Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution.

The aquaporins (formerly called the major intrinsic protein family) are transmembrane channel proteins. The family includes the CHIP group, which are functionally characterised as water channels and the GLP group, which are specialised for glycerol transport. The present study reports the identification and characterisation of a novel GLP family member in a teleost fish, the sea bream Sparus auratus. A sea bream aquaporin (sbAQP) cDNA of 1047 bp and encoding a protein of 298 amino acids was isolated from a kidney cDNA library. Functional characterization of the sbAQP using a Xenopus oocyte assay revealed that the isolated cDNA stimulated osmotic water permeability in a mercury-sensitive manner and also stimulated urea and glycerol uptake. Northern blotting demonstrated that sbAQP was expressed at high levels in the posterior region of the gut, where two transcripts were identified (1.6 kb and 2 kb), and in kidney, where a single transcript was present (2 kb). In situ hybridisation studies with a sbAQP riboprobe revealed its presence in the lamina propria and smooth muscle layer of the posterior region of the gut and in epithelial cells of some kidney tubules. sbAQP was also present in putative chloride cells of the gill. Phylogenetic analysis of sbAQP, including putative GLP genes from Fugu rubripes, revealed that it did not group with any of the previously isolated vertebrate GLPs and instead formed a separate group, suggesting that it may be a novel GLP member.

Peptides and ATP binding cassette peptide transporters.

In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed. Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment. In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed. Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.

Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis has the unique capacity to mediate the transport of peptides from 4 up to at least 18 residues. The substrate specificity of this binding protein-dependent ATP-binding cassette transporter is determined mainly by the receptor protein OppA. To study the specificity and ligand-binding mechanism of OppA, the following strategy was used: (i) OppA was purified and anchored via the lipid moiety to the surface of liposomes; (ii) the proteoliposomes were used in a rapid filtration-based binding assay with radiolabeled nonameric bradykinin as a reporter peptide; and (iii) combinatorial peptide libraries were used to determine the specificity and selectivity of OppA. The studies show that (i) OppA is able to bind peptides up to at least 35 residues, but there is a clear optimum in affinity for nonameric peptides; (ii) the specificity for nonameric peptides is not equally distributed over the whole peptide, because positions 4, 5, and 6 in the binding site are more selective; and (iii) the differences in affinity for given side chains is relatively small, but overall hydrophobic residues are favored-whereas glycine, proline, and negatively charged residues lower the binding affinity. The data indicate that not only the first six residues (enclosed by the protein) but also the C-terminal three residues interact in a nonopportunistic manner with (the surface of) OppA. This binding mechanism is different from the one generally accepted for receptors of ATP-binding cassette-transporter systems.

On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis.

Lactococcus lactis degrades exogenous proteins such as beta-casein to peptides of 4-30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppA(Ll)) of the oligopeptide transport system (Opp) of L.LACTIS: has the unique capacity to bind peptides from five up to at least 20 residues. To study the binding mechanism of OppA(Ll), nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non-fluorescent or bulky and fluorescent groups. Also, nonameric peptides with a non-natural residue, azatryptophan, at positions 3 or 7 were used. The fluorescence of azatryptophan reports on the polarity of the environment. The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein. The peptide binding mechanism of OppA(Ll) is discussed in relation to known three-dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.

Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis.

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.

Purification and characterization of coenzyme F390 synthetase from Methanobacterium thermoautotrophicum (strain delta H).

Coenzyme F390 synthetase catalyzes the formation of 8-hydroxyadenylylated-coenzyme F420 (coenzyme F390-A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 synthetase from Methanobacterium thermoautrophicum was purified from a cell-free extract and its catalytic properties were determined. The synthetase was purified 150-fold to a specific activity of 0.45 mumol.min-1.mg protein-1. The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co-elution during purification of GTP-dependent coenzyme F390 synthetase activity suggested that the synthetase is also capable of 8-hydroxyguanylylated-coenzyme F420 (coenzyme F390-G) formation. Initial-velocity measurements of the two-substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary-complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 microM and a Km for ATP of 1.7 mM. In contrast to the enzyme in the cell-free extract, the isolated enzyme was active under aerobic and anaerobic conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5-dihydro-coenzyme F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 microM) with respect to coenzyme F420. The latter findings may explain why the enzyme could only be detected in crude extracts that had been exposed to air, i.e. treatment with air causes the oxidation of reduced coenzyme F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.