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. 2012 Jul 13;91(1):146–151. doi: 10.1016/j.ajhg.2012.05.004

Figure 5.

Figure 5

PIGO Activity Is Required for Linking GPI-Anchored Substrates to the Cell Membrane

(A) PIGO-deficient CHO cells were transiently transfected with human wild-type (dotted lines), p.Thr788Hisfs∗5 (dashed line), or p.L957F (solid lines) PIGO cDNA expression constructs. Restoration of the levels of CD59 at the cell surface and of uPAR was assessed 2 days later. Wild-type PIGO efficiently restored levels of CD59 at the cell surface and of uPAR, whereas Thr788Hisfs∗5 PIGO did not restore the level of CD59 at all and the Leu957Phe PIGO induced only very low levels of CD59 and uPAR. The shadowed area indicates an empty-vector transfectant (control).

(B) PIGO levels. The level of the truncated Thr788Hisfs∗5 PIGO (lane 3) was about 2.5× higher than that of wild-type PIGO (lane 2), and the level of Leu957Phe PIGO (lane 4) was slightly lower than that of wild-type PIGO (lane 2).

(C) The level of PLAP at the cell surface after cotransfection with PIGO into PIGO-deficient CHO cells. PIGO-deficient CHO cells were transiently transfected with pME HA-PLAP together with pME PIGO (dotted line) or an empty vector (solid line). The level of PLAP at the cell surface was analyzed by fluorescence-activated cell sorting. PLAP activity was measured in culture medium and cell lysates after cotransfection of PLAP and PIGO cDNAs into PIGO-deficient CHO cells. Relative ALP activity was measured in culture medium (black bars) and in cell lysates (dark gray bar) against the total ALP activity in PIGO-restored CHO cells. Restoration of PIGO activity reduces ALP activity in the medium and increases activity at the cell membrane.