Localization of neutral N-linked carbohydrate chains in pig zona pellucida glycoprotein ZPC.
(9/4049)
Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays important roles in fertilization and consists of three glycoproteins; ZPA, ZPB and ZPC. In pig, neutral complex-type N-linked chains obtained from a ZPB/ZPC mixture possess sperm-binding activity. We have recently reported that among neutral N-linked chains triantennary and tetraantennary chains have a sperm-binding activity stronger than that of diantennary chains. Triantennary and tetraantennary chains are localized at the second of the three N-glycosylation sites of ZPB. In this study, we focused on the localization of neutral N-linked chains in ZPC. ZPB and ZPC can not be separated from each other unless the acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-beta-galactosidase digestion. A large part of the acidic N-linked chains becomes neutral by the digestion, but the main neutral N-linked chains are not susceptible to the enzyme. N-glycanase digestion indicated that ZPC has three N-glycosylation sites. Three glycopeptides each containing one of the N-glycosylation sites were obtained by tryptic digestion of ZPC and the N-glycosylation sites were revealed as Asn124, Asn146 and Asn271. The carbohydrate structures of the neutral N-linked chains from each glycopeptide were characterized by two-dimensional sugar mapping analysis taking into consideration the structures of the main, intact neutral N-linked chains of ZPB/ZPC mixture reported previously. Triantennary and tetraantennary chains were found mainly at Asn271 of ZPC, whereas diantennary chains were present at all three N-glycosylation sites. Thus, ZPC has tri-antennary and tetra-antennary chains as well as ZPB, but the localization of the chains is different from that in ZPB. (+info)
N-glycan structures of matrix metalloproteinase-1 derived from human fibroblasts and from HT-1080 fibrosarcoma cells.
(10/4049)
Matrix metalloproteinase-1 (MMP-1) is a collagenolytic metalloproteinase capable of cleaving native triple-helical forms of several collagen subtypes, as well as a number of non-collagenous substrates. The role of MMP-1 in various diseases affecting the connective tissue is well characterized. MMP-1 is secreted as both glycosylated and unglycosylated species, and the two forms have been shown to be identical with respect to substrate specificity, specific activity and inhibitory profile. No function for the glycan moiety of the enzyme has been ascribed to date. In the present study, we report on the detailed characterization of MMP-1-derived oligosaccharides. Using strategies based on sequential exoglycosidase digestion combined with matrix-assisted laser desorption ionization-time of flight MS and electrospray tandem MS, we have characterized the N-glycan structures of MMP-1, derived from human dermal fibroblasts and from the HT-1080 fibrosarcoma cell line. MMP-1 derived from fibroblasts was found to carry mainly alpha 2,3-sialylated complex-type diantennary glycans. On the other hand, HT-1080 cells produce MMP-1 that has a heterogeneous glycosylation pattern, comprising diantennary glycans carrying Lewis X, LacdiNAc, sialylated LacdiNAc and GalNAc beta 1,4 (Fuc alpha 1,3)GlcNAc (LacdiNAc analogue of Lewis X) as terminal elements. We also show that, of the two potential glycosylation sites in the MMP-1 sequence, only Asn120 is used. (+info)
Crystal structure of the beta-glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability.
(11/4049)
The glycosyl hydrolases are an important group of enzymes that are responsible for cleaving a range of biologically significant carbohydrate compounds. Structural information on these enzymes has provided useful information on their molecular basis for the functional variations, while the characterization of the structural features that account for the high thermostability of proteins is of great scientific and biotechnological interest. To these ends we have determined the crystal structure of the beta-glycosidase from a hyperthermophilic archeon Thermosphaera aggregans. The structure is a (beta/alpha)8 barrel (TIM-barrel), as seen in other glycosyl hydrolase family 1 members, and forms a tetramer. Inspection of the active site and the surrounding area reveals two catalytic glutamate residues consistent with the retaining mechanism and the surrounding polar and aromatic residues consistent with a monosaccharide binding site. Comparison of this structure with its mesophilic counterparts implicates a variety of structural features that could contribute to the thermostability. These include an increased number of surface ion pairs, an increased number of internal water molecules and a decreased surface area upon forming an oligomeric quaternary structure. (+info)
Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.
(12/4049)
Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p. (+info)
Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells.
(13/4049)
The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors. (+info)
Latency of some glycosidases of rat liver lysosomes.
(14/4049)
The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited. (+info)
Partial characterization of a major autolysin from Mycobacterium phlei.
(15/4049)
Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene. (+info)
Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis.
(16/4049)
Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo. (+info)