Abstract
The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.
Highlights
An oligosaccharide arising from theATIII-bind- et al, 1988; Fransson et al, 1990; Turnbull and Gallagher, ing site precursor was found that comprised only 0.8 1991) that are aimed at sequencing the glycosaminoglycan mol % of the oligosaccharideproductmixture
Oligosaccharide was only slightly enriched in heparin Biosynthetic studies representa second, important having a low affinity for ATIII anodnly slightly disen- approach toward understandingheparin’s structure
The biosynthesis of the heparin these ATIII-binding site precursors, found in unfrac- proteoglycan in mastocytomabeginswith the assembly of tionated and fractionated heparins, suggests the exisct-ore protein and the 0-glycosylation of multiple serine resience of a low ATIII affinity heparinmay not be dues to form a linkage region. From this linkage region the the result of the incomplete action of 3-0-sulfotrans- polymer chain grows by the alternate additionof 1-4-linked ferase in the final step in hepbairoinsynthesis. Rather these data suggest that some earlier step, involved in the formation or placement of these precursor sites, may be primarily responsible for high andlow ATIII affinity heparins
Summary
Heparin isa polydispersesulfated copolymer of 1-4-linked would result in a simple repeatingpolymer of heparin’s major trisulfated disaccharide. Thestructuralvariation found in glucosamine and uronic acid residues (Casu1,985) It hasbeen heparin, which complicates its structure,is thought tobe the medically important as an anticoagulant since its introductiornesult of incomplete modification during itsbiosynthesis. Despiteboth medical and scientific interest in heparin, fied, sequences are 3-0-sulfated (Lindahl et al, 1986; Kusche et al, 1988). This step is noteworthyas it generits precise chemical structure remains unclear. An improved ally resultsinsites a t which theserineproteaseinhibitor understanding of heparin’s structure has resulted from two ATIIIbinds(Pejler et al, 1987; Kusche et al, 1988).
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