Thermodynamic stability of human lipocalin-type prostaglandin D synthase under various pH conditions.

Lipocalin-type prostaglandin D synthase (L-PGDS) binds various hydrophobic small molecules. Since we aim to use human L-PGDS as a carrier in a drug delivery system (DDS) for poorly water-soluble drugs, quality control of the protein is indispensable. In this study, we investigated the thermodynamic stability of human L-PGDS under various pH conditions. Differential scanning calorimetry revealed that the thermal unfolding of L-PGDS was an almost-reversible two-state transition between the native and unfolded states over the pH range from 2.5 to 7.4. The linear relationship of ΔH(Tm) to Tm in this pH range gave a heat capacity change (ΔCp) of 4.76 kJ/(K·mol), which was small compared to those commonly found in globular proteins. The temperature-dependent free energy of unfolding, ΔG(T), specified by Tm, ΔH(Tm) and ΔCp, showed a pH dependence with the highest value at pH 7.4 closest to the isoelectric point of 8.3. The small value of Cp resulted in a large value of ΔG(T), which contributed to the stability of the protein. Taken together, these results demonstrated that human L-PGDS is sufficiently thermostable for storage and practical use and can be useful as a delivery vehicle of protein-based DDS.

Zinc mediates the interaction between ceruloplasmin and apo-transferrin for the efficient transfer of Fe(III) ions.

Fe(II) exported from cells is oxidized to Fe(III), possibly by a multicopper ferroxidase (MCF) such as ceruloplasmin (CP), to efficiently bind with the plasma iron transport protein transferrin (TF). As unbound Fe(III) is highly insoluble and reactive, its release into the blood during the transfer from MCF to TF must be prevented. A likely mechanism for preventing the release of unbound Fe(III) is via direct interaction between MCF and TF; however, the occurrence of this phenomenon remains controversial. This study aimed to reveal the interaction between these proteins, possibly mediated by zinc. Using spectrophotometry, isothermal titration calorimetry, and surface plasmon resonance methods, we found that Zn(II)-bound CP bound to iron-free TF (apo-TF) with a Kd of 4.2 µM and a stoichiometry CP:TF of ∼2:1. Computational modeling of the complex between CP and apo-TF predicted that each of the three Zn(II) ions that bind to CP further binds to an acidic amino acid residue of apo-TF to play a role as a cross-linker connecting both proteins. Domain 4 of one CP molecule and domain 6 of the other CP molecule fit tightly into the clefts in the N- and C-lobes of apo-TF, respectively. Upon the binding of two Fe(III) ions to apo-TF, the resulting diferric TF [Fe(III)2TF] dissociated from CP by conformational changes in TF. In human blood plasma, zinc deficiency reduced the production of Fe(III)2TF and concomitantly increased the production of non-TF-bound iron. Our findings suggest that zinc may be involved in the transfer of iron between CP and TF.

Absence of binding between the human transferrin receptor and the transferrin complex of biological toxic trace element, aluminum, because of an incomplete open/closed form of the complex.

Human transferrin (Tf) very tightly binds two ferric ions to deliver iron to cells. Fe(III)(2)Tf (Fe(2)Tf) binds to the Tf receptor (TfR) at pH 7.4; however, iron-free Tf (apoTf) does not. Iron uptake is facilitated by endocytosis of the Fe(2)Tf-TfR complex. Tf can also bind aluminum ions, which cause toxic effects and are associated with many diseases. Since Al(III)(2)Tf (Al(2)Tf) does not bind to TfR, the uptake of aluminum by the cells does not occur through a TfR-mediated pathway. We have studied the absence of binding between Al(2)Tf and TfR by investigating the physicochemical characteristics of apoTf, Al(2)Tf, Fe(2)Tf, and TfR. The hydrodynamic radius of 38.8 A for Al(2)Tf obtained by dynamic light scattering was between that of 42.6 A for apoTf and 37.2 A for Fe(2)Tf. The zeta potential of -11.3 mV for Al(2)Tf measured by capillary electrophoresis was close to -11.2 mV for apoTf as compared to -11.9 mV for Fe(2)Tf, indicating that the Al(2)Tf surface had a relatively scarce negative charge as the apoTf surface had. These results demonstrated that the structure of Al(2)Tf was a trade-off between the closed and open forms of Fe(2)Tf and apoTf, respectively. Consequently, it is suggested that Al(2)Tf cannot form specific ionic interresidual interactions, such as those formed by Fe(2)Tf, to bind to TfR, resulting in impossible complex formation between Al(2)Tf and TfR.

Computational structure models of apo and diferric transferrin-transferrin receptor complexes.

Complexation of transferrin (Tf) and its receptor (TfR) is an essential event for iron uptake by the cell. Much data has been accumulated regarding Tf-TfR complexation, such as results from mutagenesis. We created 3D structural models of apo-human Tf-TfR (apoTf-TfR) and Fe(III)(2)Tf-TfR (Fe(2)Tf-TfR) complexes by computational rigid body refinement. The models are consistent with published mutagenesis experiments. In our models, the C-lobes of apoTf and Fe(2)Tf bind to the helical domain of TfR, and the N-lobes are sandwiched between the ectodomain of TfR and the cell membrane as previously reported. Further, the molecules of apoTf and Fe(2)Tf are not forced to undergo large conformational changes upon complexation. The creation of the models led a new and important finding that a residue of TfR, R651, which is called a hot spot for Tf-TfR binding, interacts with Tf E385 when either apoTf or Fe(2)Tf bind to TfR. The models rationally interpret the iron release from Fe(2)Tf-TfR upon acidification, dissociation of apoTf from TfR at slightly alkaline pH, and metal specific recognition of TfR.

Mutagenicity and PAH contents of soil in forests or planted areas in Japan.

We investigated the behavior of mutagenic substances in the soil of forests or planted areas. Mutagenicity and concentration was examined for 16 types of PAHs in soil samples collected at a depth of 1 m in 10 forests in Iwate, Ibaraki, Tokyo, Kanagawa, Yamanashi and Shizuoka prefectures in Japan. Mutagenicity and PAHs were detected mostly in soil from the surface to a depth of 30 cm when strains TA100, TA98 and YG1024 were used. In addition, a significant correlation was not found between the concentration of BaP, and specific mutagenic activity (TA98 without S9mix, r = 0.285).