Chemometric analysis for comparison of heparan sulphate oligosaccharides

Heparan sulphate (HS) is a glycosaminoglycan present in all metazoan organisms. It is an unbranched chain made up of repeating disaccharide units of uronic acid and glucosamine sugars, and is present in both cells and the extracellular matrix. It is one of the most structurally diverse biological molecules and its biosynthesis involves a variety of enzymic modification steps. Unlike the genome and the transcriptome, HS synthesis is not template driven. Nevertheless, the HS structure and function are highly regulated with modification steps occurring in discrete regions of the polysaccharide chain to give rise to diverse structures interacting with, and regulating, many different proteins. The resulting variation leads to diverse biological roles of HS. To study this structural diversity, rapid isolation and characterization of HS from small amounts of tissues, followed by digestion with bacterially derived enzymes (heparitinases) and chromatography techniques can be used to separate HS oligosaccharides of different size and charge. However, this leads to complex datasets where comparison of just a few samples leads to difficulties in data analysis. Using automatically integrated peak data obtained from chromatographic software, one can apply the effective disc technique to the data points to obtain the centre of mass in each dataset, for example from different murine tissues. This allows facile comparative analysis of different datasets. When the cloud of points displays some preferential direction (anisotropy), it is preferable to compute its effective ellipse. Analysis of the dynamics of the cloud of points for repeated experiments allows the quantification of their reproducibility through evaluation of an average Lyapunov exponent characterizing the area-preserving nature of a sequence of effective ellipses. These basic mathematical approaches allow a more systematic comparison of datasets derived from structural analysis using basic spreadsheet software calculations and contribute to the development of system biology strategies for tackling biocomplexity of HS polysaccharides.

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Structural differences and the presence of unsubstituted amino groups in heparan sulphates from different tissues and species

Biochemical Journal ◽

10.1042/bj3220499 ◽

1997 ◽

Vol 322 (2) ◽

pp. 499-506 ◽

Cited By ~ 115

Author(s):

Toshihiko TOIDA ◽

Hisao YOSHIDA ◽

Hidenao TOYODA ◽

Ichiro KOSHIISHI ◽

Toshio IMANARI ◽

...

Keyword(s):

Acid Content ◽

Heparan Sulphate ◽

Kidney Medulla ◽

Isolation And Characterization ◽

Iduronic Acid ◽

Structural Differences ◽

Liver And Kidney ◽

High Degree ◽

Different Tissues

This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain → 4)-β-d-GlcAp-(1 → 4)-α-d-GlcNp-(1 → 4)-β-d-GlcAp-(1 → low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.

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Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2

Mechanisms of Development ◽

10.1016/s0925-4773(96)00549-7 ◽

1996 ◽

Vol 58 (1-2) ◽

pp. 15-26 ◽

Cited By ~ 83

Author(s):

J. Klingensmith ◽

Y. Yang ◽

J.D. Axelrod ◽

D.R. Beier ◽

N. Perrimon ◽

...

Keyword(s):

Structure And Function ◽

Isolation And Characterization ◽

And Function

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Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1

Biochemical Journal ◽

10.1042/bj3170203 ◽

1996 ◽

Vol 317 (1) ◽

pp. 203-212 ◽

Cited By ~ 6

Author(s):

Georg STÖCKER ◽

Zofia DRZENIEK ◽

Ursula JUST ◽

Wolfram OSTERTAG ◽

Barbara SIEBERTZ ◽

...

Keyword(s):

Cell Line ◽

Stromal Cells ◽

Progenitor Cell ◽

Heparan Sulphate ◽

Proteoglycan Synthesis ◽

Heparan Sulphate Proteoglycan ◽

Haematopoietic Progenitor Cell ◽

Sulphate Proteoglycan ◽

Isolation And Characterization

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.

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ISOLATION AND CHARACTERIZATION OF AMBER SUPPRESSORS IN YEAST

Genetics ◽

10.1093/genetics/82.2.251 ◽

1976 ◽

Vol 82 (2) ◽

pp. 251-272

Author(s):

Susan W Liebman ◽

Fred Sherman ◽

John W Stewart

Keyword(s):

Amino Acids ◽

Cytochrome C ◽

Yeast Strain ◽

Normal Amount ◽

Amber Mutation ◽

Genetic Analyses ◽

Isolation And Characterization ◽

And Function ◽

Nonsense Suppressors

ABSTRACT Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome cin cyc1-179 strains suppressed with these efficient amber suppressors.

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Isolation and characterization of lung connective-tissue glycoproteins

Biochemical Journal ◽

10.1042/bj2030593 ◽

1982 ◽

Vol 203 (3) ◽

pp. 593-601 ◽

Cited By ~ 9

Author(s):

C Lafuma ◽

M Moczar ◽

L Robert

Keyword(s):

Proteinase Inhibitors ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Lung Parenchyma ◽

Isolation And Characterization ◽

Isoelectric Points ◽

And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

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Structure and Function of L-Lactate Dehydrogenases from Thermophilic and Mesophilic Bacteria. I) Isolation and Characterization of Lactate Dehydrogenases from Thermophilic and Mesophilic Bacilli

Hoppe-Seyler´s Zeitschrift für physiologische Chemie ◽

10.1515/bchm2.1979.360.2.795 ◽

1979 ◽

Vol 360 (2) ◽

pp. 795-808 ◽

Cited By ~ 46

Author(s):

Hans-Peter SCHÄR ◽

Herbert ZUBER

Keyword(s):

Structure And Function ◽

Mesophilic Bacteria ◽

Isolation And Characterization ◽

And Function ◽

Lactate Dehydrogenases

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070 Isolation and characterization of heparan sulphate proteoglycan from human articular cartilage

Fresenius Journal of Analytical Chemistry ◽

10.1007/bf00332064 ◽

1992 ◽

Vol 343 (1) ◽

pp. 128-128

Author(s):

G. St�cker ◽

D. -C. Fischer ◽

S. Handt ◽

H. Greiling ◽

H. -D. Haubeck

Keyword(s):

Articular Cartilage ◽

Heparan Sulphate ◽

Human Articular Cartilage ◽

Heparan Sulphate Proteoglycan ◽

Sulphate Proteoglycan ◽

Isolation And Characterization

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Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors

Blood ◽

10.1182/blood-2002-03-0974 ◽

2003 ◽

Vol 101 (4) ◽

pp. 1453-1459 ◽

Cited By ~ 115

Author(s):

Meredith O'Keeffe ◽

Hubertus Hochrein ◽

David Vremec ◽

Bernadette Scott ◽

Paul Hertzog ◽

...

Keyword(s):

Human Blood ◽

Ex Vivo ◽

Mouse Blood ◽

Surface Phenotype ◽

Isolation And Characterization ◽

Tnf Α ◽

Close Relationship ◽

Factor Α ◽

And Function

Immature and predendritic cells (pre-DCs) of human blood are the most readily accessible human DC sources available for study ex vivo. Murine homologues of human blood DCs have not been described. We report the isolation and characterization of 2 populations of precursor DCs in mouse blood. Mouse blood cells with the surface phenotype CD11cloCD11b−CD45RAhi closely resemble human plasmacytoid cells (or pre-DC2) by morphology and function. On stimulation with oligonucleotides containing CpG motifs (CpG), these cells make large amounts of type 1 interferons and rapidly develop into DCs that bear CD8, though they may be distinct from the CD8+ DCs in the unstimulated mouse. A second population of cells with the surface phenotype CD11c+CD11b+CD45RA− closely resembles the immediate precursors of pre-DC1, rapidly transforming into CD8− DCs after tumor necrosis factor-α (TNF-α) stimulation. These findings indicate the close relationship between human and mouse DCs, provided cells are obtained directly from equivalent source materials.

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