Mikkel Holmen Andersen

C-terminal Modification of Osteopontin Inhibits Interaction with the αVβ3-Integrin

Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the αVβ3-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the αVβ3-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the αVβ3-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the αVβ3-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the αVβ3-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the αVβ3-integrin by modification of the highly conserved C-terminal region of the protein.

Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice.

OBJECTIVE Uremia markedly increases the risk of atherosclerosis. Thus, effective anti-atherogenic treatments are needed for uremic patients. This study examined effects of non-lipidated recombinant human apoA-I (h-apoA-I) and a recombinant trimeric apoA-I molecule (TripA-I) on lipid metabolism and atherosclerosis in uremic apoE-/- mice. METHODS AND RESULTS Upon intraperitoneal injection, h-apoA-I and TripA-I rapidly associated with plasma HDL and reduced mouse apoA-I plasma levels without affecting total or HDL cholesterol concentrations. The plasma half-life was approximately 36 h for TripA-I and approximately 16 h for h-apoA-I. Injection of h-apoA-I (100mg/kg) or TripA-I (100mg/kg) twice weekly for 7 weeks did not affect the cross-sectional area of atherosclerotic lesions in the aortic root, or the en face lesion area and cholesterol content in the thoracic aorta in uremic apoE-/- mice. Also, the treatments did not affect expression of selected inflammatory genes in the thoracic aorta or plasma concentrations of soluble ICAM-1 and VCAM-1. However, h-apoA-I-treated mice had larger smooth muscle cell-staining areas in aortic root plaques than PBS-treated mice (4.8+/-0.8% vs. 2.5+/-0.6%, P<0.05). CONCLUSIONS The data suggest that long-term treatment with non-lipidated h-apoA-I or TripA-I might affect plaque composition but does not reduce atherosclerotic lesion size in uremic apoE-/- mice.

Trimerization of apolipoprotein A-I retards plasma clearance and preserves antiatherosclerotic properties.

An increased plasma level of the major high-density lipoprotein (HDL) component, apolipoprotein A-I (apoA-I) is the aim of several therapeutic strategies for combating atherosclerotic disease. HDL therapy by direct intravenous administration of apoA-I is a plausible way; however, a fast renal filtration is a major obstacle for this approach. Using protein engineering technology, we have fused apoA-I to the trimerization domain of human tetranectin and thus constructed a high-mass recombinant trimeric apoA-I variant. The recombinant fusion protein was stable and expressed well; upon purification and intravenous injection into mice, it exhibited prolonged plasma retention time compared to wild type apoA-I. Trimeric apoA-I was biologically active in terms of promoting cholesterol efflux, stimulation of lecithin cholesterol acyltransferase-mediated cholesterol esterification, and reducing progression of atherosclerosis in cholesterol-fed low-density lipoprotein receptor-deficient mice. Direct administration of recombinant high-mass apoA-I analogues with retarded clearance is therefore a potential novel therapeutic approach for atherosclerotic plaque stabilization.

Selection of a Novel and Highly Specific Tumor Necrosis Factor α (TNFα) Antagonist INSIGHT FROM THE CRYSTAL STRUCTURE OF THE ANTAGONIST-TNFα COMPLEX

Inhibition of tumor necrosis factor α (TNFα) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFα inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFα as a test target to validate this new technology because of the extensive experience available with protein-based TNFα antagonists. Here, we present a novel and highly specific TNFα antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFα complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFα.Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.

Selection of a novel and highly specific TNF{alpha} antagonist: insight from the crystal structure of the antagonist-TNF{alpha} complex

Abstract Inhibition of TNFα is a favourable way of treating several important diseases such as rheumatoid arthritis, Crohns disease and psoriasis. Therefore an extensive range of TNFα inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutic. We have developed a novel technology platform to using C-type lectins as vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties compared to current protein therapeutics. We chose TNFα as a test target to validate this new technology because of the extensive experience available in protein based TNFα antagonist. Here we present a novel and highly specific TNFα antagonist developed using this technology. Furthermore, we have solved the 3 dimensional structure of the antagonist-TNFα complex by X-ray crystallography and this structure is presented here. The structure has given us a unique insight in how the selection procedure works at a molecular level. Surprisingly little change is seen in the CTLD structure outside of the randomized regions, where as substantial change is seen within the randomized loops. Thus the overall integrity of the CTLD is maintained where as the specificity and binding affinity is change by the introduction of a range of specific contact with TNFα.