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Exp Eye Res. 2014 Oct;127:14-9. doi: 10.1016/j.exer.2014.06.015. Epub 2014 Jul 2.

Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

Author information

1
Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada.
2
Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada.
3
Schulich School of Engineering - Centre for Bioengineering Research & Education, University of Calgary, Calgary, AB, Canada.
4
TearLab Research Inc, San Diego, CA, USA.
5
Schepens Eye Research Institute, Massachusetts Eye and Ear, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
6
Chemical Engineering, McMaster University, Hamilton, ON, Canada.
7
Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada; Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada; Schulich School of Engineering - Centre for Bioengineering Research & Education, University of Calgary, Calgary, AB, Canada. Electronic address: tschmidt@ucalgary.ca.

Abstract

Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 μg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear.

KEYWORDS:

lubrication; lubricin; ocular surface; rhPRG4

PMID:
24997456
DOI:
10.1016/j.exer.2014.06.015
[Indexed for MEDLINE]

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