Publisher Summary This chapter discusses the application of high-resolution, 1H-nuclear magnetic resonance (NMR) spectroscopy to the structural analysis of carbohydrates related to glycoproteins. Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the O-glycosylic chains are linked to the hydroxyl group of amino acid residues such as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). The high-resolution, 1H-NMR spectroscopy technique, in conjunction with methylation analysis, is extremely suitable for the structural studies of N-, as well as on O-, glycosylic glycans. For the interpretation of the 1H-NMR spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structural reporter groups” was developed. This means that the chemical shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit the assigning of the primary structure. This chapter presents literature data on the high-resolution, 1H-NMR spectroscopy of carbohydrates derived from glycoconjugates. It also discusses the results for carbohydrates related to the glycoproteins of N-glycosylic type.

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High-Resolution, 1H-Nuclear Magnetic Resonance Spectroscopy as a Tool in the Structural Analysis of Carbohydrates Related to Glycoproteins

The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques. Previous results indicate that complete inactivation of the RB gene is required for tumour formation. As a 'cancer suppressor' gene, RB thus functions in a manner opposite to that of most other oncogenes. Sequence analysis of RB complementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function. To further substantiate and identify the RB protein, we have prepared rabbit antisera against a trypE-RB fusion protein. The purified anti-RB IgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000-114,000. The specific protein(s) are present in all cell lines expressing normal RB mRNA, but are not detected in five retinoblastoma cell lines examined. The RB protein can be metabolically labelled with 32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that the RB gene product may function in regulating other genes within the cell.

The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity

Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 micrograms/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis- and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40 degrees C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures greater than or equal to 20 degrees C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.

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Brefeldin A Redistributes Resident and Itinerant Golgi Proteins to the Endoplasmic Reticulum

Publisher Summary This chapter provides an overview of the primary structure of glycoprotein glycans. There exists a certain unity of structure among glycans. All are derived by the substitution of common, core oligosaccharides ( inv fractions) by variable oligosaccharide structures ( var fractions), which are the basis of their specificity. The var fractions present a limited number of the types of structure. In the case of N -glycosylproteins, in which the glycan-protein bond is asparaginyl-2-acetamido-2-deoxyglucose, the glycans are of three fundamental types: the oligomannosidic, the N -acetyllactosaminic, and the mixed, oligomannosidic- N -acetyl-lactosaminic. The determination of the structures of oligosaccharides from the urines of glycoproteinoses, diseases characterized by a deficit in lysosomal enzymes catabolizing the glycans of glycoprotein, allows reconstruction of the different stages of glycoprotein catabolism, and presentation of the hypothesis that the catabolism of N -acetyl-lactosainine glycans starts by the action of endo-2-acetamido-2-deoxy-β- D -glucosidases in liberating oligosaccharides that are then degraded by exoglycosidases. This chapter elaborates the primary structure and metabolism of the N -linked glycans. Development and improvement of procedures for the study of the primary structure of glycans is also discussed.

M Ogata-Arakawa

Papers

Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.