The Saccharomyces cerevisiae CWH8 gene is required for full levels of dolichol-linked oligosaccharides in the endoplasmic reticulum and for efficient N-glycosylation.
The Saccharomyces cerevisiae mutant cwh8 was previously found to have an anomalous cell wall. Here we show that the cwh8 mutant has an N -glycosylation defect. We found that cwh8 cells were resistant to vanadate and sensitive to hygromycin B, and produced glycoforms of invertase and carboxypeptidase Y with a reduced number of N -chains. We have cloned the CWH8 gene. We found that it was nonessential and encoded a putative transmembrane protein of 239 amino acids. Comparison of the in vitro oligosaccharyl transferase activities of membrane preparations from wild type or cwh8 Delta cells revealed no differences in enzyme kinetic properties indicating that the oligosaccharyl transferase complex of mutant cells was not affected. cwh8 Delta cells also produced normal dolichols and dolichol-linked oligosaccharide intermediates including the full-length form Glc3Man9GlcNAc2. The level of dolichol-linked oligosaccharides in cwh8 Delta cells was, however, reduced to about 20% of the wild type. We propose that inefficient N -glycosylation of secretory proteins in cwh8 Delta cells is caused by an insufficient supply of dolichol-linked oligosaccharide substrate. (+info)
Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin. (+info)
The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection.
Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one alpha1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and alpha1,3 fucosyltransferases in the human bronchial mucosa. (+info)
Re-entering the translocon from the lumenal side of the endoplasmic reticulum. Studies on mutated carboxypeptidase yscY species.
Misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded by the cytosolic ubiquitin-proteasome system. This requires their retrograde transport from the ER lumen into the cytosol, which is mediated by the Sec61 translocon. It had remained a mystery whether ER-localised soluble proteins are at all capable of re-entering the Sec61 channel de novo or whether a permanent contact of the imported protein with the translocon is a prerequisite for retrograde transport. In this study we analysed two new variants of the mutated yeast carboxypeptidase yscY, CPY*: a carboxy-terminal fusion protein of CPY* and pig liver esterase and a CPY* species carrying an additional glycosylation site at its carboxy-terminus. With these constructs it can be demonstrated that the newly synthesised CPY* chain is not retained in the translocation channel but reaches its ER lumenal side completely. Our data indicate that the Sec61 channel provides the essential pore for protein transport through the ER membrane in either direction; persistent contact with the translocon after import seems not to be required for retrograde transport. (+info)
Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor.
For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor. (+info)
N-Linked glycosylation and sialylation of the acid-labile subunit. Role in complex formation with insulin-like growth factor (IGF)-binding protein-3 and the IGFs.
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability. (+info)
Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells.
The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG. (+info)
The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex.
In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex. (+info)