H-80) against neutrophils. This assay revealed similar levels of CD16 in neutrophils in PMM2-CDG and in controls (Figure 4B).

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