scholarly journals Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3.

1995 ◽  
Vol 181 (1) ◽  
pp. 115-125 ◽  
Author(s):  
M Matsumoto ◽  
F Yamashita ◽  
K Iida ◽  
M Tomita ◽  
T Seya
Keyword(s):  
Membrane Protein ◽  
Human Cells ◽  
Alternative Complement Pathway ◽  
Regulatory Proteins ◽  
Complement C3 ◽  
Parent Cell ◽  
Complement Pathway ◽  
Single Chain ◽  
Complement Regulatory Proteins ◽  
Alternative Complement

A human myeloid cell subline, P39+, is found to be a target for human complement (C) via the alternative pathway and to allow the deposition of multiple C3 fragments on its membranes, though expressing the complement regulatory proteins decay-accelerating factor and membrane cofactor protein. The parent cell line, P39-, which is phenotypically similar to the P39+ subline, does not allow the deposition of homologous C3 fragments. In this study, we established a monoclonal antibody, M161 Ab, which reacted with P39+ but not P39- cells. This Ab recognized a 43-kD protein in P39+ cell lysate transblotted onto nitrocellulose. Using this Ab as a probe, we purified the 43-kD protein, namely, M161 antigen (Ag). M161 Ag had a basic isoelectric point (pI), 9.3-9.4 by chromatofocusing, and was precipitated as an insoluble material at the pI point. The purified M161 Ag was a single-chain protein and did not possess N- or O-linked carbohydrates. When the purified M161 Ag was transblotted onto nitrocellulose and incubated with Mg(2+)-EGTA serum, human C3 fragments were efficiently deposited on M161 Ag. The major species of the deposited C3 fragments was C3b. Furthermore, the C3 fragments bound to the M161 Ag were detached by 1 M hydroxylamine, suggesting that a covalent ester linkage sustains M161 Ag-C3b interaction. NH2-terminal amino acid analysis revealed that M161Ag is a novel membrane protein. Hence, it appeared that M161 Ag is a potent activator of human alternative complement pathway on human cells that activates homologous C3 and allows the deposition of C3b on itself. Thus, under some conditions, homeostasis of complement is maintained even on human cells, not only by the complement regulatory proteins, but also by membrane C3-activating molecules on which C3b is deposited.

Membrane protein Crry prevents excessive turnover of the alternative complement pathway in vivo

2007 ◽  
Vol 44 (1-3) ◽  
pp. 258
Author(s):  
Xiaobo Wu ◽  
Dirk Spitzer ◽  
Dailing Mao ◽  
Stanford Peng ◽  
Hector Molina ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Alternative Complement Pathway ◽  
Complement Pathway ◽  
Alternative Complement

scholarly journals Activation of the alternative complement pathway with rabbit erythrocytes by circumvention of the regulatory action of endogenous control proteins

1977 ◽  
Vol 146 (1) ◽  
pp. 22-33 ◽  
Author(s):  
DT Fearon ◽  
KF Austen
Keyword(s):  
Alternative Pathway ◽  
Fluid Phase ◽  
Fold Increase ◽  
Alternative Complement Pathway ◽  
Regulatory Proteins ◽  
Low Grade ◽  
Phase Reaction ◽  
Complement Pathway ◽  
Alternative Complement ◽  
Grade Fluid

Cleavage of C3 by the alternative complement pathway occurs in at least two distinct phases: continuous low grade generation of C3b by the interaction of native C3, B, D, and P, and subsequent amplified cleavage of C3 by the interaction of C3b, B, D, and P which forms the amplification convertase, P,C3b,Bb. Transition to C3b-dependent amplification is necessary to achieve substantial C3 cleavage and is normally limited by the combined action of C3b inactivator (C3bINA) and βlH. An activator of the alternative pathway, such as rabbit erythrocytes (E(r)), provides sites that protect bound C3b and P,C3b,Bb from the action of these regulatory proteins and permits C3b deposited by the low grade fluid phase reaction to assemble a membrane-associated amplification convertase which can deposit additional protected C3b. Under conditions in which the control proteins, C3bINA and β1H, almost completely inactivated C3b bound to sheep erythrocytes (E(s)), which does not activate the alternative pathway, the function of C3b bound to E(r) was diminished by less than one-fifth. Further, the P- stabilized amplification convertase on E(r) was 10-fold less sensitive to β1H-mediated decay-dissociation than the convertase on E(s). The addition of E(r) to a regulated mixture of purified C3, B, D, P, C3bINA, and β1H resulted in amplified inactivation of C3 and B by formation of the amplification convertase on E(r) as indicated by its lysis with subsequent exposure to C3-C9. In contrast, E(s) did not advance the low grade fluid phase inactivation of C3 and B to amplified inactivation and the cell was not converted to an intermediate susceptible to lysis by C3- C9. Since E(r) and E(s) did not differ in their inefficient fixation of C3b generated during an unregulated fluid phase reaction, the activating capacity of E(r) must reside in its protection of bound C3b and P, C3b,Bb from the regulatory proteins rather than in enhanced capacity to bind C3b from the fluid phase. When the reaction is limited to low grade fluid phase turnover, introduction of E(r) but not E(s) results in a 100-fold increase in the deposition of C3b, indicating that surface-dependent activation of the alternative pathway is characterized by efficient deposition of C3b on the initiating surface. Thus, the activating surfaces advance the interaction of the alternative pathway proteins to the amplification phase because of the selective inability of the regulatory proteins to deal with their substrates when deposited on these surfaces and results in a specificity that is not necessarily dependent on adaptive immunity.

scholarly journals Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway

2017 ◽  
Author(s):  
Rosario Fernandez-Godino ◽  
Kinga M. Bujakowska ◽  
Eric A. Pierce
Keyword(s):  
Extracellular Matrix ◽  
Macular Degeneration ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Alternative Complement Pathway ◽  
Complement C3 ◽  
Matrix Metalloproteinase 2 ◽  
Complement Pathway ◽  
Rpe Cells ◽  
Alternative Complement

ABSTRACTThe design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch’s membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in AMD, we generated ARPE-19 cells with the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The ARPE-19-EFEMP1R345W/R345W cells make abnormal extracellular matrix (ECM) that binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. hfRPE cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 (MMP-2) activity. Therapies that target ECM synthesis and turnover and activation of C3 could be effective for early AMD.

scholarly journals A patient with a homozygous diacylglycerol kinase epsilon (DGKE) gene mutation with atypical haemolytic uraemic syndrome and low C3 responded well to eculizumab: a case report

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Muneera Alabdulqader ◽  
Khalid Alfakeeh
Keyword(s):  
Case Report ◽  
Gene Mutation ◽  
Haemolytic Uraemic Syndrome ◽  
Diacylglycerol Kinase ◽  
Alternative Complement Pathway ◽  
Complement C3 ◽  
Complement Pathway ◽  
Atypical Haemolytic Uraemic Syndrome ◽  
Alternative Complement ◽  
Nephrotic Range Proteinuria

Abstract Background Atypical haemolytic uraemic syndrome (aHUS) is a rare systemic syndrome characterized by non-immune haemolytic anaemia, thrombocytopenia, and kidney injury. In most cases, alternative complement pathway dysregulation is the identifying cause. Recently, other genetic causes have been identified, including a mutation in the diacylglycerol kinase epsilon (DGKE) gene, which theoretically affect the coagulation pathway and does not affect the complement pathway. Data about the management of these patients are limited. Ideal management and definitive treatment protocols have not yet been established. Case presentation A three-year-old boy presented with features of atypical haemolytic uraemic syndrome (aHUS) and low complement C3. He was presumed to have complement-mediated aHUS and was managed empirically with eculizumab. Two weeks after starting eculizumab, his haemoglobin levels, platelet count, and complement C3 level normalized but he continued to have non-nephrotic range proteinuria. His genetic testing revealed a homozygous DGKE mutation, with no other mutation detected. Six months after presentation, the patient was still in remission with no features of aHUS, a trial of weaning eculizumab by increasing dose interval was followed by nephrotic range proteinuria and severe oedema. His proteinuria improved and his oedema resolved after resuming his recommended eculizumab dose. Conclusions DGKE gene mutation can lead to aHUS with theoretically no complement dysregulation. However, some patients with this mutation show alternative complement pathway activation. This case report describes a patient with aHUS due to a DGKE gene mutation and low C3 levels who responded to eculizumab, adding to the previously reported cases of patients with DGKE gene mutations who had complete remission with no relapse with C5 blockers and/or plasma exchange. A randomized controlled study on patients with DGKE mutations might be beneficial in understanding the disease and generating a management protocol.

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