SEQUENCE-ANALYSIS OF PARA-HYDROXYPHENYL-O-BETA-D-XYLOSIDE INITIATED AND RADIOIODINATED DERMATAN SULFATE FROM SKIN FIBROBLASTS

To generate xyloside-primed dermatan sulfate suitable for sequence ana lysis, skin fibroblasts were incubated with p-hydroxyphenyl-beta-D-xyl opyranoside and [H-3]galactose, and free [H-3]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromat ography. After I-125 labelling of their reducing-terminal hydroxphenyl groups, chains were subjected to various chemical and enzymatic degra dations, both partial and complete, followed by gradient polyacrylamid e gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Re sults of periodate oxidation-alkaline scission indicated that the xylo se moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment DELTA-HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments DELTA-HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or witho ut sulfate on the N-acetylgalactosamine. Partial digestions with testi cular hyaluronidase or chondrotin B lyase indicated that glucuronic ac id was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustere d GlcA-GalNAc repeats which was previously observed in proteoglycan de rived dermatan sulfate [Fransson L-angstrom, Havsmark B, Silverberg I (1990) Biochem J 269:381-8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during g lycosaminoglycan assembly.