Sequence analysis of heparan sulphate and heparin oligosaccharides.
The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides.
Vivès
R R
RR
CRC section of Drug Development and Imaging, Department of Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX, UK.
Pye
D A
DA
Salmivirta
M
M
Hopwood
J J
JJ
Lindahl
U
U
Gallagher
J T
JT
D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism; D007066:Iduronate Sulfatase / Q000378:metabolism; D007068:Iduronidase / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009092:Mucous Membrane; D009608:Nitrous Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017421:Sequence Analysis / Q000379:methods*; D013329:Structure-Activity Relationship; D013429:Sulfatases / Q000378:metabolism; D013552:Swine; D013997:Time Factors
D004187:Disaccharides; D006493:Heparin; D006497:Heparan Sulfate; D007066:Iduronate Sulfatase; D007068:Iduronidase; D009608:Nitrous Acid; D009844:Oligosaccharides; D013429:Sulfatases; C028553:disulfoglucosamine-6-sulfatase