A yeast mutant capable of producing Man 5 GlcNAc 2 human compatible sugar chains on glycoproteins was constructed. An expression vector for ␣-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/ retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro ␣-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an ␣-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man 5 GlcNAc 2 . This is the first report of a recombinant S. cerevisiae able to produce Man 5 GlcNAc 2 -oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.Saccharomyces cerevisiae is useful for the production of recombinant proteins of biological interest because of the established expression system, and it can be easily grown in large quantities. Moreover, yeast share the early steps of the mammalian Asn-linked glycosylation pathway. However, the mature Asn-linked oligosaccharides of yeast are mannan glycans and are highly antigenic against mammals. Thus, it would be necessary to eliminate the antigenicity of the sugar chains when recombinant therapeutic glycoproteins are produced in yeast.Several genes concerned with the biosynthesis of yeast sugar chains have been cloned, and the glycosylation pathway of yeast has been clarified. The OCH1 gene encodes an ␣-1,6-mannosyltransferase that initiates ␣-1,6-polymannose outer chain formation on the Asn-linked inner oligosaccharide Man 8 GlcNAc 2 in S. cerevisiae (1). MNN1 has been proposed as the structural gene for the ␣-1,3-mannosyltransferase that elongates the outer chain and the inner core oligosaccharide (2, 3). The ⌬och1 mnn1 double mutant accumulated a single oligosaccharide moiety, Man 8 GlcNAc 2 , a high mannose-type structure (1). This mutant may be useful to produce recombinant therapeutic glycoproteins without any antigenicity toward humans.On the other hand, some glycoproteins of therapeutic value require complex-type sugar chains for their efficacy. Erythropoietin (EPO), 1 a hematopoietic glycoprotein factor produced in the kidney, has three complex-type Asn-linked sugar chains and one mucin-type sugar chain. It is reported that the composition and structure of each sugar chain affected the biological activity, the efficiency of secretion, and had profound effects on the half-life of EPO in the blood circulation (4). It seems that the most active form of the EPO molecule requires tetraantennary Asn-linked sugar chains (5) with full sialylation, to prevent serum clearance by the action of the hepatic asialoglycoprotein binding protein (6, 7). When EPO was expressed in the ⌬och1 mnn1 mutant yeast, the recombinant EPO should have high mannose-type oligosaccharides, which are trapped by the...
Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.Methane fermentation is a complex microbiological process and involves metabolic interactions among a variety of microorganisms. These microorganisms are classified into at least four trophic groups, namely, primary fermenting bacteria, secondary fermenting bacteria, hydrogenotrophic methanogenic archaea, and acetoclastic methanogenic archaea (39). Among these microorganisms, secondary fermenting bacteria oxidize volatile fatty acids and alcohols in syntrophic association with methanogenic archaea and are thus called syntrophic bacteria (or syntrophs). Despite plenty of genetic and genomic information being available for primary fermenting bacteria (18, 30) and methanogenic archaea (11, 13, 41), such information is currently scarce for syntrophic bacteria.A significant feature of syntrophic bacteria is their growth at the thermodynamic limit. For instance, the Gibbs free energy (⌬G°) change in syntrophic propionate oxidation is approximately Ϫ25 kJ mol Ϫ1 , which is less than the energy needed for synthesizing one ATP molecule (44). Syntrophs and methanogens share this energy for their growth. More surprisingly, Jackson and McInerney (22) have shown that substrate oxidation by syntrophs proceeds at values close to the thermodynamic equilibrium (⌬G°Ϸ 0 kJ mol Ϫ1 ). Therefore, one can deduce that these bacteria should have extremely efficient catabolic systems (22), and their c...
Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.
The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is ϳ40-fold better than serine as the glycosylated amino acid and a proline at position ؉3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions ؉1 and ؉3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position ؊1 to ؉3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions ؊1, ؉2, and ؉4 was analyzed. Uncharged amino acids were preferred at position ؊1, and small or positively charged amino acids were preferred at position ؉2. No preference was observed at position ؉4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.