STUDY ON THE BIOLOGICAL FUNCTIONS OF SPECIFIC HEPARAN SULFATE STRUCTURES GENERATED BY HEPARAN SULFATE 6-O-SULFOTRANSFERASES

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Generation of mutant animals with loss-of-function of HS6STs: 1) Drosophila HS6ST was disrupted with dsRNAi. RNA interference experiments indicated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These findings suggest dHS6ST play crucial roles on tracheal development and on FGF signaling. 2) Zebrafish HS6ST was knockdown by HS6ST morpholino injections in embryos. These morphants displayed the curled tail, the drastic reduction of the size of fins, the stronger reduction of the white matter in brain and the impaired muscle differentiation. These results suggest that HS6ST may regulate the wnt signaling as well. 3) We generated mice with a targeted deletion of the HS6ST-1 gene. Most of HS6ST-1-/- shows the lethality at perinatal stage and runting. Less than 5% of the homozygoutes survive after birth and fertile, but the growth was severely retarded. We are now analyzing whether the lethality results from runting or from any other defects. 4) Mouse HS2ST, HS6ST-1, -2, and -3 were characterized on the substrate specificities.Analysis of chick HS2ST and HS6STs on chick limb bud development: the expression patterns of cHS6ST-1 and cHS6ST-2 transcripts were preferentially localized at the anterior proximal region and at the posterior proximal region of the limb bud, respectively, while HS2ST transcript was localized rather uniformly throughout the bud. Analysis of the fine structure of heparan sulfate in each regions showed the significant correlation between the expression patterns of HS-O-ST isoforms and HS structures.Functions of HS-O-STs on Patho-physiological phenomena: we characterized human HS6STs. Human HS6ST-2 have alternative spliced form, HS6ST-2S. These isoforms showed different substrate specificities and very different expression among various tissues. We found that 6-O-sulfate groups in HS appeared to mediate the infection of Chlamydia trachomatis into host cells.