PMC

NMR structure of the protein backbone of human CD59. The figure shows the 20 lowest energy structures of human CD59 with all lysine side chains and H44 (PDB Id: 1CDQ) (). The structures were superimposed for the backbone of the β turn 41–44. The square highlights the K41–H44 glycation motif. K41 is within 5.91 ± 1.44 Å of the D1 imidazolic nitrogen of H44.

Glycation abrogates the homologous restriction activity of human CD59. A 10% cell suspension of GPE was incubated with immunoaffinity-purified hRBC CD59 previously exposed for different time intervals to glucose (▵, 0.5 M) or ribose (□, 0.5 M), or to nonglycating sorbitol (■, 0.5 M). The sensitivity of GPE to human MAC was then tested by using purified human C5b6 and C7, C8, and C9. Each point represents the mean of triplicate determinations (SEM smaller than data points). Representative of three experiments with comparable results. (Inset) Number of CD59 molecules incorporated per GPE, determined with ¹²⁵I-CD59 before (C) and after (R) glycation with ribose.

Site-directed mutagenesis of K41 or H44 abrogates the sensitivity of human CD59 to glycation–inactivation. (A) WT and CD59–Gln-41 or CD59–Gln-44 mutants were expressed in CHO cells. Expression was confirmed by immunocytochemistry by using the anti-human CD59 monoclonal Ab YTH53.1 and fluorescence (Texas red)-labeled anti-rat IgG secondary Ab: ■, transfection vector only; □, WT CD59; ▵, Gln-41 mutant CD59; and ○, Gln-44 mutant CD59. (B) Recombinant WT and mutant CD59 were immunoaffinity purified from CHO cells, and their activity was tested in the GPE hemolytic assay (symbols as in A). (C) Activity of immunoaffinity-purified WT and mutant CD59s before and after glycation with ribose for different time intervals. The points represent the mean of triplicate determinations (SEM smaller than the data points). Representative of three experiments with comparable results.

Glycated CD59 in human urine. (A) Urine samples from diabetic (lanes 2 and 3) and nondiabetic (lanes 1, 4, and 5) subjects were concentrated by ultrafiltration and separated by anion exchange chromatography, and fractions were dot-blotted for the presence of CD59 with anti-CD59-specific Ab. CD59-positive fractions were pooled and immunoprecipitated with the HC1 anti-CD59-specific Ab. Immunoprecipitates were spun down and boiled for 30 min; then the immunocomplexes were separated by SDS/PAGE and immunoblotted with the monoclonal YTH53.1 anti-CD59 Ab (A, Upper), and with the anti-hexitol–lysine Ab (A, Lower). The Upper samples were separated by using a 20-cm-long gel that resolved the multiple CD59 bands (mw = molecular weight markers). The Lower samples were separated by using a minigel. Immunoaffinity-purified hRBC CD59 was included in the Upper gel as a control (lane 6). The signal intensity of the immunoblot bands marked by the arrows was quantified by BIOQUANT image analysis software. HbA1c levels were measured at the clinical laboratory of The Joslin Diabetes Center by HPLC. (B) Immunoblot of glycated (G) and nonglycated (NG) albumin (Upper) and immunoaffinity-purified hRBC CD59 (Lower) with anti-hexitol–lysine Ab.

Glycation increases the sensitivity of hRBC to MAC-mediated lysis and of HUVEC to MAC-induced release of growth factors. (A) hRBC were incubated without or with 50 mM ribose for 48 h at room temperature followed by reduction with NaBH₃CN (open column, cells not exposed to NaBH₃CN; filled columns, cells exposed to NaBH₃CN). Cell volume was adjusted in glycated and nonglycated control cells, and aliquots of cells were separated for rescue with immunoaffinity-purified human CD59 (4 μg) or incubation with neutralizing anti-CD59 Ab (YTH53.1), before exposure to purified human C5b6, C7, C8, and C9 to form the MAC. The last column to the right represents cells that were first exposed to the anti-CD59 Ab and then rescued with affinity-purified human CD59. (Inset) Similar osmotic fragility of control (□) and glycated (■) cells after volume adjustment. (B) HUVEC were incubated with or without 50 mM ribose (24 h, 37°C), followed by reduction with NaBH₃CN. Cells were then exposed to purified human C5b6, C7, C8, and C9 to form the MAC, and the mitogenic activity in the conditioned medium was measured as in ref. . Results are expressed as the ratio of mitogenic activity released into the conditioned media in the presence or absence of MAC.