Sequence analysis of p-hydroxyphenyl-O-beta-D-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts.
To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated with p-hydroxyphenyl-beta-D-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After 125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose 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 without sulfate on the N-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid 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 clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-A, 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 glycosaminoglycan assembly.
Fransson
L A
LA
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Havsmark
B
B
Sakurai
K
K
Suzuki
S
S
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003871:Dermatan Sulfate / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry; D006025:Glycosaminoglycans / Q000138:chemical synthesis / Q000302:isolation & purification; D006027:Glycosides / Q000737:chemistry*; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D012867:Skin / Q000737:chemistry* / Q000166:cytology / Q000196:embryology
D006025:Glycosaminoglycans; D006027:Glycosides; D007457:Iodine Radioisotopes; C077140:4-hydroxyphenyl-O-xyloside; D003871:Dermatan Sulfate