Molecular pathogenesis of human CD59 deficiency

摘要

Objective To characterize all 4 mutations described for CD59 congenital deficiency. Methods The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. Results Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. Conclusions All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis. Complement activation triggers membrane attack complex (MAC) assembly to form pores in cell membrane lipid bilayers of susceptible bacteria. 1 However, unregulated MAC formation may cause host tissue damage. 2 The glycosyl phosphatidylinositol (GPI)-anchored cell surface membrane glycoprotein CD59 inhibits the final step of MAC formation to protect host cells from MAC-mediated injury. 3 Several mutations in the CD59 -coding sequence are known in human patients ( figure 1A ). We and others have previously reported of 13 patients 4 , – 11 aged 1–4.5 years who suffered from chronic hemolysis and recurrent episodes of Guillain-Barré syndrome (GBS)-like disease from early infancy, suggesting chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and recurrent strokes (table e-1, links.lww.com/NXG/A87 ). The mutations included p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, all leading to CD59 loss of function. In all mutations, no surface protein was detected by anti-CD59 antibody staining. We were interested, in the current study, to verify whether indeed no proteins were produced, whether any proteins that were produced reached the membrane, and whether proteins or truncated proteins exist intracellularly or secreted outside the cells. These potential differences may have functional implications and clinical manifestations. Open in a separate window Figure 1 Sequence and structure of CD59 WT and mutants (A) The sequences of WT CD59, point mutations Cys64Tyr and Asp24Val, and frameshift mutations Asp24Valfs* and Ala16Alafs * : The mature membrane surface CD59 primary sequence consists of 77 residues after removal of a 25-residue N-terminal signal sequence and a C-terminal GPI-anchoring signal (not shown). Colored arrows and boxes represent β-strands and α-helices, respectively, as observed in WT CD59 (see B). Point mutations are marked with an asterisk. The sequence of the 2 frameshift mutants reveals significantly shortened proteins that lack most of the CD59 sequence, in particular the GPI-anchoring signal. (B) The structure of CD59 highlights the vicinity of the point mutations to known sites of activity and interaction. The mutated Cys64 and Asp24 positions are highlighted as spheres and labeled. The 3 characterized interfaces of CD59 are marked by numbered crescents: (1) The classic site characterized originally described by Bodian et al. 21 with the central Trp40 residue shown in sticks, (2) a loop spanning residues 20–24 that modulates CD59 activity 20 (colored in white), and (3) the edge β-strand that interacts with ILY 18 (to the left of the crescent). Disulfide bridges that stabilize CD59 are shown in sticks. Two solved structures (2j8b and 2uwr) are shown, highlighting the relative flexibility of the C-terminal helix (colored in magenta). (C) The frameshift mutants contain only a small part of the original CD59 sequence: The structure of CD59 is shown, with the region with a sequence in common with the Ala16Alafs * and Asp24Valfs * mutants colored in yellow and yellow-white, respectively. The I-TASSER model for the Asp24Valfs * sequence aligns 2 additio