H. van Halbeek

Bio: H. van Halbeek is an academic researcher from Utrecht University. The author has contributed to research in topics: Oligosaccharide & Glycopeptide. The author has an hindex of 26, co-authored 40 publications receiving 2469 citations. Previous affiliations of H. van Halbeek include Centre national de la recherche scientifique & French Institute of Health and Medical Research.

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

Abstract: 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.

The applicability of 500-MHz high-resolution 1H-NMR spectroscopy for the structure determination of carbohydrates derived from glycoproteins

Abstract: This report describes the application of 500-MHz 1H-NMR spectroscopy for the determination of the primary structures of the carbohydrate chains of glycoproteins. For this purpose partial structures, i.e. , glycopeptides, oligosaccharides or oligosaccharide-alditols, of the biopolymers were investigated. The spectra, recorded in deuteriumoxide at ambient temperature, contain easily accessible information suitable for the elucidation of primary structures of virtually pure compounds as well as for the analysis of mixtures of closely related components. The key to this information is found in the resonances of the so-called structural reporter groups which are the signals of individually resonating protons. The chemical shifts and coupling constants of the structural reporter groups and the line widths of their resonances are the characteristic parameters which reflect the sugar composition and substitution pattern of the constituting monosaccharides. The suitability of this approach is illustrated for a wide variety of carbohydrate chains. With regard to the N-glycosidically linked type of chains the asialo afuco N—acetyllactosaminic mono-, hi-, tnand tetra-antennary structures were studied, as well as several of their analogues terminated with different types of sialic acid or fucose residues. The structure of oligomannoside type asparagine-bound carbohydrate chains can now completely be defined in terms of NMR parameters of their structural reporter groups. Furthermore, the characterization of several representatives of the O—glycosidically linked mucin type of carbohydrate chains could be achieved. Finally it should be stressed that a high-resolution 'H-NMR spectrum is highly valuable even in cases that no complete assignment of the primary structure is possible. The spectrum can i.a. be used as an identity card to compare carbohydrate chains obtained from different sources. 1H—NMR spectroscopy appears to be significantly more powerful at 500 MHz than at 360 MHz. By this non-destructive method samples as small as 25 nmoles could be adequately analyzed. INTRODUCTION During the last decade the interest in the structure and function of glycoproteins increased greatly. It could be shown that the carbohydrate chains of these biopolymers are involved in several important biochemical processes. In particular their roles in recognition phenomena, in immunological events and in determining the life-span of cells and glycoproteins have to be mentioned. Significant progress has been made in the characterization of the primary structures of the carbohydrate chains. This holds for the chains N-glycosidically linked to asparagine, as well as for the O-glycosidically linked moieties which may be attached to serine, threonine or hydroxylysine. The refinement of enzymic methods and chemical techniques, like permethylation analysis, for the structure determination contributed to a large extent to these results. Nevertheless, the classical approach has several disadvantages and shortcomings as pointed out before (Ref. 1). Especially it is highly time and material consuming and has severe limitations in the analysis of mixtures of closely related compounds. It should be noted that the structure analysis is carried out at the level of partial structures like cjlycopeptides, oligosaccharides and oligosaccharide-alditols. It is the (micro-)heterogeneity of a single carbohydrate chain of the glycoprotein which may give rise to a mixture of partial structures difficult to fractionate. 1 In 1977 we introduced the application of high-resolution H-NMR spectroscopy as a new approach for the structure elucidation of underivatized carbohydrate chains obtained from qlycoproteins. First of all the 1H-NMR spectrum of the compound provides us with a structural PAAC 53:1 D 45 46 JOF[ANNES F. G. VLIEGENTHART, HERMAN VAN FIALBEEK and LANBERTUS DORLAND identity card which, even if the spectrum can not be interpreted, renders possible comparison with the spectra obtained from other sources, allowing the conclusion whether or not compounds are identical. The relative intensities of the signals in the NMR spectrum can be used as markers for the purity of the compound. Often it can be deduced from the spectrum whether or not the sample consists of more than one carbohydrate structure and in which molar ratios the components occur. It could be shown that 360-MHz 1H-NMR spectroscopy in conjunction with methylation analysis is excellently suited for elucidation of the primary structures of Nor O-glycosidically linked carbohydrate chains of glycoproteins (Ref. 1 17) . For the interpretation of the NMR spectrum the concept of structural reporter groups was developed (Ref. 1) , i.e. , the chemical shifts and coupling constants of protons resonating at clearly distinguishable positions in the spectrum bear the essential information to assign the primary structure. Recently a 500-MHz 1H-NMR spectrometer became available for our studies, concomitant with a generally applicable computer resolution enhancement routine (Ref. 18). The increased sensitivity of this spectrometer system with respect to the 360-MHz apparatus affords spectra with a more favourable signal to noise ratio. Therefore the gain in spectral resolution, inherent to the stronger magnetic field, can be optimally utilized in Lorentzian to Gaussian transformation. As could be demonstrated, also the line widths of the structural reporter group signals can in several cases be used to derive pertinent information on primary structures (Ref. 18). In this report the 500-MHz 'H-NMR spectra of some Nand O-glycosidically linked carbohydrate chains will be discussed in detail. RESULTS AND DISCUSSION N-acetyllactosamine type glycopeptides and oligosaccharides derived from N-glycoproteins The best defined asialo representatives of the N-acetyllactosamine type asparagine-bound carbohydrate chains of glycoproteins are shown in Fig. 1.

How proteolysis drives the cell cycle

Abstract: Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity—by degrading CDK activators or inhibitors—and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells

Abstract: Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Isolation and characterization of plant cell walls and cell wall components

Abstract: Publisher Summary This chapter describes the methods used for isolating and characterizing the noncellulosic polysaccharides of the primary walls of suspension-cultured sycamore cells These procedures are applicable to the study of other types of cell walls Cell walls form the basic structural framework of the plant, defining the shape and size of plant cells and tissues Cell walls are classified as either primary or secondary, depending upon their mechanical properties and chemical composition The primary cell wall is a mechanically dynamic structure encasing the cell during the period of rapid expansion that follows cell division The secondary cell wall is, relative to the primary cell wall, a mechanically static structure that determines the shape and size of the mature cell The chapter presents the experiments for the isolation of plant cell walls and the isolation of polysaccharides from cell walls and from extracellular polysaccharides of suspension-cultured plant cells and the chemical methods used for characterizing polysaccharides