The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin

The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle.     

Peptide crystal growth via sulfonate salt formation: The structure of bis-glycylglycine-1,5-naphthalenedisulfonate hydrate

Summary Continuing a line of investigations on methods for formation and growth of high-quality crystals of peptides, the glycylglycine sequence has been crystallized by evaporation methods as a salt with 1,5-naphthalenedisulfonic acid. The structure of the peptide is highly extended, and is conformationally quite similar to the structures which have been characterized for other zwitterionic and salt forms of this sequence. Thus, crystallization as a salt with this sulfonic acid has imposed no undue influence upon the molecular conformation. These results offer further indication that the preparation of peptide sulfonate salts, particularly with arene templates, may have broad general utility for crystallization of interesting sequences which until now have not been approachable in their zwitterionic forms.     

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells

During studies on the fucosylation of endogenous proteins inparental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferaseI-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observedthat Lec1 cells incorporate     

Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.     

Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase

An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.     

Negative Regulation of Notch Signaling by Xylose

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

O-fucosylation is required for ADAMTS13 secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.     

Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.     

O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase

O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.