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Keratan sulfate phenotype in the β-1,3-N-acetylglucosaminyltransferase-7-null mouse cornea

Littlechild, Stacy, Young, Robert D., Caterson, Bruce, Yoshida, H., Yamazaki, M., Sakimura, K., Quantock, Andrew J. and Akama, T.O. 2018. Keratan sulfate phenotype in the β-1,3-N-acetylglucosaminyltransferase-7-null mouse cornea. Investigative Ophthalmology and Visual Science 59 , pp. 1641-1651. 10.1167/iovs.17-22716

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Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine–transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Optometry and Vision Sciences
Publisher: Association for Research in Vision and Ophthalmology
ISSN: 0146-0404
Funders: NIH grant EY014620, JSPS grant 25462741, Cardiff University International Scholarship, Sir Martin Evans President's Research Scholarship
Date of First Compliant Deposit: 9 January 2018
Date of Acceptance: 27 December 2017
Last Modified: 19 Sep 2019 09:02

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