The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (K_d = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.     

Reversible monomer-oligomer transition in human prion protein

The structure and the dissociation reaction of oligomers PrP^oligo from reduced human prion huPrP^C(23–231) have been studied by ¹H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The ¹H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105∼210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrP^C*, a rare metastable form of PrP^C stabilized at high pressure (Kachel et al., BMC Struct Biol 6:16). The results strongly suggest that the oligomeric form PrP^oligo is in dynamic equilibrium with the monomeric forms via PrP^C*, namely huPrP^C ⇆ huPrP^C* ⇆ huPrP^oligo.Key words: human prion, oligomer structure, pressure dissociation, reversible monomer-oligomer transition, circular dichroism, high pressure NMR, atomic force microscopy     

The rough endoplasmic reticulum-resident FK506-binding protein FKBP65 is a molecular chaperone that interacts with collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and B?chinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.     

ATP hydrolysis and synthesis of a rotary motor V-ATPase from Thermus thermophilus

Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (approximately 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 microm, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated ( approximately 1.1 x 10(6) m(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP ( approximately 1.4 x 10(7) m(-1) s(-1)). The slower k(on)(ATP) than k(on)(ADP) and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.     

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic Mn complexes: a fluorine-19 NMR study of the reconstitution process

Reconstitution of Mn-depleted photosystem II (PSII) particles was examined with synthetic trinuclear Mn complexes of newly developed tripod ligands. Rates of the electron transfer and oxygen evolution were up to 74-86 and 52-56% of those measured in native PSII. These values are higher than those for the PSII reconstituted by MnCl(2). The role of the tripod ligands during the reconstitution process was examined by (19)F NMR. Due to the high NMR sensitivity of the (19)F nucleus and the low abundance of fluorine atoms in natural PSII, it was possible to selectively observe the fluorine atoms on the tripod ligand. It was shown that the tripod ligands were released from the Mn complex after the reconstitution. We propose that the primary step in the reconstitution process is the prebinding of the Mn complex to the hydrophobic part of the PSII particle.     

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.     

Use of a recycle-type SEC method as a powerful tool for purification of thiosialoside derivatives

An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5Ac2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity determinations and structural elucidation of the isolated compounds were performed by a combination of elemental analyses and spectroscopic analyses, including IR, ¹H, and ¹³C NMR, and mass spectroscopic analyses.