Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23.

The orphan receptor ChemR23 is a G-protein coupled receptor (GPCR) with homology to neuropeptide and chemoattractant receptors. Tazarotene, a synthetic retinoid activating retinoic acid receptor (RAR), up-regulates tazarotene-induced gene-2 (TIG2). The function and molecular target of this protein are now described. By means of reverse pharmacology screening using a peptide library generated from human hemofiltrate, we have isolated and identified TIG2 as the natural ligand of ChemR23 and report the specific molecular form of the bioactive, circulating TIG2, representing the amino-acid residues 21 to 154 of the 163 amino acid-containing prepropeptide. Based on the expression pattern of ChemR23 and TIG2, the physiological role in bone development, immune and inflammatory responses and the maintenance of skin is now being investigated.

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.

Urea-based beta-amyloid (Abeta) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Abeta peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Abeta peptides, namely, 1-40/42 and carboxyl-terminal-truncated 1-37, 1-38, and 1-39, was found. Besides Abeta1-42, we also observed a consistent elevation of amino-terminal-truncated Abeta2-42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Abeta2-42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different gamma-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Abeta peptides, we overexpressed beta-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1-/- neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1-/- neurons and PS1+/+ neurons overexpressing APP-Deltact (a slow-internalizing mutant) show a decrease of all secreted Abeta peptide species, as expected, because this mutant is processed mainly by alpha-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Abeta2-42 is significantly less affected in PS1-/- neurons and in neurons transfected with the endocytosis-deficient APP-Deltact construct. Our data confirm that PS1 is closely involved in the production of Abeta1-40/42 and the carboxyl-terminal-truncated Abeta1-37, Abeta1-38, and Abeta1-39, but the amino-terminal-truncated and carboxyl-terminal-elongated Abeta2-42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Abeta peptide species could be generated by a beta(Asp/Ala)-secretase activity.

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences.

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.

Synthesis and folding of native collagen III model peptides.

Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (P). Among the protected tripeptides FmocP(tBu)PG, FmocPP(tBu)G, and FmocGPP(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP(tBu)Gly was best suited for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(3) trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39. 4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PPG(6-8) follows a two-state, pseudomonomolecular reaction.

IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching.

A systematic membrane study investigating different neutral, cationic derivatized, and hydrophilic PVDF membranes for their suitability to carry out on-membrane tryptic digestions and to obtain infrared-matrix-assisted laser desorption/ionization (IR-MALDI) mass information on the proteolytic fragments directly from the membrane was performed. Clearly, the Immobilon CD membrane (Millipore) showed the most reproducible results over a protein mass range from 12 to 66 kDa. Typical protein load to SDS-PAGE was in the 1-2 micrograms range. The protein amount used for enzymatic treatment was estimated to be in the low picomole range. Now both the intact protein mass and the masses of the specific proteolytic fragments are available directly from the membrane. Protein databases can be searched via search algorithms on the Internet using the information on the intact protein mass and the masses, e.g., of its tryptic fragments. Investigations were performed to search for neutral, enzyme-compatible IR matrixes which allow the enzymatic treatment (on-membrane digestion) while the membrane is matrix-incubated. Thiourea could be tolerated during enzymatic cleavage in solution in concentrations of 15 g/L and resulted in high-quality spectra of intact protein signals and turned, therefore, out to be the most promising candidate.

Analysis of proteins by direct-scanning infrared-MALDI mass spectrometry after 2D-PAGE separation and electroblotting.

A novel approach is reported for the analysis and identification of proteins separated by 2D-PAGE with scanning infrared matrix-assisted laser desorption/ionization mass spectrometry (scanning IR-MALDI-MS). The proteins of human blood plasma were separated by 2D-PAGE, electroblotted onto PVDF membranes, incubated in matrix solution, and then scanned by IR-MALDI-MS. Mass contour plots of selected spots were obtained. Protein separation is shown to be conserved by comparison with silver-stained gels. The sensitivity for the protein detection is comparable if not better than that of silver-stained gels. Posttranslational modifications were identified by comparing the measured mass to the one calculated from the known DNA sequence. Adduct formation to unprotected cysteine residues during gel separation is demonstrated for selected proteins.