Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage

Allan R Shepard; Nasreen Jacobson; Ruifang Sui; H Thomas Steely; Andrew J Lotery; Edwin M Stone; Abbot F Clark

doi:10.1186/1471-2156-4-5

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Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage

Journal article

Open access

Peer reviewed

Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage

Allan R Shepard, Nasreen Jacobson, Ruifang Sui, H Thomas Steely, Andrew J Lotery, Edwin M Stone and Abbot F Clark

BMC genetics, Vol.4(1), pp.5-5

04/02/2003

DOI: 10.1186/1471-2156-4-5

PMCID: PMC156599

PMID: 12697062

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Published (Version of record) Open Access

Abstract

Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. In addition, myocilin has been associated with glucocorticoid-induced ocular hypertension and steroid-induced glaucoma. To better understand the role myocilin plays in steroid-induced glaucoma and open-angle glaucoma, we examined rabbit myocilin for use in the rabbit animal model of steroid-induced glaucoma. We have cloned the rabbit ortholog of human MYOC. Rabbit MYOC consists of three exons and an open reading frame encoding a 490 amino acid, 54,882-Da protein, which is 14 amino acids shorter at the N-terminus than human myocilin but 84% identical overall. Rabbit myocilin migrates as a single electrophoretic band, vs. double-banded human myocilin, by SDS-PAGE/immunoblot analysis. We determined that the differential migration exhibited is due to an N-glycosylation site that is present in human (Asn57), monkey and mouse myocilin but absent in rabbit (Ser43), rat and bovine myocilin. Rabbit myocilin is secreted in vitro in trabecular meshwork cell culture and in vivo in aqueous humor. Secretion of human myocilin is shown to be dependent on the signal peptide and independent of the extra 14 amino acids not found in rabbit myocilin. Many of the amino acids in myocilin that are mutated in glaucoma patients are conserved across species. We have cloned the rabbit MYOC cDNA and determined that rabbit myocilin is secreted but not N-linked glycosylated. Knowledge of the rabbit MYOC cDNA sequence will facilitate future studies in the rabbit animal model examining the role of myocilin in steroid-induced glaucoma and the gain-of-function hypothesis in open-angle glaucoma.

Cytoskeletal Proteins

Aqueous Humor - secretion

Humans

DNA, Complementary - genetics

Glycoproteins - metabolism

Molecular Sequence Data

Immunoblotting

Cattle

Base Sequence

Eye Proteins - genetics

Trabecular Meshwork - cytology

Glycoproteins - genetics

Amino Acid Sequence

Rabbits

Electrophoresis, Polyacrylamide Gel

Cells, Cultured

Rats

Glycosylation

Sequence Analysis, DNA

Haplorhini

Sequence Homology, Amino Acid

Sequence Alignment

Animals

Eye Proteins - metabolism

Protein Sorting Signals - genetics

Trabecular Meshwork - metabolism

Mice

DNA, Complementary - chemistry

Mutation

Details

Title: Subtitle: Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptide usage
Creators: Allan R Shepard - Alcon Research, Ltd, 6201 South Freeway, Fort Worth, TX 76134, USA. allan.shepard@alconlabs.com
Nasreen Jacobson
Ruifang Sui
H Thomas Steely
Andrew J Lotery
Edwin M Stone
Abbot F Clark
Resource Type: Journal article
Publication Details: BMC genetics, Vol.4(1), pp.5-5
DOI: 10.1186/1471-2156-4-5
PMID: 12697062
PMCID: PMC156599
NLM abbreviation: BMC Genet
ISSN: 1471-2156
eISSN: 1471-2156
Publisher: England
Grant note: R01-EY-10564 / NEI NIH HHS R01 EY010564 / NEI NIH HHS
Language: English
Date published: 04/02/2003
Academic Unit: Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9983980010402771

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