DAF in diabetic patients is subject to glycation/inactivation at its active site residues☆
Introduction
Decay accelerating factor (DAF or CD55) is a cell-associated regulatory protein initially characterized as a control protein that circumvents activation of autologous complement on self- cell surfaces (Medof et al., 1984). It recently was shown to be a key modulator of T cell activation (Heeger et al., 2005, Strainic et al., 2008). It functions by rapidly dissociating C3 and C5 convertases that assemble on self-cells thereby blocking C3a/C5a generation. In the context of serum complement activation, it not only shields against self-cell injury but also prevents amplification of C3b deposition (that enables interaction with C3b-bearing leukocytes) and C5b deposition [which triggers assembly of the membrane attack complex (MAC)] (Medof et al., 1996). In the context of adaptive T effector cell responses, DAF suppresses the endogenous generation of C3a/C5a by interacting with dendritic cells (DCs) and T cells. This latter function inhibits autocrine signaling through C3a and C5a receptors (C3ar1/C5ar1) in both partners needed for the costimulation and viability of T cells (Strainic et al., 2008, Lalli et al., 2008) as well as for the activation of DCs, macrophages and other antigen presenting cells (APCs) (Liu et al., 2008, Strainic et al., 2008).
In diabetes mellitus hyperglycemia leads to nonenzymatic modification of proteins by glucose and by glucose-derived reactants such as methylglyoxal and glyoxal (reviewed in Monnier et al., 1996). Similar modifications can occur in other conditions including cancer, and Alzheimer’s disease, as well as in aging. These modifications, termed advanced glycation end products (AGEs), have been implicated in the development of the chronic complications of diabetes, i.e., nephropathy, neuropathy, and retinopathy. The AGEs can form extracellularly or intracellularly via oxidation reaction of sugars, dicarbonyls or ascorbate oxidation products with protein amines. Among reported AGEs are N−ε-carboxymethyl lysine (CML) (Ahmed et al., 1986), pentosidine (Sell and Monnier, 1989) and argpyrimidine (Shipanova et al., 1997).
If DAF were to be chemically modified in any of the above conditions so as to lose functionality, several deleterious processes might result. Autologous C3b and its immediate (factor I) cleavage product, iC3b, bound to self-cells could serve as ligands for C3b and iC3b receptors (CR1 and CR3) on leukocytes, (polymorphonuclear cells, monocytes and macrophages) potentially inducing the release of proteolytic enzymes and inflammatory mediators from these cells (reviewed in van Lookeren Campagne et al., 2007). The local generation of autologous C3a and C5a anaphylatoxins that accompanies C3 cleavage by dysregulated C3 convertases could enhance recruitment of these leukocytes as well as cause vasodilation and aggregation of the incoming cells (reviewed in Liszewski and Atkinson, 2015). In addition, local C3a/C5a generation by interacting DCs and T cells could be potentiated and consequent C3ar1/C5ar1 signaling augmented thereby favoring the generation of Th1/Th17 cells (Liu et al., 2008) rather than the generation of Foxp3+ T regulatory cells (Tregs) (Strainic et al., 2013). This would increase the production of inflammatory cytokines by DCs, macrophages, and other APCs (Strainic et al., 2008, Liu et al., 2008).
DAF is composed of 4 tandem ∼60 amino acid long homologous repeats termed short consensus repeats (SCRs) or complement control repeats (CCPs). The four CCPs are attached on a long heavily O-glycosylated stalk which suspends them at an appropriate distance above the cell membrane (Medof et al., 1987, Stafford et al., 1988, Kuttner-Kondo et al., 1996). Structure function analyses of the four CCPs have shown that DAF’s regulatory activity resides principally in residues at and near the junction of CCP2 and CCP3 with some contribution from residues in CCP4 (Kuttner-Kondo et al., 2001, Kuttner-Kondo et al., 2007). NMR analyses and crystallography have shown that CCP4 is located in proximity to the active site residues in CCPs2-3 (Kuttner-Kondo et al., 2001, Kuttner-Kondo et al., 2007, Uhrinova et al., 2002, Uhrinova et al., 2003, Harris et al., 2005, Lukacik et al., 2004).
Herein, we report that DAF proteins isolated from erythrocytes of diabetic patients contain AGEs. Functional and structural analyses of DAF showed that glucose (and ribose) incubation inactivated it by chemical modification of its critical active site residues. That DAF glycation could enhance complement activation as well as promote T effector responses in diabetes and other conditions argues that this process could contribute to the development of complications.
Section snippets
Proteins and antibodies
DAF protein was purified from extracts of erythrocyte (Ehu) stroma as detailed in (Medof et al., 1986). Murine anti-DAF monoclonal antibodies (mAb) IIH6 and IA10 were prepared as described (Kinoshita et al., 1985). Rabbit polyclonal anti-GXL-AGE antibodies (Sady et al., 2000), glyoxal-modified bovine serum albumin (GXL-BSA), and murine anti-argpyrimidine mAb were similarly prepared as described (Oya et al., 1999). Rabbit polyclonal anti-pentosidine antibodies and pentosidine-modified bovine
Identification of AGEs on DAF expressed on erythrocytes of diabetic patients
To establish whether DAF is subject to glycation in vivo and is modified by AGEs analogous to findings reported for other proteins (Monnier et al., 1996), we first affinity purified DAF protein from erythrocytes of diabetic patients with long-standing disease and healthy controls, and probed immunoblots of the immuno-isolates with antibodies against different AGEs. In contrast to DAF recovered from erythrocytes of nondiabetic control subjects, which showed reactivity with anti DAF mAb (Fig. 1A)
Discussion
DAF was originally characterized as a cell surface, i.e. “intrinsic” complement regulatory protein that protects self- tissues from injury that could be induced by autologous complement activation on their surfaces (Medof et al., 1984). In this context, it acts on systemic complement and functions as an inhibitor of the central C3/C5 amplification convertases of the complement cascade. More recent studies unexpectedly found that DAF also plays a pivotal role in regulating adaptive T cell
Conclusions
Taken together, our results indicate that augmented systemic complement activation that could result from impairment of DAF’s function by nonenzymatic glycation could participate in the local inflammation that has been implicated in diabetes (Lukacik et al., 2004, Tang and Kern, 2011) cancer, aging, and other conditions. Since activated monocytes/macrophages serve as professional antigen presenting cells, and vascular endothelial cells can serve as nonprofessional antigen presenting cells in
Acknowledgements
The authors thank Dr. V. Monnier (Institute of Pathology, Case Western Reserve University) for providing the anti-pentosidine antibody and Dr. Koji Uchida (Nagoya University, Japan) for providing the argpyrimidine antibody. We thank Dr. Jose Halperin for supporting RF for this work and helpful suggestions, Dr. Judith Hettinga and Dr. Nicole Krumrei for establishing the hemolytic assay, Dr. John Rush from the Howard Hughes Medical Institute, Harvard Medical School for performing the LC/MS
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This work was supported by National Institutes of Health grants EY11288, AI071125, and HL109561, AR067182 (MEM), EY022938, EY024864 and a Merit grant from the Department of Veteran Affairs (TSK), EY023286 (RHN), and EY11373 (Core), DK52855 (RF).
- 1
Current Address: Novacule, LLC, 2587 Albany Ave, West Hartford, CT 06117, USA.
- 2
Current address: University of Colorado School of Medicine, Aurora, CO 80045, USA.