Human intestinal TFF3 forms disulfide-linked heteromers with the mucus-associated FCGBP protein and is released by hydrogen sulfide.

TFF3 is a secretory peptide belonging to the trefoil factor family with a predicted size of 59 amino acid residues containing seven cysteine residues. It is predominantly expressed in intestinal goblet cells where it plays a key role in mucosal regeneration and repair processes. In the course of these studies, human colonic TFF3 was shown to exist mainly as a high molecular weight heteromer. Purification of this heteromer and characterization by LC-ESI-MS/MS analysis identified the IgG Fc binding protein (FCGBP) as the disulfide-linked partner protein of TFF3. FCGBP is a constituent of intestinal mucus secreted by goblet cells. Furthermore, low amounts of TFF3/monomer and only little TFF3/dimer were detected in human colonic extracts. Here, we show that these TFF3 forms can be released from the purified TFF3-FCGBP heteromer complex in vitro by reduction with hydrogen sulfide (H(2)S). Such a mechanism would be in line with the high H(2)S concentrations reported to occur in the lumen of the colon. Of special note, this points to intestinal mucus as a reservoir for a biologically active peptide. Also proteolytic processing of FCGBP was observed which is in line with multiple autocatalytic cleavages as proposed earlier by Johansson et al. (J. Proteome Res. 2009 , 8 , 3549 - 3557).

Biosynthesis of gastrokine-2 in the human gastric mucosa: restricted spatial expression along the antral gland axis and differential interaction with TFF1, TFF2 and mucins.

UNLABELLED: Gastrokine-2 (GKN2) is a secretory peptide of human gastric surface mucous cells (SMCs). It forms disulfide-linked heterodimers with the trefoil factor family (TFF) peptide TFF1. Binding with TFF2 was also reported. Antral SMCs differ from those of the corpus by their TFF3 expression. The aim of this study was to localize GKN2 expression along the antral gland axis, to characterize the continuous regeneration of antral glands, and to investigate the interactions of GKN2 with TFF1, TFF2 and mucins. METHODS: The spatial expression of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 was determined using laser microdissection and RT-PCR analysis. Furthermore, antral extracts were separated by gel chromatography and the association of GKN2 with TFF1, TFF2, and mucins was investigated. RESULTS: Differential GKN2 expression was localized along the rostro-caudal axis of the stomach. Laser microdissection revealed characteristic differential expression profiles of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 along the antral gland axis. Both GKN2 and TFF1 were expressed in superficial SMCs. Surprisingly, the TFF1-GKN2 heterodimer did not associate with the mucin fraction; whereas TFF2 showed exclusive association with mucins. CONCLUSIONS: Maturation of antral SMCs occurs stepwise via trans-differentiation of TFF3 expressing progenitor cells. The TFF1-GKN2 heterodimer and TFF2 differ characteristically by their binding to gastric mucins. This points to different physiological functions of TFF1 and TFF2, the latter maybe acting as a 'link peptide' for stabilization of the gastric mucus.

Distinct mono- and dinucleotide-specific P2Y receptors in A549 lung epithelial cells: different control of arachidonic acid release and nitric oxide synthase expression.

P2Y nucleotide receptors activated by mono- and dinucleotides have already been found in lung tissue. Here, we compare effects of dinucleotides and mononucleotides on arachidonic acid release, intracellular calcium mobilization, and inducible nitric oxide synthase (iNOS) expression in the alveolar lung cell line A549. Both types of nucleotides were effective. Diadenosine polyphosphates (Ap(n)A, n=2 to 5) increased arachidonic acid release and raised intracellular calcium concentration ([Ca(2+)](i)), albeit with lower potency than mononucleotides (ATP, UTP, UDP). Among the dinucleotides only diadenosine tetraphosphate (Ap(4)A) was a potent agonist. Arachidonic acid release induced by Ap(4)A was almost completely abolished in the presence of the P2 receptor antagonists suramin and Reactive blue 2, whereas arachidonic acid release evoked by ATP, UTP or UDP was hardly reduced by these antagonists. Both, the mononucleotides ATP and UDP and the dinucleotide Ap(4)A induced the expression of iNOS in the cytoplasm around the nucleus, similar to the expression of iNOS evoked by lipopolysaccharide. iNOS is barely detectable in unstimulated cells. Suramin selectively blocked the capacity of Ap(4)A to induce iNOS, but not that of ATP or UDP. Thus, we find the same pharmacology for nucleotide-induced arachidonic acid release and iNOS expression. Therefore, we suggest that a distinct P2Y receptor subtype specifically activated by Ap(4)A exists in A549 cells, which is sensitive to the antagonist suramin, in contrast to other P2Y receptor subtypes activated by mononucleotides which are suramin-insensitive. Distinct P2Y receptors activated by mononucleotides or by Ap(4)A could play a role in inflammatory conditions by affecting the release of arachidonic acid and the expression of iNOS. Therefore, these receptors present a promising target in inflammatory diseases.

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes.

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.

P2Y receptor specific for diadenosine tetraphosphate in lung: selective inhibition by suramin, PPADS, Ip5I, and not by MRS-2197.

Extracellular dinucleotides, which act as signaling molecules in a variety of systems, may regulate fluid homeostasis in the human lung by activation of a specific P2Y receptor subtype. Previously, we presented evidence for a G protein-coupled P2Y receptor with high affinity for dinucleotides in both rat and human lung tissue. In a human bronchial epithelial cell line (HBE-1), diadenosine polyphosphates (Ap(n)A, n=2-6) increase intracellular Ca(2+). The aim of the present work was to find additional evidence that, in these cells, the receptors selectively activated by diadenosine polyphosphates are distinct from already known P2Y receptors, which are activated by the mononucleotides ATP or UTP. We tested antagonists suitable to classify P2Y receptor subtypes. The P2Y(1) receptor-selective antagonist 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS-2197) did not affect Ca(2+) mobilization induced by diadenosine tetraphosphate (Ap(4)A). However, suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) and diinosine pentaphosphate (Ip(5)I) inhibited the Ca(2+) response by 96%, 92% and 32%, respectively. Moreover, these results were confirmed by assessing the specific binding of [3H]Ap(4)A to membranes from human and rat lung. Suramin (100 microM), PPADS (400 microM) and Ip(5)I (200 microM), reduced [3H]Ap(4)A binding in lung membrane preparations by 66%, 77% and 80%, respectively. The Ap(4)A-induced Ca(2+) response in HBE-1 cells was inhibited to a much greater extent by these antagonists than the ATP- or UTP-evoked Ca(2+) rise. Thus, Ap(4)A in lung epithelial cells also activates a still unidentified P2Y receptor that is specific for dinucleotides over mononucleotides.