Abstract
Nonsulfated, monosulfated, and disulfated glycopeptides containing the entire carbohydrate sequence of the glycosaminoglycan-specific linkage region were isolated after exhaustive enzymatic digestions of Swarm rat chondrosarcoma proteoglycans with chondroitinase ABC, papain, and Pronase. Their structures were examined by 500 MHz 1H NMR spectroscopy. The nonsulfated compound has the following structure with trace amounts of a few additional amino acids: delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The monosulfated compound has an ester sulfate on C-4 of the GalNAc residue and the disulfated compound has an additional hitherto unrecognized ester sulfate on C-4 of the second galactose residue which is remote from the innermost xylose. This new structure was confirmed by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The molar ratio of the isolated nonsulfated, monosulfated, and disulfated compounds was 53:37:10 based on the serine contents. Biological significance of the newly found sulfated linkage structure is discussed.
Highlights
Structural Studies on Sulfated Glycopeptides fromthe CarbohydrateProtein Linkage Region of Chondroitin4-Sulfate Proteoglycans of Swarm Rat Chondrosarcoma
Swarm rat chondrosarcoma proteoglycanws ith chon- reported by Rodbn and co-workers
The considerable complexity of proteoglycan structure and composition has been recognized, they are usually classified into several categories based on the attached glycosaminoglycan species including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate [1]. these glycosaminogly
Summary
Preparation of Linkage Glycopeptides-Proteoglycan monomers (497 mgof dry weight) were repeatedly digested with chondroitinase ABC as described under “Experimental Procedures” until no furtherunsaturated oligosaccharide was formed. When chromatographed on a Sephadex G-50 column, the papain digest showed a single carbazole positive peak, in which essentially all orcinol positive materials were recovered This peak (52.5 pmol of uronic acid) was further digested with Pronase and chromatographed on a column of Sephadex G-25 (Fig. 1).The major fraction, which contained 82% of the applied uronic acid, was redigested with Pronase. A major fraction containing 91% of the applied uronic acid was recovered from the second Pronase digest This fraction turned outot contain a small yet significant amount of sialic acid (2.5 pmo1/30.9 pmol of uronic acid), which was indicative of the contamination with mucin-type glycopeptides. D-4 gave two major spots; the faster (Rserine= 2.45) and slower (Rserine= 2.05) migrating spots were referred to as D-4F and D-4S,respectively These glycopeptides were isolated by preparative borate paperelectrophoresis followed by removal of borate as described under “Experimental Procedures.”.
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