Structural and Metabolic Studies of Glycosaminoglycans (Sulfatase, Hydrazine, Mucopolysaccharidosis)
Shaklee, Patrick Neil
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https://hdl.handle.net/2142/70543
Description
Title
Structural and Metabolic Studies of Glycosaminoglycans (Sulfatase, Hydrazine, Mucopolysaccharidosis)
Author(s)
Shaklee, Patrick Neil
Issue Date
1985
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
A method for chemical cleavage of glycosaminoglycans (GAGs) at N-acetylhexosamine residues was developed. The reaction scheme involved N-deacetylation of the N-acetylhexosamine residues with hydrazine (N(,2)H(,4)) followed by nitrous acid (HONO) cleavage of the polymer at pH 4.0. Initial studies with heparin-derived model compounds indicated that ester and N-sulfate substituents were stable during N(,2)H(,4) treatment but that uronic acids were converted to hydrazides which were epimerized at C-5 during the hydrazinolysis reaction. Subsequently, it was found that uronic acid hydrazides could be oxidized to uronic acids by HONO or iodic acid (HIO(,3)) treatment. The chemical cleavage method was used to convert reference GAGs to disaccharides which were reduced with NaB('3)H(,4) and separated by paper chromatography and anion exchange HPLC. Mono- and disulfated disaccharides from keratan sulfate (KS), the chondroitin sulfates (CS), and dermatan sulfate (DS) were identified. The relative amounts of CS and DS disaccharides released following a 10 hr N(,2)H(,4) treatment were found to be comparable to the corresponding products released by treatment of these polymers with chondroitinase ABC or chondroitinase AC. A study of the rates of disaccharide release during the N(,2)H(,4)/HONO treatment of reference GAGs indicated that 4-sulfation of CS significantly decreased the overall N-deacetylation rate.
The side reactions discovered in the initial phases of the work were found not to effect the results with the reference GAGs significantly. However, the epimerization reaction was useful for generation of GlcA(2-SO(,4))-AMan(,R)(6-SO(,4)) from IdoA(2-SO(,4))-AMan(,R)(6-SO(,4)). Both the former disaccharide and GlcA(2-SO(,4))-ATal(,R)(6-SO(,4)), a disaccharide obtained from treatment of CS with N(,2)H(,4)/HONO, were used to show the existence of a glucurono-2-sulfatase in extracts of chick embryo chondrocytes and human fibroblasts. This enzyme, which had a lysosomal pH optimum, was also present in idurono-2-sulfatase-deficient human fibroblasts, and is proposed to have a role in the catabolism of CS and heparan sulfate. The N(,2)H(,4)/HONO reaction scheme was also used to characterize the structure of the KS on the cartilage-specific proteoglycan (PCS-H) produced by chick embryo tibial chondrocytes isolated from histologically distinct zones of the tibia. The KS structure was found not to change significantly from one zone of chondrocytes to the next, and the structure was nearly identical in cell-associated and culture medium PCS-H. (Abstract shortened with permission of author.)
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