Collagen binding activity of recombinant and N‐terminally modified annexin V (anchorin CII)

Abstract

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.

Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice

“…26 In one set of experiments Balb/c mice were assigned randomly to three treatment groups (n=6), intraperitoneally injected with viable cells, apoptotic cells or AxVtreated apoptotic cells, respectively. In another set of experiments the cell suspension was mixed with FA (140 ml/ml) immediately before injection (n=4).…”

Expression of Anchorin CII (Cartilage Annexin V) in Human Young, Normal Adult, and Osteoarthritic Cartilage

“…It has been proposed that a stable membrane-associated variant of Anx V exists that differs from the cytosolic soluble variant in the three-dimensional arrangement of a tryptophan residue at position 187 (Sopkova et al 1993). Many characteristics have been demonstrated for Anx V, such as the binding of collagen (Mollenhauer and von der Mark 1983;von der Mark et al 1991;Kirsch and Pfäffle 1992;Turnay et al 1995), formation of calcium channels (Rojas et al 1990;Berendes et al 1993), inhibition of protein kinase C (Schlaepfer et al 1992;Raynal et al 1993), promotion of urokinasetype plasminogen activator synthesis and migration of keratinocytes (Nakao et al 1994), binding of human platelets (Andree et al 1985;Tait et al 1995), and binding to the small hepatitis B virus envelope protein (Hertogs et al 1993). In addition, elevated levels of Anx V have been shown in the pathology of cystic fibrosis (Gaspera et al 1995).…”

Changes in the expression of annexin A5 gene during in vitro chondrocyte differentiation: Influence of cell attachment

“…Autoradiograhs were scanned and the densitometric analysis of the gels was performed obtaining volumograms on a photodocumentation system from UVItec (Cambridge, UK) and by using the UVIBand V.97 software. The cDNA probe for chicken annexin A5 (pACII) was obtained in our laboratory [Turnay et al, 1995]; that for the chicken a1 type X [SPLX; LuValle et al, 1988], and a1 type II [pCS1; Young et al, 1984] collagen chains were kindly provided by Dr. P. LuValle (Department of Biochemistry and Molecular Biology, University of Calgary, Canada) and Dr. M.E. Sobel (National Cancer Institute, Bethesda, MD), respectively.…”

“…Since annexin A5 binds speci®cally to native collagens II and X [Kirsch et al, 1994;Turnay et al, 1995;von der Mark and Mollenhauer, 1997], a model was developed proposing that these properties may be of particular relevance for the role of matrix vesicles in cartilage calci®cation. Matrix vesicles, which are released from hypertrophic chondrocytes in the cartilage growth plate, are rich in annexin A5 and alkaline phosphatase among other proteins [Anderson, 1995].…”