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Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.

The astrocyte-derived protein S100B stimulates production of inducible nitric oxide synthase and nitric oxide (NO) in astrocytes [Hu et al., 1996, J. Biol. Chem. 271:2543], but its effect on microglia is not known. In addition, S100B's ability to modulate the activity of other glial activating agents has not been defined. In this study, we compared the ability of S100B to stimulate NO in cultures of rat primary astrocytes and the BV-2 murine microglial cell line, and investigated the effect of the combined action of S100B and other stimuli known to activate glial cells. S100B itself stimulated the production of NO in astrocytes, and did not modify or potentiated only weakly the NO production induced by interleukin-1 beta, tumor necrosis factor alpha, dibutyryl cyclic AMP, zymosan A or lipid A. In contrast, S100B alone did not induce NO in BV-2 cells but strongly potentiated NO production in the presence of lipid A but not zymosan A. The deletion of eight C-terminal amino acid residues in S100B leads to a loss of the effect of S100B on microglia but not on astrocytes. These results demonstrate that responses of glial cells to extracellular S100B can vary depending on the cell type, and suggest that different structural features of S100B are important for the protein's effects on microglia and astrocytes.

Calcium vector protein target (CaVPT), a 26-kDa endogenous target of calcium vector protein from Amphioxus (CaVP), contains three distinct regions: a N-terminal Pro-Ala-Lys-rich motif, segment 36-50 displaying sequence similarity to the calmodulin-binding site in neuromodulin and neurogranin where they are designated as the IQ domain; and two immunoglobulin-like folds. The phosphorylation by protein kinase C of Ser-43 in the IQ domain drastically decreases the affinity of CaVPT for CaVP and CaVP protects CaVPT from phosphorylation. Phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase has a similar effect, but in addition to Ser-43 four other phosphorylated sites were identified. Removal of the Pro-Ala-Lys-rich region and the IQ domain in CaVPT by trypsin leads to the loss of binding to CaVP, whereas the chymotryptic fragment, containing these regions and first immunoglobulin-like domain, retained the ability to interact with CaVP. A synthetic IQ domain alone interacts strongly with calmodulin, but not with CaVP. Two main conclusions can be drawn from this study: 1) the regulation of interaction between CaVP and CaVPT is very similar to the mechanism observed in the complex between neuromodulin or neurogranin and calmodulin; 2) in spite of this similarity the entire CaVP-binding site is not restricted to the IQ domain; in addition the Pro-Ala-Lys-rich motif may be necessary for high affinity binding to CaVP.

Calcium vector protein (CaVP) of amphioxus shares some common structural features with Ca(2+)-dependent activators such as troponin C and calmodulin, and is associated in vivo with a 26 kDa (CaVPT), a multidomain protein with one IQ- and two IgII-motifs. Isolated CaVP binds two Ca2+ ions with very different intrinsic affinity constants: K'Ca1 = 4.9 x 10(6) M-1 and K'Ca2 = 7.3 x 10(3) M-1, respectively. In the complex with CaVPT, CaVP also binds two Ca2+, but with strong positive cooperativity (nH = 1.9) and with distinctly higher affinity: K'Ca1 = 2.4 x 10(5) M-1 and K'Ca2 = 1.0 x 10(8) M-1. Since neither in the isolated CaVP nor in the complex Ca2+ binding is influenced by 2 mM MgCl2, both sites can be considered as Ca(2+)-specific. In the absence of Ca2+, the complex is stable under physiological conditions, but the interaction is governed by the principle of linked functions and Ca2+ binding to CaVP reinforces the affinity between CaVP and CaVPT 70-fold. Both proteins interact with the hydrophobic probe 2 p-toluidinylnaphthalene-6-sulfonate (TNS), but CaVPT enhances the fluorescence 45-fold, CaVP-Ca2 and metal-free CaVP only 10- and 5-fold, respectively. Complex formation between CaVPT and CaVP leads to a 3-fold reduction of the fluorescence enhancement, suggesting that a strong solvent-shielded hydrophobic core is formed. CaVP contains two highly reactional thiols (kSH > 0.3 s-1) for 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); CaVPT contains three thiols, two of them also with kSH > 0.3 s-1 in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)