We have cloned a 2-kb cDNA for rabbit MYOC that encodes a 490 amino acid, 55-kDa protein with a calculated pI of 5.25 (Fig. 1). Rabbit myocilin amino acid sequence is 84% identical to human myocilin 28 and highly homologous across species (Table 2) with an overall 64% homology. Rabbit myocilin is 14 amino acids shorter at the N-terminus than human myocilin and the same length as monkey 15, mouse 2930, and bovine 31 myocilin (Fig. 2). As in all myocilin orthologs cloned to date, rabbit myocilin contains a leucine zipper motif (aa 103 to 152) (Fig. 2). A potential peroxisomal targeting signal is encoded on the C-terminus (SKI, aa 488–490). Unlike human, monkey and mouse myoclin, rabbit myocilin does not contain a consensus N-linked glycosylation site (N-X-S/T; human, aa 57–59; monkey, aa 43–45; mouse, aa 43–45) (Fig. 2). The nucleotide sequence for rabbit MYOC reported in this manuscript has been submitted to GenBank (AY191317).

Rabbit, monkey, mouse and bovine myocilin open reading frames begin at amino acid 15 relative to human myocilin. The function, if any, of the extra N-terminal 14 aa encoded by human myocilin is not presently known. Rat myocilin also contains an extra 13 aa N-terminal sequence 32. To determine if rabbit myocilin is secreted despite the absence of the additional human-like N-terminal 14 aa, we examined rabbit aqueous humor. We were able to detect myocilin in rabbit aqueous humor (Fig. 3A). This suggests that the human N-terminal 14 aa is dispensible for secretion and that the rabbit myocilin sequence is sufficient to direct secretion.

Next we created a human myocilin expression construct where the N-terminal 14aa were deleted (pcDNA3.hMYOC.ΔN14) forcing translation to begin at the second methionine codon (Fig. 2). GTM66 cells were chosen for this study based on their ability to grow and transfect well and are a glaucomatous TM cell line derived from a 92-year-old female donor 1117. Transfection of GTM66 cells with the N-terminal deletion mutant resulted in a distribution pattern similar to wild-type human myocilin (Fig. 3B). The fact that we see no discernable difference in the relative mobility between transfected human wild-type myocilin and 14 amino acid amino-terminal deleted myocilin (Fig. 3B) and human vs. monkey myocilin in AH (Fig. 3A) may be due to the use of small format gels or possibly to the usage of the second N-terminal methionine by the cell for synthesizing human myocilin, making human and monkey molecular weights comparable.

Nonetheless, an N-terminal signal peptide is likely necessary for directing synthesis of myocilin, via the ER-Golgi pathway, for secretion outside the cell. To determine what actually constitutes the signal sequence for myocilin, we conducted an analysis of signal peptide cleavage sites using the Center for Biological Sequence Analysis SignalP v1.1 Database http://www.cbs.dtu.dk/services/SignalP/. A signal peptide cleavage site was predicted to exist between rabbit aa 18 and 19 (human aa 32 and 33) 33. We directly tested the importance of the signal peptide in myocilin secretion by replacing the first 32 aa of human myocilin with a single methionine (pcDNA3.hMYOC.ΔN32). Transfection of GTM66 cells with pcDNA3.hMYOC.ΔN32 resulted in intracellular but not extracellular myocilin distribution (Fig. 3B). This is the result expected if the signal peptide were necessary for secretion. This suggests that the first 18 aa of rabbit myocilin may function as a signal peptide and are necessary for myocilin secretion.

Human myocilin has been shown to migrate as a 55/57-kDa doublet by SDS-PAGE/immunoblot analysis 1011121617. The modification accounting for this molecular weight difference is hypothesized to be due to differential glycosylation. N-linked glycosylation sites were predicted using the Center for Biological Sequence Analysis NetNGlyc 1.0 Prediction Server http://www.cbs.dtu.dk/services/NetNGlyc/. Human, monkey and mouse myocilin contain a consensus asparagine (N)-linked glycosylation site (NES) near the N-terminus, whereas rabbit, rat and bovine code for SES at this position (Fig. 2). Having a serine present at this position would eliminate N-linked glycosylation and would be predicted to result in a difference in relative gel mobility between human/monkey and rabbit myocilin.

To test this hypothesis we analyzed human, monkey, and rabbit AH myocilin side-by-side in SDS-PAGE/immunoblots. The anti-human myocilin antibody 129 used in these studies (generated to human amino acids 156–1711217) cross-reacts with rabbit and monkey myocilin. With human and monkey myocilin we see a clear doublet, whereas rabbit myocilin migrates as a single band with the same relative mobility as the lower band of human and monkey myocilin (Fig. 3A), suggesting that the asparagine vs. serine amino acid difference is responsible for the differential glycosylation.

To directly test this theory, we mutated human myocilin amino acid 57 from Asn to Ser, as found in rabbit myocilin. Media and cell lysate from transfected GTM66 cells contained a single immunoreactive myocilin band that migrated with the same relative mobility as the lower wt myocilin band (Fig. 3B). This suggests that the 55/57-kDa human myocilin doublet is due to differential glycosylation and that the site of N-linked glycosylation on human myocilin is Asn 57.

To determine the effects of deglycosylation on myocilin, we treated human, monkey, and rabbit AH with a cocktail of glycosidases (N- and O-linked). Our results show that the upper band of human and monkey myocilin is eliminated whereas the lower band remains unaltered (Fig. 3A). This indicates that the upper human and monkey myocilin band is glycosylated. In addition, the single rabbit myocilin band shifted downward slightly with deglycosylation. This may be due to removal of O-linked glycosylation.

Total RNA was extracted from rabbit eye and heart tissue (Pel Freez) using Trizol (Invitrogen). Primers derived from conserved regions of human and mouse MYOC (Table 1, primers Exon1F/Exon1R) were used to amplify a 250-bp section of rabbit MYOC exon 1 (Fig. 1, nts 184–433) by PCR as described 15. 5' and 3' RACE (Invitrogen) was used to obtain the 5' and 3' cDNA ends. Essentially, two micrograms rabbit total RNA was converted into first strand cDNA with adapter primer and Superscript II reverse transcriptase. Primary and nested 5' and 3' RACE primers were all designed within the 250-bp region of rabbit MYOC exon 1 (Table 1). PCR products were subcloned into TOPO™ TA cloning vectors (Invitrogen) and subjected to cycle sequencing (Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center).

Due to difficulties in amplifying the rabbit MYOC coding region from cDNA, we pieced together the entire contiguous open-reading frame using rabbit genomic DNA (Clontech; New Zealand White) as a template with a multi-step PCR/ligation process. In the first step, exons 1 and 2 were amplified from genomic DNA with primers P357F/P368R to exon 1 and P355F/P365R to exon 2. Primers P368R and P355F directly abut each other at the exon 1/2 boundary. PCR amplification was performed in a 50 ul volume with the PfuTurbo Hotstart DNA Polymerase system (Stratagene) and included 0.2 mM dNTPs, 100 ng each primer, 2.5 units PfuTurbo DNA polymerase, 100 ng genomic DNA, and 1X cloned Pfu DNA polymerase reaction buffer. Cycling conditions consisted of 2 min at 95°C followed by 15 cycles at 95°C 30 sec, 60°C 30 sec, and 72°C 1 min in a Perkin Elmer 9700 thermalcycler. PCR products were restriction digested with BamH1 (exon 1) or Not1 (exon 2), purified from an agarose gel by Qiaquick extraction (Qiagen), and simultaneously subcloned into a BamH1/Not1 digested pcDNA3 (Invitrogen) vector to create a directional clone of rabbit MYOC exons 1 and 2 spliced together (pcDNA3.RbMYOCex1–2). Sequence integrity of the cloned DNA was verified by cycle sequencing and restriction digestion.

Exons 1–2 of plasmid pcDNA3.RbMYOCex1–2 was then amplified with primers P357F/P370R and exon 3 was amplified from genomic DNA with primers P356F/P356R. Primers P370R and P356F directly abut each other at the exon 2/3 boundary. PCR was performed as above and PCR products digested with BamH1 (exons 1–2) or Not1 (exon 3) and processed/cloned as above to create a directional clone of rabbit MYOC exons 1–2 and 3 spliced together (pcDNA3.RbMYOCex1–3). Plasmid DNA integrity was confirmed by restriction analysis and cycle sequencing.

Alignment of DNA sequence contigs, protein sequences, and plasmid DNA sequence/construction was performed with Vector NTI 7.1 software (Informax).

Human MYOC cDNA was cloned into the pcDNA3 (Invitrogen) mammalian expression vector as described 12. Human myocilin signal sequence deletions were generated by PCR. A 5' PCR primer, with a 5' HindIII site added, was designed to eliminate either the first 42- (P235; ΔN14) or 96- (P236; ΔN32) nucleotides of the human MYOC ORF and used in conjunction with a 3' PCR primer (P237) covering the internal Bsu36I restriction site (Table 1). Primer P236 replaced the first 32 amino acid codons with a methionine codon. PCR and processing were as described above with the exception that the 5' human MYOC deletion products were cloned into a HindIII/Bsu36I digested pcDNA3.hMYOC wild-type vector.

Substitution of human myocilin Asn 57 with Ser was accomplished using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primer pair P316F/P316R was designed to replace the Asn57 A

A

G

GTM66 cells were derived from a 92-year old woman with glaucoma and were cultured in low glucose DMEM (HyClone Laboratories) with 10% FBS (HyClone Laboratories) and antibiotics (Invitrogen) as described 1117. Cells were transfected with Lipofectamine 2000 reagent according to the manufacturers recommendations (Invitrogen). Transfected plasmid DNA included wild-type human MYOC (pcDNA3.hMYOC; 12), wild-type rabbit MYOC (pcDNA3.RbMYOC.ex1–3), N-terminal 14 amino acid deleted human MYOC (pcDNA3.hMYOC.ΔN14), N-terminal 32 amino acid deleted human MYOC (pcDNA3.hMYOC.ΔN32), or N57S human MYOC (pcDNA3.hMYOC.N57S). Cells were switched to serum-free media 24 h post-transfection and media or cell lysates were collected after overnight incubation. Equivalent cell lysate protein amounts (5 μg) were loaded for reduced SDS-PAGE analysis (Fig. 3B). Cell media was not concentrated and was loaded at 16 ul per lane in Fig. 3B.

All experimental animals used in this study were treated in compliance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in research. Rabbit aqueous humor samples (135–190 μl) were taken from anesthetized New Zealand Albino (NZA) rabbits and mixed with protease inhibitor cocktail (Complete; Roche) to prevent myocilin degradation. Human aqueous humor samples were obtained as previously described 12. The equivalent of 4 μl AH was used per lane in Fig. 3A.

Cells were rinsed with PBS and solubilized in a commercial mammalian extraction buffer (M-Per™; Pierce, Rockford, IL) supplemented with a protease inhibitor cocktail (Complete; Roche) followed by centrifugation at 12,000 × g for 5 min. Protein concentration of the supernatant was determined with Coomassie Plus Protein Assay Reagent (Pierce). Cell extracts were stored at -20°C.

Aqueous humor, cell media, and cell extracts were analyzed using pre-cast Criterion 7.5% Tris-HCl polyacrylamide gels (Bio-Rad) and the Bio-Rad electrophoresis system (Bio-Rad). Proteins were electroblotted to Hybond-P PVDF membranes (Amersham Pharmacia Biotech), blocked with gelatin, and probed with affinity purified rabbit anti-human myocilin antibody 129 (AB129; generated to human myocilin amino acids 156–171) 1217 and an anti-rabbit IgG secondary antibody (Amersham). Immunoreactivity was detected with the ECL Plus detection system (Amersham). Blots were exposed to BioMax MR film (Eastman Kodak) and scanned with a HP ScanJet 7400 c scanner (Hewlett Packard).

Aqueous humor from human, monkey and rabbit was subjected to deglycosylation with a mixture of N-linked and O-linked glycosidases (PNGase F, sialidase, β-galactosidase, glucosaminidase and O-glycosidase) using the Enzymatic CarboRelease Kit according to the manufacturers instructions (QA-Bio). AH samples in buffer were boiled for 5 min in the presence of denaturant followed by incubation for 3 hr at 37°C with the glycosidase mixture. NuPAGE sample buffer (Invitrogen) was added to stop the reaction, samples were heated for 10 min at 70°C and subjected to NuPAGE electrophoresis on 10% NuPAGE gels (Invitrogen). Samples were transferred to PVDF, immunoblotted versus AB129, and developed using ECL Plus.