Search

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers., Competing Interests: Conflict of interest C. Q. S., H. S., B. H., A. D. and M. H.-L. are inventors of (a) patent application(s) that describes the use of complement inhibitors for therapeutic applications. C. Q. S. has received research funding from pharmaceutical companies. M. H.-L., C. Q. S., B. H. and H. S. received honoraria for speaking at symposia organized by Alexion Pharmaceuticals. H. S. served on advisory committees for Alexion AstraZeneca Rare Diseases, Sanofi, Sobi, Novartis, Amgen and received research funding from Alexion Pharmaceuticals (all to the University of Ulm). The other authors have disclosed no relevant conflict of interest with the contents of the article. B. K. G. and R. B. were/are employees at Takeda Pharmaceuticals., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing lesion on the surface of infected cells causes significant remodeling of the host cell surface; however, limited information is available about changes at the protein level. Here we employed plasma membrane profiling, a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor cluster of differentiation 55 (CD55) exhibited the greatest reduction in cell-surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Furniss et al.)

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Cyclooxygenase-derived prostaglandin E(2) (PGE(2)) stimulates tumor progression by modulating several proneoplastic pathways. The mechanisms by which PGE(2) promotes tumor growth and metastasis through stimulation of cell migration, invasion, and angiogenesis have been fairly well characterized. Much less is known, however, about the molecular mechanisms responsible for the immunosuppressive effects of PGE(2). We identified PGE(2) target genes and subsequently studied their biologic role in colorectal cancer cells. The complement regulatory protein decay-accelerating factor (DAF or CD55) was induced following PGE(2) treatment of LS174T colon cancer cells. Analysis of PGE(2)-mediated activation of the DAF promoter employing 5'-deletion luciferase constructs suggests that regulation occurs at the transcriptional level via a cyclic AMP/protein kinase A-dependent pathway. Nonsteroidal anti-inflammatory drugs blocked DAF expression in HCA-7 colon cancer cells, which could be restored by the addition of exogenous PGE(2). Finally, we observed an increase in DAF expression in the intestinal mucosa of Apc(Min+/-) mice treated with PGE(2) in vivo. In summary, these results indicate a novel immunosuppressive role for PGE(2) in the development of colorectal carcinomas.

Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum.

Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.