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

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

doi:10.1186/1471-2156-4-5

Abstract

BackgroundMutations 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.ResultsWe 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.ConclusionWe 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.

Highlights

Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma
We show that an N-terminal signal peptide is necessary for myocilin secretion and we identify the Nlinked glycosylation site found in human but not rabbit myocilin
As in all myocilin orthologs cloned to date, rabbit myocilin contains a leucine zipper motif (Fig. 2)

Summary

Introduction

Mutations in the gene encoding human myocilin (MYOC) have been shown to cause juvenile- and adult-onset glaucoma. Several groups have shown that myocilin is glycosylated [10,16,17,18,19,20,21] whereas one group suggests that myocilin is not glycosylated [22] In our hands, both an N-linked glycosylated (≈57-kDa) and unglycosylated (≈55-kDa) form of myocilin is detected in TM cell culture media [17] and aqueous humor [12]. Both an N-linked glycosylated (≈57-kDa) and unglycosylated (≈55-kDa) form of myocilin is detected in TM cell culture media [17] and aqueous humor [12] The basis for both secretion and N-linked glycosylation of myocilin is part of the focus of this paper

Methods

Results

Discussion

Conclusion