ABSTRACT
Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.
Subject(s)
Conserved Sequence , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Heparin/metabolism , Poxviridae/immunology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Binding Sites , Complement Inactivator Proteins/chemistry , Complement Inactivator Proteins/genetics , Complement Inactivator Proteins/immunology , Complement Inactivator Proteins/metabolism , Endothelium, Vascular/cytology , Hemolysis , Histocompatibility Antigens Class I/immunology , Humans , Models, Molecular , Molecular Sequence Data , Poxviridae/chemistry , Poxviridae/genetics , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion/genetics , Static Electricity , Structure-Activity Relationship , Surface Properties , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
Traumatic brain injury can lead to locally destructive secondary events mediated by several inflammatory components. Following lateral fluid-percussion (FP) brain injury in rats, we examined cortical and hippocampal sections for neutrophil infiltration and accumulation of complement component C3. Neutrophil influx into the brain after injury was detected by an improved myeloperoxidase (MPO) microassay and manual cell counting, while C3 accumulation was detected using immunocytochemistry. MPO levels were elevated in the injured cortical tissue, whereas C3 immunoreactivity was increased in both injured cortical and ipsilateral hippocampal sections. These results show that the FP model of head injury leads to an intense local inflammatory reaction and subsequent tissue destruction.
