June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Beta 1,3-N-acetylglucosaminyltransferse-7 is necessary for normal keratan sulfate proteoglycan phenotype in mouse cornea.
Author Affiliations & Notes
  • Stacy L Littlechild
    Biophysics Group, Cardiff University School of Optometry and Vision Sciences, Cardiff, United Kingdom
    Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom
  • Robert D Young
    Biophysics Group, Cardiff University School of Optometry and Vision Sciences, Cardiff, United Kingdom
    Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom
  • Andrew J Quantock
    Biophysics Group, Cardiff University School of Optometry and Vision Sciences, Cardiff, United Kingdom
    Cardiff Institute of Tissue Engineering and Repair, Cardiff, United Kingdom
  • Tomoya O Akama
    Department of Pharmacology, Kansai Medical University, Osaka, Japan
    Tumor Microenvironment Program, Sanford-Burnham Medical Research Institute, La Jolla, United Kingdom
  • Footnotes
    Commercial Relationships Stacy Littlechild, None; Robert Young, None; Andrew Quantock, None; Tomoya Akama, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1940. doi:https://doi.org/
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      Stacy L Littlechild, Robert D Young, Andrew J Quantock, Tomoya O Akama; Beta 1,3-N-acetylglucosaminyltransferse-7 is necessary for normal keratan sulfate proteoglycan phenotype in mouse cornea.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1940. doi: https://doi.org/.

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

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Abstract

Purpose: In vitro studies have demonstrated the importance of glycosyltransferases in corneal keratan sulfate glycosaminoglycan (KS-GAG) biosynthesis; however, few studies have shown corneal ultrastructural ramifications if glycosyltransferase activity is abnormal. Here, we studied keratan sulfate proteoglycan (KSPG) in the absence of B3gnt7, the gene encoding β1,3-N-acetylglucosaminyltransferase-7 (β3GnT7), in a knockout mouse model. We hypothesize that KSPG phenotype and corneal ultrastructure will significantly change without the action of β3GnT7, as this enzyme has function to elongate KS-GAG repeating structure.

Methods: B3gnt7 heterozygous mutant (HTZ) mice were created on C57BL/6J background using a knockout vector obtained from trans-NIH Knock-Out Mouse Project (KOMP) repository by a standard protocol, and the homozygous mutant (NULL) mice were produced by intercross of B3gnt7 HTZ mice. Corneas were collected from 5-month old mice with wild type (WT), HTZ, and NULL genotypes. Protein extract was prepared from ten corneas of each group by overnight extraction with 8 M guanidine-HCl, and subjected to Western blot analysis for evaluating expression level and carbohydrate chain length of KSPG. Two corneas of each group were processed for electron microscopy (EM) using cupromeronic blue, a dual fixative and proteoglycan stain. Images were collected from control and chondroitinase ABC-treated tissue.

Results: Western blot data indicated untreated NULL corneal extract had shorter KS-GAG than HTZ or WT untreated extracts. Normal lumican production and KS-sulfation pattern of HTZ and NULL corneas, relative to WT tissue, was also observed. Finally, EM images of WT and HTZ showed similar ultrastructure, whereas NULL corneas contained notably elongated PGs susceptible to chondroitinase ABC. KSPGs were unobservable in NULL tissue after enzymatic digestion.

Conclusions: Expectedly, the complete absence of β3GnT7 decreased the length of KS-GAG chains, suggesting this glycosyltransferase is essential for normal KS-GAG chain elongation. In addition, EM data demonstrated a possible compensatory mechanism to preserve corneal architecture via pronounced elongation of chondroitinase ABC-digestible PGs when sufficient KS-GAG synthesis is inhibited by defunct β3GnT7.

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