High Mannose-type Glycoproteins Research Articles

The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

Macrophages have long been known to secrete a Phospholipase A(2) with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A(2) (LPLA(2)) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA(2). LPLA(2) is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA(2) produced by HEK293 cells was applied to LPLA(2)-deficient (LPLA(2)(-/-)) mouse alveolar macrophages. The uptake of exogenous LPLA(2) into LPLA(2)(-/-) alveolar macrophages occurred in a concentration-dependent manner. The LPLA(2) taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of alpha-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA(2) is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA(2)(-/-) alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA(2) rescued the LPLA(2)(-/-) alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA(2) revealed that the enzymatic activity of LPLA(2) was required for the phospholipid reduction. These studies identify LPLA(2) as a high m.w.-secreted Phospholipase A(2).

Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VIP36

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca(2+) and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel beta-sandwich and binds one Ca(2+) adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca(2+) ion orients the side chains of Asp(131), Asn(166), and His(190) for carbohydrate binding. This result explains the Ca(2+)-dependent carbohydrate binding of this protein. The Man-alpha-1,2-Man-alpha-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca(2+)-dependent and D1 arm-specific manner.

Structures of the Carbohydrate Recognition Domain of Ca2+-independent Cargo Receptors Emp46p and Emp47p

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

The H,K-ATPase β Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.

Glycosylation affects the protein stability and cell surface expression of Kv1.4 but Not Kv1.1 potassium channels. A pore region determinant dictates the effect of glycosylation on trafficking.

Kv1.1 and Kv1.4 potassium channels are plasma membrane glycoproteins involved in action potential repolarization. We have shown previously that glycosylation affects the gating function of Kv1.1 and that a pore region determinant of Kv1.1 and Kv1.4 affects their cell surface trafficking negatively or positively, respectively. Here we investigated the role of N-glycosylation of Kv1.1 and Kv1.4 on their protein stability, cellular localization pattern, and trafficking to the cell surface. We found that preventing N-glycosylation of Kv1.4 decreased its protein stability, induced its high partial intracellular retention, and decreased its cell surface protein levels, whereas it had little or no effect on these parameters for Kv1.1. Exchanging a trafficking pore region determinant between Kv1.1 and Kv1.4 reversed these effects of glycosylation on these chimeric channels. Thus it appeared that the Kv1.4 pore region determinant and the sugar tree attached to the S1-S2 linker showed some type of dependence in promoting proper trafficking of the protein to the cell surface, and this dependence can be transferred to chimeric Kv1.1 proteins that contain the Kv1.4 pore. Understanding the different trafficking programs of Kv1 channels, and whether they are altered by glycosylation, will highlight the different posttranslational mechanisms available to cells to modify their cell surface ion channel levels and possibly their signaling characteristics.

Distribution of endo-beta-N-acetylglucosaminidase amongst enterococci.

Enterococci are becoming increasingly important nosocomial pathogens, a fact mainly attributed to their antimicrobial resistance profiles. However, the enzymic activities required for these organisms to proliferate in vivo have received little attention. Enterococcus faecalis has been shown previously to produce an endo-beta-N-acetylglucosaminidase activity which cleaves high mannose-type glycans in glycoproteins between the N-acetylglucosamine residues of the pentasaccharide core. This study investigated the distribution of this endoglycosidase activity amongst the other enterococcal species. Ribonuclease B, a high mannose-type glycoprotein, was used as a substrate and endoglycosidase activity was demonstrated by a combination of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and high pH anion-exchange chromatography. Endo-beta-N-acetylglucosaminidase activity was present in 10 of the 18 enterococcal species isolated from both human and animal sources, including all E. faecalis strains. The most notable exception was the lack of this activity in all E. faecium isolates tested. All enterococcal species possessing endoglycosidase activity utilised the liberated glycans to support bacterial growth.

8] Analysis of galactolipids and UDP-galactose: Ceramide galactosyltransferase

Publisher Summary Glycosphingolipids are enriched in the outer leaflet of the plasma membrane of most eukaryotic cells where they are thought to be involved in cell recognition and signaling. Although glycosphingolipids constitute only a few mol% of the lipids in most membranes, they are major components of the myelin sheath, where GalCer and SGalCer are involved in axonal insulation, myelin function, and stability. The apical plasma membrane of epithelial cells in the gastrointestinal and urinary tracts is enriched in glycosphingolipids. In rodents, these are typically glucolipidsy whereas in humans most are galactolipids. Glycosphingolipids play a structural role in rigidifying and protecting the apical cell surface. Their role in sorting lipids and proteins to various membranes along the exocytotic and endocytotic transport routes is not fully understood. The foremost enzyme involved in the biosynthesis of galactosphingolipids is the UDP-galactose:ceramide galactosyltransferase, CGalT or GalT-1. CGalT catalyzes the transfer of galactose from UDP-galactose to Cer yielding GalCer and has relatively promiscuous substrate specificity. Whether there are one or more CGalT enzymes with distinct specificity and cellular localization has been a controversial issue. The localization of CGalT has long been enigmatic. The enzyme is exclusively localized to the endoplasmic reticulum (ER) by immunogold electron microscopy on ultrathin cryosections. CGalT is a high mannose-type glycoprotein that is N-glycosylated at Asn-78 and Asn-333 and contains a putative carboxy terminal Lys-Lys-Val-Lys ER-retrieval signal. The conceptual translation product exhibits no amino acid sequence similarity with other glycosyltransferases. Instead, CGalT is related to the superfamily of UDP-glucuronosyltransferases.

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl2 extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees.

Procedure for selection of cell wall-associated glycoproteins

A novel, sequential extraction-affinity chromatographic procedure for the selection of high mannose-type glycoproteins from the ionically bound proteins of plant cell walls is reported. Ionically bound proteins were extracted using a modified binding buffer for Concanavalin-A, the extract passed through Concanavalin-A Sepharose and high-mannose type glycoproteins selectively desorbed using a-methyl mannoside. Extraction with the ConA-binding buffer compared favourably with previously used extraction methods in terms of the purity of peroxidase and the range of peroxidase isozymes recovered from tobacco cell walls. The sequential affinity chromatography step cleanly selected peroxidase activity and provided a significant purification via a reduction in bulk protein. This procedure has great potential as a convenient and robust, initial step in the purification of a wide range of cell wall-associated high-mannose type glycoproteins.

Changes in high mannose-type glycoproteins of peripheral blood mononuclear cells in cirrhosis patients

Glycoproteins of peripheral blood mononuclear cells (PBMC) in patients with liver cirrhosis (LC) have not yet been studied. In the present study, we investigated high mannose-type glycoproteins of PBMC from patients with LC. We showed that [2- 3H]mannose uptake by PBMC and binding of glycoproteins from PBMC to concanavalin A (Con A)-Sepharose were significantly lower in patients with LC than in healthy subjects. These results indicate that high mannose-type glycoproteins of PBMC are decreased in patients with LC. High mannose-type glycoproteins play an important role in the formation of functional high-affinity interleukin-2 (IL-2) receptors. Thus, the decrease of high mannose-type glycoproteins may be one of the factors affecting the function of T or natural killer (NK) cells, which require IL-2 receptors to exert various immunological functions, in patients with LC.

Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.

Chinese Hamster Ovarian Cell Glycoproteins that Mediate Type 1 Piliated Gram-negative Bacterial Adherence

We used Chinese hamster ovary (CHO) cell lines to define the structures of glycoproteins responsible for Type 1 piliated bacterial adherence. CSH 50 Escherichia coli, a Type 1 piliated bacteria, adhered significantly better than an isogenic nonpiliated E. coli to all CHO lines tested. CSH 50 E. coli adhered least well to CHO cells expressing intact complex type oligosaccharides on cell surface glycoproteins. CSH 50 adherence increased when shorter oligosaccharides were present and was maximal when mannose groups were present in terminal, nonreducing positions. Five high mannose type glycoproteins, with molecular weights of 79, 75, 55, 50, and 37 kD, were identified as high affinity ligands for Type 1 piliated bacteria. Our results suggest that alterations in cell surface carbohydrates may increase adherence of Type 1 piliated gram-negative bacteria to cells.

Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies

A spatio-temporal analysis of the differentiation of a group of specialized (secretory) ependymal cells in the subcommissural organ (SCO) of the brain was undertaken in the bovine using a monoclonal antibody (C1B8A8) which is specific of the secretory process in this organ. In addition, lectins (concanavalin agglutinin (Con A), Lens culinaris agglutinin (LCA), wheat germ agglutinin (WGA), and Phaseolus vulgaris agglutinin (PHA)) were used to analyse the maturation of the carbohydrate moieties of the secretory product (subcommissuralin). Monoclonal antibody NC-1 specific to a complex carbohydrate epitope including a terminal 3-sulfoglucuronyl residue similar to HNK-1 was also tested to compare the reactivity of the SCO with that of other brain structures. These cells express a specific antigen related to the known secretory activity of the SCO during early embryogenesis (2 months). This antigen is recognized by C1B8A8 antibody and by Con A suggesting that high mannose-type glycoproteins are synthesized at this stage. Later on ( ∼ 3.5 months), appearance of C1B8A8, WGA, LCA, L- and E-PHA-positive material in the apical lining of the ependymal cells, close to the ventricular cavity, suggests that maturation of the complex-type glycoproteins (Asn-linked) occurs at this stage. Presence of secretory material in the CSF and Reissner's fibre could be detected using the same probes at a stage of 4 months. As early as 2 months NC-1-positive material was detected in the ependyma of the mesencephalic roof, while no reaction occurred in the SCO epithelium. This suggests that the carbohydrate moieties of subcommissuralin is different from that of ependymins β and γ. Using specific monoclonal antibodies, molecular characterization of subcommissuralin and experimental analyses on its accurate role in brain development will further our tentative comparison with ependymins. The secretory ependymal cells in the SCO express a particular phenotype and could represent an interesting model to study cell differentiation in the brain.

High Level of Expression of the Myelin Protein P0 in Chinese Hamster Ovary Cells

The major PNS myelin protein, P0, has been expressed in Chinese hamster ovary cells by transfection with a plasmid containing the P0-cDNA. The expression of P0 at both the RNA and the protein level was greatly increased, without detriment to the cell, by the dihydrofolate reductase-methotrexate strategy of gene amplification. The P0 expressed by these cells was glycosylated (containing approximately equal amounts of the complex and high-mannose type glycoproteins) and reached the plasma membrane. This system is suitable not only for addressing the function of P0 directly, but it also applicable to any protein of which an abundance is needed.

Neoglycoenzymes: A versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial β-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein-carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.

Stabilization of glycoenzymes by cross-linking of their carbohydrate chains.

The results presented here demonstrate the potential applicability of the described cross-linking method for preparation of soluble glycoenzyme derivatives. The high level of the retained enzyme activity supports the assumption that this approach is superior to the cross-linking through the protein part. In this study, high mannose-type glycoproteins were used. However, the complex-type glycoproteins that are spread among glycoproteins of higher eukaryotes also can be cross-linked by this procedure because, at the least, the terminal monosaccharide will be oxidized by periodate. Moreover, this approach may be applicable when dealing with partially purified glycoenzymes because the protein impurities are not expected to interfere with the cross-linking reaction. Besides cross-linking, other kinds of chemical modifications of glycoenzymes through the carbohydrate part are possible. This, in turn, could lead to changes in the physicochemical and catalytic properties of the enzymes, thereby opening a new field of glycoenzyme engineering.

Differential glycosylation of the components of the bovine trophoblast protein-1 complex

The bovine trophoblast protein-1 complex, a major secretory product of the day 17 to 18 conceptus, has been implicated in extension of luteal lifespan during early pregnancy. This glycoprotein complex, identifiable by immunoprecipitation procedures utilizing rabbit antiserum to ovine trophoblast protein-1, exists as seven isomers of two size classes (22 and 24 kDa). Culture of embryos with tunicamycin demonstrated that the isomers are N-linked glycoproteins, as deglycosylated products migrate as a single band (18 kDa) during electrophoresis. Culture with deoxymannojirimycin indicated that the 24 kDa form is complex in nature, whereas treatment with endoglycosidase H and lectin chromatography indicated that the 22 kDa form is a high-mannose type glycoprotein. These results indicate that molecular weight variants of bovine trophoblast protein-1 arise as a single translation product that undergoes differential post-translational glycosylation.

Preparation of the stabilized glycoenzymes by cross-linking their carbohydrate chains

Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.e., the oxidation degree and the concentrations of glycoproteins, cross-linker, and hydrogen ions during the cross-linking reaction. It was found that the quantity and size of polymers increased with oxidation degree and protein concentration and by lowering the pH. When the protein concentration was above and pH below certain values, depending on the glycoenzyme, insoluble polymers formed. The soluble cross-linked polymers retained a high level of original activity, and the minor decrease in specific activity noticed was shown to occur during the periodate oxidation step. The cross-linked glycoenzymes are much more resistant to denaturation by high temperature and by changes in pH, demonstrating the usefulness of this method in preparation of the stabilized glycoprotein derivatives.

Studies on Turbatrix aceti β- N-acetylglucosaminidase : 1. Purification and physicochemical characterization

N-Acetyl-β- d-glucosaminidase was purified, from the culture medium of the nematode Turbatrix aceti, to homogeneity, as judged by electrophoresis in polyacrylamide gel and ultracentrifugation. The purification scheme involved the following steps: (i) concentration of the culture medium by ultra-filtration by an Amicon PM-30 membrane; (ii) ammonium sulfate precipitation; (iii) DEAE-Sephadex and (iv) Sephadex G-200 chromatography; and (v) affinity chromatography on succinyldiaminopropyl amino-Sepharose bearing the ligand p-aminophenyl 2-acetamido-2-deoxy-1-thio-β- d-glucopyranoside. The molecular weight of the enzyme was 112,000 ± 4800 and 124,000 as determined by polyacrylamide gel electrophoresis and by gel filtration through Sephacryl S-200, respectively. The enzyme showed a pH optimum of 4.8 for N-acetylglucosaminidase and 5.4 for N-acetylgalactosaminidase. The detailed substrate specificity studies were carried out on both synthetic and natural oligosaccharides and glycopeptides. The chitin oligosaccharides and asialo-agalacto complex type as well as high mannose-type glycoproteins such as fetuin and ovalbumin, respectively, were good substrates for the enzyme. Substrate analogs in which the oxygen atom of the acetamido group was replaced by sulfur atom proved to be poor substrates.

1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes.

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.