Glycoproteins: carbohydrates to cloning.

This article is dedicated to Professor Saul Roseman and briefly outlines some of the early studies on sialyltransferases, on glycoproteins such as pig submaxillary mucins and, more recently, on a series of unusual proteins and glycoproteins high in proline, the so-called proline-rich proteins. Hopefully, it represents, in an inadequate manner, my appreciation for 'The man and his works'. During the Roseman Symposium at the 11th International Symposium on Glycoconjugates in Toronto, several of his former students and postdocs tried to describe what it was like in the Roseman laboratory. Clearly, the time I was in Saul's lab was like no other time in my career. Thanks for everything, Saul.     

Glycoproteins: glycan presentation and protein-fold stability.

Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks to recent advances in obtaining conformational constraints across glycosidic linkages, structural characterisation of glycoproteins has improved considerably. It is now becoming apparent that N-glycosylation of a folded protein can have a significant stabilising effect on large regions of the backbone structure.     

Mucin-Type Glycoproteins

Abstract

Considerable advances have been made in recent years in our understanding of the biochemistry of mucin-type glycoproteins. This class of compounds is characterized mainly by a high level of O-linked oligosaccharides. Initially, the glycoproteins were solely known as the major constituents of mucus. Recent studies have shown that mucins from the gastrointestinal tract, lungs, salivary glands, sweat glands, breast, and tumor cells are structurally related to high-molecular-weight glycoproteins, which are produced by epithelial cells as membrane proteins. During mucin synthesis, an orchestrated sequence of events results in giant molecules of M_r 4 to 6·10⁶, which are stored in mucous granules until secretion. Once secreted, mucin forms a barrier, not only to protect the delicate epithelial cells against the extracellular environment, but also to select substances for binding and uptake by these epithelia. This review is designed to critically examine relations between structure and function of the different compounds categorized as mucin glycoproteins.     

Glycoproteins and glycolipids of oxyntic cell microsomes I. Glycoproteins: Carbohydrate composition,analytical and preparative fractionation

Isotopically-labeled sugars were incorporated into glycoproteins of isolated bullfrog gastric mucosa. The majority of the label was found in gastric microsomal fractions which were shown to contain membranes derived from the oxyntic cell tubular membrane system and were not significantly contaminated with mucus. The tubular membranes contained exceptionally large quantities of carbohydrate (approx. 260 μg/mg protein). Most of the sugar (73%) was associated with protein in the following molar ratios: hexose, 1.0: fucose, 0.42; hexosamine, 0.62; sialic acid, <0.02. The remaining sugar, predominantly hexose, could be extracted into lipid solvents and was presumably glycolipid.Gastric microsomes were dissolves in sodium dodecyl sulfate and subjected to acrylamide gel electrophoresis and Sephadex G-200 fractionation. The latter preparative procedure yielded several molecular weight classes, each of which contained different sets of proteins and/or glycoproteins; however, the molar ratios of the sugars found in the two carbohydrate containing classes were quite similar.Significant quantities of carbohydrate were also found in gastric microsomal fractions from other species, e.g. pig and rabbit. Furthermore, characteristic proteins and glycoproteins were not present in tadpole gastric microsomes until the later stages of metamorphosis when HCl secretory capability had been established. The above findings suggest that glycoproteins may play an important role in oxyntic cell functions; the possibility of a membrane protective role is discussed.     

Glycoproteins. 48. Method for fraction of acetolyzates

Acetolysis, followed by quantitative de-O-acetylation with sodium methylate of the chloroform extract of the acetolyzates and chromatographic fractionation, was applied to the sialoglycopeptide α and asialoglycopeptide β obtained by pronase hydrolysis of ovomucoid. The acetolysis yielded small amounts of monosaccharides and a large proportion of oligosaccharides without transglycosylation. It does not split off the acetamido groups and, on the other hand, the sialosyl and the 2-acetamido-N-(L-aspart-4-oyl)-2-deoxy-β-D-glucopyranosylamine bonds are protected to a high degree. After de-O-acetylation, three fractions are obtained from the chloroform phase in the case of a sialoglycoprotide and two in the case of an asialoglycoprotide by chromatography on ion-exchange resins. The first fraction, not retained, contains neutral oligosaccharides from the median portion of the carbohydrate moieties. The second fraction, present only in the acetolyzates of the sialoglycopeptides and released from the anion-exchange resin, contains sialo-oligosaccharides from the outer part of the carbohydrate moieties. The last fraction, eluted from the cation-exchange resin, contains the glycopeptides and represents the carbohydrate components near the site of attachment to the peptide chain.     

Glycoproteins of measles virus under reducing and nonreducing conditions.

Measles virus has two glycoproteins. The larger glycoprotein (HA) is composed of 76,000-dalton subunits that are bound by disulfide bonds. The smaller glycoprotein (F) appears to contain a glucosamine-rich portion that is linked to an unglycosylated protein by disulfide bonds.     

分子伴侣与病毒糖蛋白

分子伴侣(molecular chaperone)的概念由Lasky于1978年首先提出[1],真核细胞内质网中的分子伴侣是由多种蛋白质组成,它们可以介导新合成蛋白质的正确折叠与装配,并在真核生物的细胞中广泛存在.     

Glycoproteins in prokaryotes

Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins. A literature survey revealed the different types of glycoproteins. They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes. Only in a few cases is structural information available. Many different structures have been observed that display much more variation than that observed in eukaryotes. A few studies have given evidence for the function of the prokaryotic glycoprotein glycans. Also from the biosynthetic point of view, information is rather scarce. Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins. However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed. Received: 24 February 1997 / Accepted: 13 May 1997     

Glycoproteins from the cell wall of Phaseolus coccineus.

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.     

Interactions and Oligomerization of Hantavirus Glycoproteins

In this report the basis for the structural architecture of the envelope of hantaviruses, family Bunyaviridae, is systematically studied by the interactions of two glycoproteins N and C (Gn and Gc, respectively) and their respective disulfide bridge-mediated homo- and heteromeric oligomerizations. In virion extracts Gn and Gc associated in both homo- and hetero-oligomers which were, at least partially, thiol bridge mediated. Due to strong homo-oligomerization, the hetero-oligomers of Gn and Gc are likely to be mediated by homo-oligomeric subunits. A reversible pH-induced disappearance of a neutralizing epitope in Gc and dissociation of the Gn-Gc complex at pH values below 6.2 provide proteochemical evidence for the fusogenicity of Gc. Incomplete inactivation of virions at acidic pH indicates that additional factors are required for hantavirus fusion, as in the case of pestiviruses of the Flaviviridae. Based on similarities to class II fusion proteins, a structure model was created of hantavirus Gc using the Semliki Forest virus E1 protein as a template. In total, 10 binding regions for Gn were found by peptide scanning, of which five represent homotypic (Gn_I to Gn_V) and five represent heterotypic (Gc_I to Gc_V) interaction sites that we assign as intra- and interspike connections, respectively. In conclusion, the glycoprotein associations were compiled to a model wherein the surface of hantaviruses is formed of homotetrameric Gn complexes interconnected with Gc homodimers. This organization would create the grid-like surface pattern described earlier for hantaviruses in negatively stained electron microscopy specimens.Hantaviruses, a genus in the family Bunyaviridae, are rodent- and insectivore-borne zoonotic viruses that are seemingly apathogenic to the carrier rodents (39, 57). A number of hantaviruses are human pathogens that in areas of endemicity are responsible for two diseases: hemorrhagic fever with renal syndrome in Eurasia and hantavirus cardiopulmonary syndrome in the Americas (49, 57, 61). Hantaviruses are enveloped viruses and have a trisegmented, single-stranded, negative-sense RNA genome that encodes an RNA-dependent RNA polymerase, two glycoproteins, and a nucleocapsid protein (22, 34, 49, 60). During assembly, the four proteins and the RNA genome are packed into a round or a pleomorphic particle enveloped with a lipid bilayer. The interactions among the structural components of hantavirus have not been described in sufficient detail to construct the basic architecture of the virus particle or to understand the mechanisms of its assembly and entry.The envelope glycoproteins are expressed as a precursor polypeptide, which is cotranslationally cleaved after a conserved pentapeptide WAASA into an N- and C-terminal portion prior to maturation of the envelope glycoproteins proteins N and C (Gn and Gc, respectively) (27). In the family Bunyaviridae the transport of newly synthesized glycoproteins from endoplasmic reticulum to the Golgi apparatus requires the presence of both Gn and Gc (36, 37, 50, 53). Recombinant coexpression of the hantavirus glycoproteins is sufficient to achieve proper folding and the expected cellular localization at the Golgi even when the glycoproteins are not expressed from a common precursor (6, 36, 50). This suggests that the expression of the precursor is not a prerequisite for interactions between Gn and Gc during maturation in which the formation of a Gn-Gc complex results in exposure of a conformational Golgi apparatus-targeting signal, present only in the heterodimeric Gn-Gc complex (6, 50).Entry of enveloped viruses via recognition of the cell surface receptors and subsequent fusion of the virus and cell membranes are accomplished by viral glycoproteins which often appear in homo- and/or heteromeric complexes. For example, the E1 and E2 of Semliki Forest virus (SFV) form a trimer of heterodimers (45), and the E protein of tick-borne encephalitis virus (TBEV) forms a homodimer (41) while the hemagglutinin of influenza A virus (67) and the S protein of severe acute respiratory syndrome coronavirus associate in homotrimers (4, 5). The mature glycoproteins extracted from virions of Uukuniemi phlebovirus exist as homodimers (44), whereas glycoprotein complex formations of many other members of the Bunyaviridae have not been defined. The viral fusion proteins can be classified into class I, class II, and class III (25). Between classes I and II, a distinguishing property is the orientation of a fusion protein in the metastable state. The class I proteins are oriented perpendicular to the viral membrane, and the class II protein is parallel to the viral membrane (7). The class II viral fusion proteins assemble in virions as metastable homo- or heterodimeric complexes which, upon exposure to low pH, fuse the viral and target cellular membranes (7). This process begins with a conformational change in the fusion protein, leading to the revelation of its fusion loop, which binds to the cellular target membrane (7). Additionally, the formation of a homotrimeric fusion protein complex and structural changes that drive the fusion into completion occur (7).Understanding the multimeric status, protein-protein interactions, and pH-dependent conformational changes of glycoproteins is paramount to our understanding of selectivity in cell receptor binding and mechanisms of virus entry. It is unknown whether higher-order oligomeric complexes are found in hantavirus particles. Many neutralizing monoclonal antibodies (MAbs) have been isolated and by MAb escape mutants shown to recognize epitopes in both Gn and Gc, typically localized at discontinuous sites (15). Different neutralization mechanisms for hantavirus MAbs have been elucidated. These range from inhibiting receptor binding to inhibition of virus fusion (2, 23, 28, 30, 65). It is known that hantaviral glycoproteins possess fusogenic activity. Glycoproteins of hantaviruses that cause hemorrhagic fever with renal syndrome can induce syncytia when subjected to low pH (32, 35), and infection by Hantaan virus was shown to use low-pH-dependent clathrin-mediated endocytosis (19). Hantavirus Gc is suggested to be a class II fusion protein (13, 55), and the N-linked glycosylation of Gc is essential for cell fusion activity (70); but no clear understanding exists of the fusion mechanism or conformational changes that mediate uncoating of virions after entry.Our study supports the hypothesis that the Gc of hantaviruses is a class II fusion protein. We show the interaction between Gn and Gc to be pH sensitive and dissociation to start at a pH below 6.4. The low-pH-induced Gc dissociation from Gn was reversible, suggesting that the conformational changes in Gc are also reversible. Both glycoproteins were found to form homodimeric and hetero-oligomeric complexes in virion extracts through thiol bridging. Interaction studies further suggested that the protruding part of the spike complex in the hantavirus virion consists of four Gn subunits and that the spike complexes interconnect with homodimeric Gc subunits. Finally, we mapped and compiled the interaction sites of Gn and Gc proteins in a class II fusion protein three-dimensional (3D) model of Gc. The identified Gn-Gn, Gn-Gc, and Gc-Gc interaction sites may play an important role in glycoprotein folding and maturation, spike assembly, virus fusion, and neutralization of infection.