Abstract
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.
Highlights
Chondroitin sulfate (CS)3 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)
The characteristic spatiotemporal distribution of IdoUA-containing Chondroitin sulfate/dermatan sulfate (CS/DS) hybrid chains in the postnatal mouse brain was demonstrated by in situ hybridization for sulfotransferases involved in the biosynthesis of DS and by chemical in situ analysis of CS/DS chains of the brain sections
Both approaches showed that IdoUA-containing CS/DS hybrid structures are more concentrated in the cerebellum when compared with whole brain, providing insight into the important biological functions of IdoUA-containing CS/DS hybrid chains during postnatal cerebellar development as discussed below
Summary
Materials—The following sugars and enzymes were purchased from Seikagaku Corp. (Tokyo, Japan): five authentic unsaturated CS disaccharides, conventional chondroitinase ABC (EC 4.2.2.4) from Proteus vulgaris, and chondroitinase AC-I (EC 4.2.2.5) from Flavobacterium heparinum. A cDNA fragment (ϳ1.3 kbp) of mouse C4ST-1 (UniGene Cluster Mm. 339052) was amplified with a forward primer 5Ј-GCA CAC CAA CTC CTG CG-3Ј and a reverse primer 5Ј-TGC CGC TCG CAG CAT CT-3Ј, followed by nested PCR with nested primers as follows: a forward primer 5Ј-AGA GCC TCG GTG AAG CTA-3Ј and a reverse primer 5Ј-ATA ACC CAG TCT CCA TAG AAT TC-3Ј. A cDNA fragment (ϳ1.4 kbp) of mouse C4ST-2 (UniGene Cluster Mm. 28934) was amplified with a forward primer 5Ј-CCA GCT GTG CAC AAG GCT GA-3Ј and a reverse primer 5Ј-TGC CTG TCA CAC CAG GAA GC-3Ј, followed by nested PCR with nested primers as follows: a forward primer 5Ј-GCA CAA GGC TGA AGT GAA GG-3Ј and a reverse primer 5Ј-AAG GAA GCC AGG AGA GAA CC-3Ј.
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