Figure 1
Figure 1. Expression of GPI-anchored proteins on patient's peripheral blood cells; and reduced activity of PIGT mutant in restoring surface expression of GPI-anchored proteins after transfection into PIGT-null cell lines. (A) Expression of (left) CD58 and CD59 on red cells, (center) CD24 and CD66b on neutrophil granulocytes, and (right) CD48 on B and T lymphocytes. The first row shows the expression of GPI-anchored protein (AP) at the time of ultradeep sequencing (4.5 years after start of eculizumab); the second row shows expression of GPI-AP in a healthy control. In healthy controls, CD58 and CD59 are expressed on >99.9% and >99.5% of red cells, respectively, and CD24/CD66b is expressed on >99.8% of neutrophil granulocytes (second row). In contrast, the patient shows a mosaic of cells with normal expression of GPI-anchored proteins and cells with reduced or completely missing expression of GPI-AP on (left) erythrocytes or (center) neutrophil granulocytes. The cell populations that completely lack expression of the respective GPI-AP are indicated by arrows; the populations with reduced GPI-AP expression are marked by asterisks. The patient did not receive any blood transfusions over a period of 3 months before this measurement. Expression of the GPI-AP CD48 on T lymphocytes was normal, whereas a subpopulation of B lymphocytes did not express the GPI-AP CD48. The percentages of cells with reduced or absent GPI-AP, ie, PNH cells, and normal range is shown in the supplemental Materials. (B) PIGT-deficient Chinese Hamster Ovary cells were transiently transfected with wild-type or a mutant version skipping exon 11 of transcript NM_0015937. (Left) Restoration of the cell surface protein levels of wild-type PIGT and the mutant PIGT lacking 28 amino acids encoded by exon 11 was assessed by flow cytometry. Wild-type PIGT efficiently restored expression levels of CD59 and CD55 at the cell surface (dotted black lines), whereas the mutant PIGT did not rescue CD59 and only partially rescued CD55 expression (solid black lines). Dark shading, empty vector; light shading, isotype-matched control. (Right) Expression levels of transfected wild-type and the mutant HA-tagged PIGT. PIGT proteins were determined by western blotting with anti-HA; GAPDH, loading control. Normalized PIGT levels are shown at the bottom.

Expression of GPI-anchored proteins on patient's peripheral blood cells; and reduced activity of PIGT mutant in restoring surface expression of GPI-anchored proteins after transfection into PIGT-null cell lines. (A) Expression of (left) CD58 and CD59 on red cells, (center) CD24 and CD66b on neutrophil granulocytes, and (right) CD48 on B and T lymphocytes. The first row shows the expression of GPI-anchored protein (AP) at the time of ultradeep sequencing (4.5 years after start of eculizumab); the second row shows expression of GPI-AP in a healthy control. In healthy controls, CD58 and CD59 are expressed on >99.9% and >99.5% of red cells, respectively, and CD24/CD66b is expressed on >99.8% of neutrophil granulocytes (second row). In contrast, the patient shows a mosaic of cells with normal expression of GPI-anchored proteins and cells with reduced or completely missing expression of GPI-AP on (left) erythrocytes or (center) neutrophil granulocytes. The cell populations that completely lack expression of the respective GPI-AP are indicated by arrows; the populations with reduced GPI-AP expression are marked by asterisks. The patient did not receive any blood transfusions over a period of 3 months before this measurement. Expression of the GPI-AP CD48 on T lymphocytes was normal, whereas a subpopulation of B lymphocytes did not express the GPI-AP CD48. The percentages of cells with reduced or absent GPI-AP, ie, PNH cells, and normal range is shown in the supplemental Materials. (B) PIGT-deficient Chinese Hamster Ovary cells were transiently transfected with wild-type or a mutant version skipping exon 11 of transcript NM_0015937. (Left) Restoration of the cell surface protein levels of wild-type PIGT and the mutant PIGT lacking 28 amino acids encoded by exon 11 was assessed by flow cytometry. Wild-type PIGT efficiently restored expression levels of CD59 and CD55 at the cell surface (dotted black lines), whereas the mutant PIGT did not rescue CD59 and only partially rescued CD55 expression (solid black lines). Dark shading, empty vector; light shading, isotype-matched control. (Right) Expression levels of transfected wild-type and the mutant HA-tagged PIGT. PIGT proteins were determined by western blotting with anti-HA; GAPDH, loading control. Normalized PIGT levels are shown at the bottom.

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