title: Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.
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.
authors.affiliation: Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Samsom
authors.affiliation: Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada.
authors.forename: Sheila
authors.initials: S
authors.lastname: Morrison
authors.affiliation: Schulich School of Engineering - Centre for Bioengineering Research & Education, University of Calgary, Calgary, AB, Canada.
authors.forename: Nemanja
authors.initials: N
authors.lastname: Masala
authors.affiliation: TearLab Research Inc, San Diego, CA, USA.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Sullivan
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear, USA; Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Sullivan
authors.affiliation: Chemical Engineering, McMaster University, Hamilton, ON, Canada.
authors.forename: Heather
authors.initials: H
authors.lastname: Sheardown
authors.affiliation: 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.
authors.forename: Tannin A
authors.initials: TA
authors.lastname: Schmidt
mesh_terms: D000368:Aged; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003315:Cornea / Q000187:drug effects*; D003412:Cricetulus; D004129:Dimethylpolysiloxanes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005143:Eyelids / Q000187:drug effects*; D017276:Friction; D006031:Glycosylation; D006801:Humans; D008153:Lubrication; D008970:Molecular Weight; D009883:Ophthalmic Solutions / Q000494:pharmacology*; D011509:Proteoglycans / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D013312:Stress, Physiological / Q000502:physiology; D013499:Surface Properties; D053719:Tandem Mass Spectrometry; D000099158:Proteoglycan 4
chemical_list: D004129:Dimethylpolysiloxanes; D009883:Ophthalmic Solutions; D011509:Proteoglycans; D011994:Recombinant Proteins; C419197:PRG4 protein, human; D000099158:Proteoglycan 4; C013830:baysilon
keywords: lubrication; lubricin; ocular surface; rhPRG4