May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Identifying Sulfotransferase and Glycosyltransferase Genes Responsible for Regulation of Corneal Keratan Sulfate Biosynthesis
Author Affiliations & Notes
  • J. L. Funderburgh
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • M. M. Mann
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • P. R. Kinchington
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • M. L. Funderburgh
    Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
  • Footnotes
    Commercial Relationships J.L. Funderburgh, None; M.M. Mann, None; P.R. Kinchington, None; M.L. Funderburgh, None.
  • Footnotes
    Support NIH Grants EY09368, P30-EY08098, Research to Prevent Blindness , Eye and Ear Foundation of Pittsburgh. JLF is a Jules and Doris Stein RPB Professor
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1482. doi:https://doi.org/
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    • Get Citation

      J. L. Funderburgh, M. M. Mann, P. R. Kinchington, M. L. Funderburgh; Identifying Sulfotransferase and Glycosyltransferase Genes Responsible for Regulation of Corneal Keratan Sulfate Biosynthesis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1482. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Corneal stroma contains a unique class of proteoglycans modified with keratan sulfate (KS). Abundance of KS is credited with maintenance of corneal transparency, and KS biosynthesis is reduced or lost during corneal healing and in chronic pathology. Glycosaminoglycans such as KS are synthesized in the Golgi by coordinated action of multiple glycosyl- and sulfo- transferases. Currently, only one sulfotransferase, coded by the gene CHST6, has been definitively associated with biosynthesis of corneal KS. This study aims to identify glycosyl- and sulfo- transferases involved in KS biosynthesis and to determine which are responsible for KS downregulation in wound healing.

Methods:: Primary bovine keratocytes synthesize high levels of KS in serum-free culture but rapidly lose KS biosynthesis in serum. Cloned keratocyte progenitor cells make no KS but upregulate KS secretion in mitogen-poor medium. qRT-PCR assessed mRNA levels of six candidate genes (CHST6, CHST1, B4GALT1, B4GALT4, B3GNT1, B3GNT7) coding for transferases. siRNA against these was used to knock down mRNA expression. Adenovirus expressing CHST6 was used to overexpress this sulfotransferase.

Results:: Knockdown of mRNA expression of six transferases potentially involved in KS synthesis each resulted in reduced KS biosynthesis. Most strongly linked were the galactose-6-sulfotransferase CHST1, N-acetylglucosamine-6-sulfotransferase CHST6, and N-acetylglucosaminlytransferase B3GNT7. Each of these three mRNAs also decreased rapidly after exposure of keratocytes to serum-containing medium. KS was markedly upregulated by overexpression of CHST6 using an adenovirus vector.

Conclusions:: These results demonstrate, for the first time, participation of galactose-6-sulfotransferase CHST1 and N-acetylglucosaminyltransferase B3GNT7, in addition to the known role of CHST6 in the synthesis of corneal KS by living cells. The coordinated reduction of mRNA from all three of these genes during downregulation of KS by serum and the restoration of KS synthesis by overexpression of CHST6 supports the hypothesis that KS biosynthesis is controlled by expression levels of transferase genes specific for elongation and sulfation of corneal KS.

Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • gene/expression 
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