Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains in the Development of Cerebellum

Chondroitin sulfate/dermatan sulfate (CS/DS) chains regulate the development of the central nervous system in vertebrates. Previously, we demonstrated that CS/DS hybrid chains from embryonic pig brain exhibit neuritogenic and growth factor binding activities, which depended on their IdoUA content defining the DS-like structure. To elucidate the distribution of such functional sugar chains during the development of the brain, in situ hybridization was performed to examine expression of three CS/DS GalNAc 4-O-sulfotransferases, D4ST-1, C4ST-1, and C4ST-2, and a single uronyl 2-O-sulfotransferase (UST) involved in the biosynthesis of DS in addition to CS intermediates. C4ST-1 and C4ST-2 were ubiquitously expressed in the postnatal mouse brain, whereas the expression of D4ST-1 and UST was restricted in the developing cerebellum and culminated at postnatal day 14 as shown by reverse transcriptase-PCR analysis. In situ analysis of the disaccharides of CS/DS in brain sections revealed that the concentration of CS/DS increases 2-fold during development (postnatal day 7 to 7 weeks). The proportions of DS-specific, principal disaccharides, IdoUA-Gal-NAc(4-O-sulfate) (iA) and IdoUA(2-O-sulfate)-GalNAc(4-O-sulfate) (iB), produced by the sequential actions of D4ST-1 and UST, were higher in the CS/DS chains from cerebellum than those from whole brain sections. A dramatic increase (10-fold) in the proportion of iB during development was noteworthy. In contrast, GlcUA/IdoUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D/iD) and GlcUA/IdoUA-GalNAc(4, 6-O-disulfate) (E/iE) decreased to 50 and 30%, respectively, in the developing cerebellum. These results suggest that the IdoUA-containing iA and iB units along with D/iD and E/iE units in the CS/DS hybrid play important roles in the formation of the cerebellar neural network during postnatal brain development. Chondroitin sulfate/dermatan sulfate (CS/DS) chains regulate the development of the central nervous system in vertebrates. Previously, we demonstrated that CS/DS hybrid chains from embryonic pig brain exhibit neuritogenic and growth factor binding activities, which depended on their IdoUA content defining the DS-like structure. To elucidate the distribution of such functional sugar chains during the development of the brain, in situ hybridization was performed to examine expression of three CS/DS GalNAc 4-O-sulfotransferases, D4ST-1, C4ST-1, and C4ST-2, and a single uronyl 2-O-sulfotransferase (UST) involved in the biosynthesis of DS in addition to CS intermediates. C4ST-1 and C4ST-2 were ubiquitously expressed in the postnatal mouse brain, whereas the expression of D4ST-1 and UST was restricted in the developing cerebellum and culminated at postnatal day 14 as shown by reverse transcriptase-PCR analysis. In situ analysis of the disaccharides of CS/DS in brain sections revealed that the concentration of CS/DS increases 2-fold during development (postnatal day 7 to 7 weeks). The proportions of DS-specific, principal disaccharides, IdoUA-Gal-NAc(4-O-sulfate) (iA) and IdoUA(2-O-sulfate)-GalNAc(4-O-sulfate) (iB), produced by the sequential actions of D4ST-1 and UST, were higher in the CS/DS chains from cerebellum than those from whole brain sections. A dramatic increase (10-fold) in the proportion of iB during development was noteworthy. In contrast, GlcUA/IdoUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D/iD) and GlcUA/IdoUA-GalNAc(4, 6-O-disulfate) (E/iE) decreased to 50 and 30%, respectively, in the developing cerebellum. These results suggest that the IdoUA-containing iA and iB units along with D/iD and E/iE units in the CS/DS hybrid play important roles in the formation of the cerebellar neural network during postnatal brain development. Chondroitin sulfate (CS) 3The abbreviations used are: CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; PG, proteoglycan; D4ST, dermatan 4-O-sulfotransferase; C4ST, chondroitin 4-O-sulfotransferase; UST, uronyl 2-O-sulfotransferase; Δ4,5HexUA, 4,5-unsaturated hexuronic acid or 4-deoxy-α-l-threo-hex-4-ene-pyranosyluronic acid; O, GlcUAβ1–3Gal-NAc; C, GlcUAβ1–3GalNAc(6S); iC, IdoUAα1–3GalNAc(6S); A, GlcUAβ1–3GalNAc(4S); iA, IdoUAα1–3GalNAc(4S); D, GlcUA(2S)β1–3GalNAc(6S); iD, IdoUA(2S)α1–3GalNAc(6S); B, GlcUA(2S)β1–3GalNAc(4S); iB, IdoUA(2S)α1–3GalNAc(4S); E, GlcUAβ1–3GalNAc(4S,6S); iE, IdoUAα1–3GalNAc(4S,6S); ΔDi-0S or ΔO, Δ4,5HexUAα1–3GalNAc; ΔDi-6S or ΔC, Δ4,5HexUAα1–3GalNAc(6-O-sulfate); ΔDi-4S or ΔA, Δ4,5HexUAα1-3GalNAc(4-O-sulfate); ΔDi-diSD or ΔD, Δ4,5HexUA(2-O-sulfate)α1–3Gal-NAc(6-O-sulfate); ΔDi-diSB or ΔB, Δ4,5HexUA(2-O-sulfate)α1–3Gal-NAc(4-O-sulfate); ΔDi-diSE or ΔE, Δ4,5HexUAα1–3GalNAc(4,6-O-disulfate); P, postnatal day; W, week; RT, reverse transcription; HPLC, high performance liquid chromatography; 2AB, 2-aminobenzamide; bFGF, by basic fibroblast growth factor; F, forward; R, reverse. and dermatan sulfate (DS) are sulfated glycosaminoglycan (GAG) chains with enormous structural diversity and are covalently attached to various core proteins to form proteoglycans (PGs). PGs show a widespread distribution in extracellular matrices and at cell surfaces. Increasing evidence suggests the importance of CS and DS chains in several biological processes (for reviews see Refs. 1Silbert J.E. Sugumaran G. Glycoconj. J. 2003; 19: 227-237Crossref Scopus (21) Google Scholar, 2Trowbridge J.M. Gallo R.L. Glycobiology. 2002; 12: R117-R125Crossref PubMed Google Scholar, 3Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (604) Google Scholar). Notably, CS chains regulate the development of the nervous system and play different roles as neuritogenic molecules (for reviews see Refs. 3Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (604) Google Scholar, 4Sugahara K. Yamada S. Trends Glycosci. Glycotechnol. 2000; 12: 321-349Crossref Scopus (96) Google Scholar, 5Bandtlow C.E. Zimmermann D.R. Physiol. 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