The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.
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C-Mannosylation is a unique form of protein glycosylation, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp. In the two examples found so far, human RNase 2 and interleukin-12, only the first Trp in the recognition motif WXXW is specifically C-mannosylated. To establish the generality of protein C-mannosylation, and to learn more about its mechanism, the terminal components of the human complement system (C6, C7, C8,and C9), which contain multiple and complex recognition motifs, were examined. Together with C5b they form the cytolytic agent, the membrane attack complex. These are the first proteins that are C-mannosylated on more than one Trp residue as follows: six in C6, four in C7, C8alpha, and C8beta, and two in C9. Thus, from the 113 Trp residues in the complete membrane attack complex, 50 were found to undergo C-mannosylation. The other important finding is that in C6, C7, C8, and C9 Trp residues without a second Trp (or another aromatic residue) at the +3 position can be C-mannosylated. This shows that they must contain an additional C-mannosylation signal. Whether this is encoded in the primary or tertiary structure is presently unknown. Finally, all modified Trp residues are part of the highly conserved core of the thrombospondin type 1 repeats present in these proteins. Since this module has been found in a large number of other proteins, the results suggest further candidates for C-mannosylation. (+info)
Interactions of alpha- and beta-avoparcin with bacterial cell-wall receptor-mimicking peptides studied by electrospray ionization mass spectrometry.
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Solution phase affinity constants of the glycopeptide antibiotic alpha- and beta-avoparcin, with a range of bacterial cell-wall receptor-mimicking model peptides, were determined by a relatively new method: affinity electrospray ionization mass spectrometry (ESI-MS). This method is relatively efficient and allows the parallel determination of several affinity constants in mixtures of antibiotics and receptors. The determined binding constants for alpha- and beta-avoparcin were compared with those of the related glycopeptide antibiotic vancomycin. The solution phase binding affinities of alpha- and beta-avoparcin on one hand, and vancomycin on the other, were found to be in the same order, at least for the range of receptor-mimicking peptides studied. However, beta-avoparcin displayed slightly higher binding affinities than alpha-avoparcin, particularly for strong binding receptor-mimicking peptides. The evidence that alpha- and beta-avoparcin and vancomycin are structurally similar, combined with the present data revealing their similar affinity for bacterial cell-wall receptor-mimicking peptides, supports the hypothesis that the appearance of vancomycin-resistant enterococci (VRE) might be linked to the widespread use of avoparcin. (+info)
Carbohydrate moieties in human secretory component.
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Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed. (+info)
Characterization of a core alpha1-->3-fucosyltransferase from the snail Lymnaea stagnalis that is involved in the synthesis of complex-type N-glycans.
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We have identified a core alpha1-->3-fucosyltransferase activity in the albumin and prostate glands of the snail Lymnaea stagnalis. Incubation of albumin gland extracts with GDP-[(14)C]Fuc and asialo/agalacto-glycopeptides from human fibrinogen resulted in a labeled product in 50% yield. Analysis of the product by 400 MHz (1)H-NMR spectroscopy showed the presence of a Fuc residue alpha1-->3-linked to the Asn-linked GlcNAc. Therefore, the enzyme can be identified as a GDP-Fuc:GlcNAc (Asn-linked) alpha1-->3-fucosyltransferase. The enzyme acts efficiently on asialo/agalacto-glycopeptides from both human fibrinogen and core alpha1-->6-fucosylated human IgG, whereas bisected asialo/agalacto-glycopeptide could not serve as an acceptor. We propose that the enzyme functions in the synthesis of core alpha1-->3-fucosylated complex-type glycans in L. stagnalis. Core alpha1-->3-fucosylation of the asparagine-linked GlcNAc of plant- and insect-derived glycoproteins is often associated with the allergenicity of such glycoproteins. Since allergic reactions have been reported after consumption of snails, the demonstration of core alpha1-->3-fucosylation in L. stagnalis may be clinically relevant. (+info)
In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains.
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A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins. (+info)
Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin.
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Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented. (+info)
Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelin-converting enzyme.
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We have determined the nucleotide sequence of the clone pAL2 obtained from Porphyromonas gingivalis 381 in the previous study [Ansai et al. (1995) Microbiology 141, 2047-20521. The DNA sequence analysis of this fragment revealed one complete ORF and one incomplete ORF. The ORF encoded a protein (PgPepO) of 690 amino acids with a calculated molecular weight of 78796. The deduced amino acid sequence exhibited a significant homology with human endothelin-converting enzyme (ECE)-1. Recombinant PgPepO was purified to homogeneity and characterized. The purified enzyme was strongly inhibited by phosphoramidon, and converted big endothelin-1 to endothelin-1. Furthermore, the purified PgPepO strongly cross-reacted with a monoclonal antibody against rat ECE-1. These results indicate that PgPepO has striking similarity to mammalian ECE in structure and function. (+info)
Sliding motility in mycobacteria.
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Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here that Mycobacterium smegmatis can spread on the surface of growth medium by a sliding mechanism. M. smegmatis spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to M. smegmatis but is also a property of the slow-growing opportunistic pathogen M. avium. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host. (+info)