Exogenously added GPI-anchored tissue inhibitor of matrix metal loproteinase-1 (TIMP-1) displays enhanced and novel biological activities

The family of tissue inhibitors of metalloproteinases (TIMPs) exhibits diverse physiological/biological functions including the inhibition of active matrix metalloproteinases, regulation of proMMP activation, cell growth, and the modulation of angiogenesis. TIMP-1 is a secreted protein that can be detected on the cell surface through its interaction with surface proteins. The diverse biological functions of TIMP-1 are thought to lie, in part, in the kinetics of TIMP-1/MMP/surface protein interactions. Proteins anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, are incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to generate a reagent that could be added directly to cell membranes and thus focus defined concentrations of TIMP-1 protein on any cell surface independent of protein-protein interaction. Unlike native TIMP-1, exogenously added GPI-anchored TIMP-1 protein effectively blocked release of MMP-2 and MMP-9 from osteosarcoma cells. TIMP-1-GP1 was a more effective modulator of migration and proliferation than TIMP-1. While control hTIMP-1 protein did not significantly affect migration of primary microvascular endothelial cells at the concentrations tested, the GPI-anchored TIMP-1 protein showed a pronounced suppression of endothelial cell migration in response to bFGF. In addition, TIMP-1-GPI was more effective at inducing microvascular endothelial proliferation. In contrast, fibroblast proliferation was suppressed by the agent. Reagents based on this method should assist in the dissection of the protease cascades and activities involved in TIMP biology. Membrane-fixed TIMP-1 may represent a more effective version of the protein for use in therapeutic expression

Decay-accelerating factor modulates induction of T cell immunity

Decay-accelerating factor (Daf) dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. We demonstrate that during primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. We show that cognate T cell–APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3beta-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions

Analysis of binding sites on complement factor I using artificial N-linked glycosylation.

Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors such as factor H, C4b-binding protein, complement receptor 1 and membrane cofactor protein. FI is composed of two chains linked by a disulphide bridge; the light chain comprises only the serine protease (SP) domain, while the heavy chain contains FI membrane attack complex domain (FIMAC), CD5 domain, low-density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based 3D models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (Km) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespectively of the cofactor used. In the other hand only few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI