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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Rabbit MYOC Sequence. Nucleotide and deduced amino acid sequence of rabbit MYOC cDNA. Open reading frame nucleotides are shown in bold. Encoded amino acids are shown by single letter code and the stop codon is indicated with an asterisk. Numbers to the left indicate nucleotide position. The consensus polyadenylation signal (AATAAA) is underlined.

Figure 2

Alignment of myocilin orthologs. Alignment of rabbit, human, monkey, mouse, rat, and bovine myocilin amino acid sequences. Identical amino acids are shaded and missing amino acids are indicated with a dash. The 14 amino acid deletion in human MYOC plasmid pcDNA3.hMYOC.ΔN14 is underlined. The putative myocilin signal peptide is boxed. Location of the predicted N-linked glycosylation site (human myocilin Asn57) is indicated with a 'n'. Locations of predicted O-linked glycosylation sites are marked with an 'o'. The leucine zipper motif is marked by dots. Disease-causing mutations identified based on experimental data and statistical arguments with glaucoma family pedigrees and documented in the Human Gene Mutation Database are indicated with a 'x'. GenBank accession numbers are as follows: rabbit, AY191317; human, NM_000261; monkey, AY190128, AY190129, AY190130; mouse, AF039869; rat, AF093567; bovine, AB027758.

Figure 3

Rabbit MYOC expression. Western immunoblot analysis of myocilin in (A) rabbit, human, and monkey aqueous humor samples treated with (+) or without (-) glycosidases and in (B) GTM66 cells transfected with expression vectors encoding wild-type human MYOC (WT), N-terminal 14 (ΔN14) or 32 (ΔN32) aa deleted human MYOC, or N57S mutated human MYOC. I, intracellular myocilin present in the cell lysate; E, extracellular myocilin secreted into the medium. 57/55-kDa myocilin doublet is indicated with arrows.