Analysis of structural signals conferring localisation of pig OST48 to the endoplasmic reticulum.

Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the co-translational transfer of GlcNAc2-Man9-Glc3 from Dol-PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ER-targeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man9-mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the C-terminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48AK) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ER-residency on OST48. The double-lysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48AK-3, when co-expressed in COS-1 cells together with myc-tagged ribophorin 1, was quantitatively retained in the ER. By contrast, co-expression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMOdeltaK-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the C-terminus of the Man9-mannosidase luminal domain. Thus ER-localisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the double-lysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in re-capture of molecules which have escaped from the ER.

Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser.

Pig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc(2)-Man(9)-Glc(3) precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH(2) as in beta,gamma-diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979-985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine beta-methyl group by OST is also specific, with serine, L-threo-beta-hydroxynorvaline and L-beta-hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH(3)-CH(OH) group.

The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation.

Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc(3)-Man(9)-GlcNAc(2) from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of approximately 48.9 kDa (OST48), approximately 68.7 kDa (ribophorin I) and approximately 69.3kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein. Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible approximately 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the -3/-5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the -3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif.

Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tü 1892.

The polyketide chains of the two ansamycin antibiotics, ansatrienin (mycotrienin) and naphthomycin produced by Streptomyces collinus are assembled using 3-amino-5-hydroxybenzoic acid (AHBA) as a starter unit. The gene encoding AHBA synthase, an enzyme which catalyzes the final step of AHBA biosynthesis in the recently discovered aminoshikimate pathway, has been used to identify two separate antibiotic biosynthetic gene clusters in S. collinus. In one of these clusters, analysis of approximately 20 kb of contiguous sequence has revealed both a cluster of six genes presumed to play a role in the AHBA pathway and the beginning of a polyketide synthase (PKS) gene containing an acyl ACP ligase domain. This domain is likely responsible for loading AHBA onto the PKS. This gene cluster also contains chcA, encoding the enzyme 1-cyclohexenylcarbonyl CoA reductase, which is essential for the biosynthesis of the cyclohexanecarboxylic acid moiety of ansatrienin from shikimic acid, and a peptide synthetase. This gene cluster thus seems to control the biosynthesis of ansatrienin, which contains a side chain of N-cyclohexanecarbonyl-d-alanine esterified to the macrocyclic lactam backbone. In the putative naphthomycin biosynthetic gene cluster approximately 13 kb of contiguous sequence has revealed a second set of the genes required for AHBA biosynthesis. In addition the end of a polyketide synthase and a gene putatively involved in termination of the chain extension process, formation of an intramolecular amide bond between the AHBA nitrogen and the carboxyl group of the fully extended polyketide chain, have been identified. Thus, despite commonality in biosynthesis, the ansatrienin and naphthomycin biosynthetic gene clusters show clear organizational differences and carry separate sets of genes for AHBA biosynthesis.

[The influence of systemic digitalis application on intraocular pressure]. / Zum Einfluss von systemischer Digitalis-Applikation auf den intraocularen Druck.

The intraocular pressure (IOP) of ten healthy subjects was measured before and after a 2-week systemic application of beta-methyldigoxin. The drug led to a highly significant decrease of IOP of on the average 2 mm Hg. The decrease was independent of the blood concentration of glycoside - at least within the therapeutic range. There was a positive correlation between IOP before digoxin application and the subsequent IOP decrease. The rate of outflow increased, but not significantly. Implications for the therapy of glaucoma are briefly discussed.