|Molecular Vision 2004;
Received 12 December 2003 | Accepted 22 June 2004 | Published 27 June 2004
In vitro and in vivo characterization of disulfide bond use in myocilin complex formation
Michael P. Fautsch, Anne M. Vrabel,
Stefanie L. Peterson, Douglas H. Johnson
Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN
Correspondence to: Michael P. Fautsch, Ph.D., Department of Ophthalmology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905; Phone: (507) 284-1913; FAX: (507) 284-8566; email: email@example.com
Purpose: Myocilin forms large complexes in aqueous humor. Part of this complex formation is due to myocilin-myocilin protein non-covalent interactions within the leucine zipper. However, additional covalent interactions also exist. We investigated the role of these covalent interactions in disulfide bond formation within myocilin.
Methods: Human aqueous humor was separated by denatured/non-reduced SDS-PAGE followed by Western blot analysis with myocilin specific antibodies. In part two of the study, site-directed mutagenesis was used to selectively mutate one, two, three, four, and all five cysteine residues in the mature myocilin protein expressed in an in vitro system. Products were immunoprecipitated with a hemagglutinin polyclonal antibody following in vitro transcription/translation and analyzed by SDS-PAGE. In part three of the study, glaucoma associated myocilin mutations Arg82Cys and Cys433Arg were created and complex formation analyzed in trabecular meshwork cells.
Results: Human aqueous humor showed myocilin in several distinct large complexes in non-reduced SDS-PAGE gels, indicating disulfide bonds occur. Similarly, in vitro expressed myocilin also produced large complexes. Mutation of all five cysteines (within the mature myocilin protein) eliminated this large complex formation. A combination of cysteine to alanine substitutions at amino acids 185, 245, and 433 had the most influence on myocilin complex formation under non-reducing conditions, however individual substitutions at each of the five cysteine amino acids had little influence on myocilin complexes. In trabecular meshwork cells, Arg82Cys was secreted but formed different sized complexes than wild type myocilin. Cys433Arg was not secreted and remained intracellular in a pattern that differed from wild type myocilin and Arg82Cys.
Conclusions: Myocilin complexes present in human aqueous humor are in part due to disulfide bond formation between cysteine amino acids. Glaucoma associated mutations that affect the number of cysteine residues may alter covalent interactions.
Myocilin, also known as the trabecular meshwork inducible gluccocorticoid response gene (TIGR), is a glaucoma associated protein linked to some forms of juvenile and late onset primary open-angle glaucoma [1-11]. Myocilin is expressed in several ocular tissues including the trabecular meshwork [2,12-22]. Myocilin is found as both an intracellular and extracellular protein, and is present in aqueous humor of bovine, monkey, and normal, JOAG and POAG human samples [23-26]. Myocilin is a 53-57 kDa protein that exists in a multimeric structure, forming complexes in human aqueous humor of 120-180 kDa and in bovine and monkey aqueous humor of 200 kDa or greater [23,26]. It is unknown whether these complexes are solely composed of myocilin-myocilin interactions, or also involve myocilin and other proteins.
The primary sequence of myocilin contains two domains: an N-terminal domain containing a leucine zipper motif and a C-terminal domain that consists of homology to olfactomedins. Although the function of myocilin is unknown, it may play a role in aqueous outflow through interactions with other extracellular proteins. Optimedin, the Hep II domain of fibronectin, and other extracellular molecules are reported as myocilin binding partners [27-29]. The intracellular protein, myosin regulatory light chain, is reported to interact with a truncated myocilin molecule .
Myocilin can also interact with itself . Crosslinking studies using recombinant myocilin suggest it may form dimer or tetramer structures. Deletion analysis and site-directed mutagenesis of selected leucine amino acids have indicated that myocilin may form dimers through interactions within the N-terminal leucine zipper motif .
Although myocilin binds to itself through non-covalent interactions within the leucine zipper, additional covalent interactions within or between myocilin molecules have been suggested [31,32]. We analyzed myocilin in human aqueous humor and in cultured trabecular meshwork cells to determine whether disulfide bond formation occurred, which cysteine amino acids were involved, and what effect glaucoma associated mutations Arg82Cys  and Cys433Arg  have on myocilin complex formation.
Collection of aqueous humor
This study was approved by the Mayo Clinic Institutional Review Board, and conforms to the Declaration of Helsinki. Human aqueous humor was collected from four patients undergoing surgery. Three specimens were obtained during cataract surgery. No evidence of glaucoma or other ocular disease was present. The fourth sample was collected from a glaucoma patient during trabeculectomy surgery. The collection of aqueous humor has been previously described . Briefly, a paracentesis was made in the peripheral cornea and the tip of a 30 gauge cannula was inserted through the paracentesis tract into the mid-anterior chamber. A small volume of aqueous humor (50-100 μl) was slowly aspirated. Aspiration was stopped as soon as the anterior chamber began to shallow. The aqueous was transferred to a vial that was frozen in liquid nitrogen immediately upon removal from the anterior segment.
Western of aqueous humor samples
Equal volumes of aqueous humor (40 μl for reduced and 75 μl for non-reduced) were resuspended in 4X Laemmli sample buffer with or without β-mercaptoethanol (reducing agent), boiled, and electrophoresed. Non-reduced samples were separated on a 7% SDS-PAGE gel while reduced samples were separated on an 11% SDS-PAGE gel. Proteins were transferred to polyvinylidene diflouride (PVDF) membrane (Millipore, Bedford, MA) in 49.6 mM Tris, 384 mM glycine, 0.01% SDS, and 20% methanol. Western blot analysis was performed with an affinity purified anti-myocilin antibody and with anti-rabbit Ig conjugated to horseradish peroxidase as previously described . Antibody:antigen complex was detected using ECL Western blotting signal detection reagent (Amersham, Piscataway, NJ). Autoradiography was performed by exposing PVDF membranes to Kodak XAR film.
Plasmid construction of myocilin and cysteine mutants
The myocilin cDNA template used in this study was described in our previous work [18,34]. Alanine was used as an amino acid substitute for cysteine since it maintained a similar hydrophobicity, steric bulk and maintenance of the dihedral angle in the peptide bond, limiting the effect of the substitution on secondary structure. This amino acid substitution has been used in various studies of disulfide bond formation [35-39].
For in vitro studies, myocilin constructs containing cysteine to alanine substitutions were prepared using the technique of Splicing by Overlap Extension (SOE ). Figure 1 details the SOE procedure in the context of a specific cysteine to alanine substitution at amino acid 47 in the myocilin sequence. In general terms, the SOE reaction was used to incorporate base pair substitutions that changed a cysteine amino acid to alanine at amino acids 47, 61, 185, 245, and 433. Overlapping oligonucleotides (forward and reverse) were designed that incorporated base pair substitutions at these amino acids (Figure 2A). The reverse oligonucleotide was used with MYOC-5' to amplify a wild type myocilin template to create a 5' intermediate PCR fragment containing the substituted base pairs. The forward oligonucleotide was used with MYOC-3' to amplify a wild type myocilin template to create a 3' fragment containing the substituted base pairs. The sequence of the oligonucleotide pairs and the lengths of the intermediate 5' and 3' fragments for each substitution are described in Figure 2A. The intermediate 5' and 3' fragments for each individual substitution were mixed together, denatured and reannealed by base pair alignment of their overlapping sequence. This initial annealing step was extended using Taq polymerase (Invitrogen, Carlsbad, CA). A subsequent PCR reaction using oligonucleotides MYOC-5' and MYOC-3' was performed with this template creating wild type myocilin containing a specific cysteine to alanine codon change. This template was digested with restriction enzymes EcoRI and BamHI (restriction sites were incorporated into MYOC-5' and MYOC-3' oligonucleotides) and cloned into pGADT7-AD (BD Biosciences, Palo Alto, CA). Single cysteine to alanine amino acid (aa) substitutions (Figure 3B) were performed in this manner to produce the following constructs: C1 (aa 47), C2 (aa 61), C3 (aa 185), C4 (aa 245), and C5 (aa 433).
For incorporation of multiple cysteine to alanine substitutions, individual mutant templates (as described above) were used in subsequent SOE reactions to produce myocilin containing 2, 3, 4, or 5 cysteine to alanine substitutions. Myocilin construct substitutions (Figure 3B) were generated containing the following cysteine to alanine amino acid (aa) changes: C1/2 (aa 47, 61); C4/5 (aa 245, 433); C2/3/5 (aa 61, 185, 433); C2/4/5 (aa 61, 245, 433); C3/4/5 (aa 185, 245, 433); C2/3/4/5 (aa 61, 185, 245, 433); and C1/2/3/4/5 (aa 47, 61, 185, 245, 433).
All constructs used for in vitro studies will translate myocilin amino acids 32-504. The signal peptide sequence of myocilin (amino acids 1-32) was not included in these studies since in vivo it is cleaved off as the protein is translocated to the endoplasmic reticulum. Furthermore, the cloning into pGADT7 will express a fusion protein by placing an N-terminal tag on all myocilin constructs. The N-terminal tag contains a hemagglutinin epitope.
For in vivo studies, full length myocilin and myocilin mutations Arg82Cys, Cys433Arg, and substitutions of all 5 cysteines (C1/2/3/4/5FL; full length) were generated. Using full length myocilin as a template (contains nucleotide sequence that will code for amino acids 1-504), Arg82Cys and Cys433Arg were created by the SOE method using oligonucleotides described in Figure 2B. Construct C1/2/3/4/5FL was created by generating a 5' fragment from full length myocilin with oligonucleotides MYOC5-H3 and Cy2-3al. This 5' fragment was combined with the DNA template of C1/2/3/4/5 and SOEn together using oligonucleotides MYOC5-H3 (contains a HindIII restriction site) and MYOC-Stop (contains a XbaI restriction site). Wild type myocilin and myocilin mutations Arg82Cys, Cys433Arg, and C1/2/3/4/5FL were cloned into the HindIII and XbaI sites of pcDNA 4/V5-His (Invitrogen).
DNA sequence of all constructs used for in vitro and in vivo studies was verified using a Perkin Elmer/Applied Biosystems (Foster City, CA) DNA sequencer. Myocilin DNA sequence and amino acid numbers are based on GenBank accession number U85257.
In vitro disulfide bond studies
Site-directed mutagenesis constructs were transcribed and translated in vitro with the TnT®T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI). Briefly, 40 μl of TnT quick master mix (contains T7 RNA polymerase, nucleotides, salts, RNase inhibitor, and rabbit reticulocyte lysate) was combined with 30 μCi of 35S-methionine (Amersham), 0.5 μg of each individual plasmid construct (DNA prepared using Qiagen Endofree® plasmid maxi kit, Qiagen; Valencia, CA), and dH2O to a final volume of 50 μl. Reactions were incubated at 30 °C for 90 min and then placed on ice for 90 min. In duplicate experiments, canine microsomal membranes (Promega) were added 60 min into the reaction. Microsomal membranes were added to ensure an endoplasmic reticulum environment that is more conducive to disulfide bond formation. These reactions were placed back at 37 °C for an additional 30 min and then placed on ice for 90 min. Control sample included in vitro transcription/translation mix minus a DNA template.
Immunoprecipitation was performed using 40 μl of in vitro transcription/translation products (as described above) diluted in 700 μl of modified RIPA buffer (50 mM Tris pH 8.0, 1% Triton, 0.1% SDS, 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, l mM KH2PO4, and TM protease inhibitors [Roche Applied Science, Indianapolis, IN]). Samples were incubated overnight at 4 °C with a hemagglutinin polyclonal antibody (BD Biosciences). After incubation, protein A-sepharose was added to each sample and incubated at 4 °C for 60 min. Immunoprecipitated products were washed three times in modified RIPA buffer. Pellets were resuspended in 4X Laemmli sample buffer without a reducing agent, boiled, electrophoresed on a 7% SDS-PAGE gel and transferred to PVDF membrane. Remaining 10 μl of in vitro transcription/translation reaction was incubated in 4X Laemmli sample buffer containing β-mercaptoethanol, boiled, electrophoresed on a 11% SDS-PAGE gel and transferred to PVDF membrane. Autoradiography was performed by exposing PVDF membranes to Kodak XAR film.
In vivo disulfide bond studies
DNA (4 μg) representing myocilin and myocilin mutants Arg82Cys, Cys433Arg and C1/2/3/4/5FL was transfected into 750,000 TM5 cells (gift from Abe Clark, Ph.D., Alcon Labs, Fort Worth, TX). TM5 cells were chosen since they are a transformed trabecular meshwork cell line and can be easily transfected using Trans-It transfection reagent (Mirrus; Madison, WI) . TM5 cells do not induce myocilin when treated with dexamethasone although a functional signaling pathway for dexamethasone is present. Forty-eight hours following transfection, media and cells were isolated. Media was centrifuged at 13,000x g for 5 min to remove any cellular debris. Supernate was collected. Cell lysates from transfected TM5 cells were made with Cell Lysis buffer (50 mM Tris pH 8.0, 0.5% Triton X-100, 0.5% sodium dodecyl sulfate, 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, l mM KH2PO4, and TM protease inhibitors [Roche Applied Science]). Protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad; Hercules, CA). Equal volumes of media (30 μl) and equal protein amounts (15 μg) were separated on either a 7.5% denatured, non-reduced SDS-PAGE gel or a 4-15% denatured, reduced SDS-PAGE gradient gel. Gels were transferred to PVDF membrane and probed with anti-myocilin antibody.
Analysis of myocilin sequence
Myocilin contains 9 cysteine amino acids in its primary sequence (Figure 3A). The first four of these (amino acids 5, 8, 9, and 25) are found in the signal peptide sequence. Because the signal peptide of secretory proteins is cleaved off as it enters the endoplasmic reticulum, myocilin only contains five cysteine amino acids in the secreted mature molecule. Cysteines at amino acids 47 and 61 are present on the N-terminal side of myocilin's leucine zipper motif while amino acids 185 and 245 are C-terminal. Only the cysteine at amino acid 433 is present in the olfactomedin homology domain.
Myocilin forms disulfide bonds in aqueous humor
Western analysis of human aqueous humor samples following separation on denatured, non-reduced SDS-PAGE showed three myocilin bands that ran larger than the 177 kDa marker (Figure 4A). This was in contrast to myocilin that was separated on denatured, reduced gels (Figure 4B) which showed a myocilin doublet at 53-57 kDa. In addition, the glaucomatous sample showed additional bands at approximately 65 kDa and 80 kDa. These additional bands appear to be specific although they are not present in the other two samples. The difference in electrophoretic mobility of myocilin when compared between the reduced and non-reduced conditions suggested that disulfide bond formation occurs in aqueous humor.
Analysis of myocilin containing cysteine mutations
The overall effect of disulfide bonds on myocilin complex formation was tested by substitution of all 5 cysteines with alanine (construct C1/2/3/4/5; Figure 3B). Wild type myocilin and C1/2/3/4/5 were transcribed and translated in vitro with and without canine microsomal membranes. Following immunoprecipitation, samples were separated on a non-reduced, denatured SDS-PAGE gel and analyzed for changes in electrophoretic mobility. Myocilin and C1/2/3/4/5 had very different migration patterns (Figure 5A). Myocilin has slower electrophoretic mobility indicating complex formation larger than 177 kDa (compared to marker). In contrast, C1/2/3/4/5 had a faster electrophoretic mobility resulting in a doublet at approximately 55 kDa. This is a similar size to that seen for non-immunoprecipitated myocilin or C1/2/3/4/5 when analyzed on denatured, reduced SDS-PAGE (Figure 5B). The presence of canine microsomal membranes in these reactions did not change the complex formation or migration of the complexes (data not shown). This indicates that loss of the five cysteines in the mature myocilin protein prevents complex formation, suggesting that covalent interactions between cysteine amino acids take place. Immunoprecipitation using an anti-myocilin antibody verified these complexes as myocilin (data not shown).
Single substitutions of alanine for cysteine were tested next. The cysteine at amino acid 185 (C3) had the greatest effect while substitution at amino acid 47 (C1), 61 (C2), 245 (C4) and 433 (C5) had little effect (Figure 6A). The C3 substitution resulted in a change in migration that differed from any of the other single cysteine substitutions. Although the majority of the protein was still found in complexes above the 177 kDa marker, several bands were present that migrated between 50-55 kDa, which is near the monomeric size of myocilin (Figure 6). This was seen when the in vitro transcription/translation was performed with or without microsomal membranes.
Multiple substitutions of the cysteines revealed the most significant complex changes occur when C3 is substituted in conjunction with the other cysteines. For example, the C3/4/5 construct resulted in several complexes with the most abundant forming at approximately 55 kDa and 100 kDa. A larger band is still present above the 177 kDa marker although this is smaller than the main wild type myocilin complex. Addition of a fourth substitution at cysteine 2 (C2/3/4/5) did not significantly change the banding pattern when compared to C3/4/5.
C4 in conjunction with C3 was also important in myocilin complexes. Substitution of cysteine 2, cysteine 3, and cysteine 5 (C2/3/5) did not alter the electrophoretic mobility of myocilin complexes significantly. However, when cysteine 4 is altered in addition to cysteines 2, 3, and 5 (C2/3/4/5), significant migration changes occur. Other combinations had little effect (C1/2, C4/5, and C2/4/5).
Glaucoma associated myocilin mutants Arg82Cys and Cys433Arg
To determine the effect of cysteine mutations in a physiologic state, we analyzed two known glaucoma associated cysteine mutations in transformed trabecular meshwork cells (TM5). Both Arg82Cys and Cys 433Arg were successfully expressed by the TM5 cells and appeared in the cell lysate (Figure 5A). Of interest, Arg82Cys was secreted by the cells, but Cys433Arg was not secreted. In the medium, myocilin with the Arg82Cys mutation formed a smaller complex than wild type myocilin when compared on denatured, non-reduced SDS-PAGE gels (Figure 7B).
Analysis of myocilin, Arg82Cys, and Cys433Arg in trabecular cell lysate separated on denatured, non-reduced SDS-PAGE gels showed that all constructs were present and capable of complex formation. Differences in the electrophoretic patterns can be seen when comparing migration patterns between these molecules (Figure 7B; see asterisks). C1/2/3/4/5FL, which was only seen in the cell lysate, migrated in a similar fashion on denatured, non-reduced gels as that seen for monomeric myocilin when analyzed on denatured, reduced gels (Figure 7A).
In eukaryotes, most secretory proteins contain disulfide bonds to maintain structure/function requirements. Our results support previous findings that myocilin forms complexes in human aqueous humor [23,26]. In addition to non-covalent interactions with the leucine zipper, we now report that myocilin can form complexes in human aqueous humor through covalent interactions involving disulfide bonds. Our results also suggest that all five of the cysteines present in the mature myocilin protein may have a role in disulfide bond formation. Our analysis does not differentiate between intramolecular or intermolecular disulfide bond interactions.
Alterations in the C-terminal cysteines have the most influence on myocilin complexes. Our data support this interpretation since construct C3/4/5, which contains cysteine to alanine substitutions at amino acids 185, 245, and 433 significantly altered complex formation. In addition, glaucoma associated mutant Cys433Arg was not secreted from the TM5 cells, while the addition of a cysteine for arginine at amino acid 82 (Arg82Cys) was secreted. Others have reported that mutations in the C-terminal domain can alter the ability of myocilin to be secreted [25,42-45]. Furthermore, transfection of several myocilin mutants in TM5 cells resulted in the inability of these products to be secreted and the formation of intracellular complexes . The mechanism by which this occurs is unknown. Our analysis of the cell lysate containing the Cys433Arg mutation showed several differences in the migration pattern when compared to wild type myocilin (Figure 7B). Alterations in disulfide bond formation may change the structure of the protein in a way that the proteasome will recognize it as mis-folded. Caballero et al have shown that expression of a truncated myocilin protein (which is not secreted) can influence secretion of wild type myocilin . Further analysis of Cys433Arg is warranted to determine the exact mechanism of its altered secretion properties.
Although the combination of cysteine to alanine substitutions at positions 185, 245, and 433 had the most influence on myocilin complex formation in vitro, substitution of all three amino acids with alanine did not reduce myocilin down to its monomeric form (compare C3/4/5 to C1/2/3/4/5 in Figure 6A). Hence, cysteine 47 and 61 are also involved in disulfide bond formation.
Several myocilin mutations that increase the number of cysteine amino acids have been reported . One of these mutations, Arg82Cys, is secreted but forms smaller complexes than wild type myocilin in vivo (Figure 7). The finding that an N-terminal mutation can be secreted further supports the importance of the C-terminal region and the maintenance of its structure, partly provided by the presence of disulfide bonds.
Disulfide bonds function to stabilize the structure of proteins. These covalent bonds may occur between cysteine amino acids within a protein or may occur between homologous or heterologous proteins. Our results do not decipher between intramolecular or intermolecular disulfide bond formation. Although complexes migrate at certain positions within a non-reduced gel, their size is arbitrary to the location of the markers since they may be forming non-linear structures. Determination of molecular weights of proteins separated on SDS-PAGE gels depends on the proteins being in a linear form. Since our in vivo and in vitro experiments are run under denatured, non-reduced conditions, the size of the myocilin complexes may be due to intramolecular disulfide bonds resulting in a non-linear form of myocilin that happens to migrate slowly through an SDS-PAGE gel. Alternatively, myocilin may form disulfide bonds with itself or other proteins resulting in a multimeric complex that migrates slowly through the SDS-PAGE gel because of not only shape but because this complex has a higher molecular weight.
Previously, we reported that myocilin forms complexes with itself through interactions between its leucine zipper motifs . Our current study shows that in addition to these non-covalent interactions, cysteine residues are involved in disulfide bond formation. Recently, protein disulfide isomerase was shown to aggregate with wild type myocilin in cultured trabecular meshwork cells . Protein disulfide isomerase catalyzes the rearrangement of disulfide bonds, accelerating the folding of newly synthesized secretory and membrane proteins in the endoplasmic reticulum. We hypothesize that non-covalent binding of the leucine zipper motifs between two myocilin molecules enables the cysteine molecules to come in close proximity to each other, resulting in disulfide bond formation that is potentially catalyzed by protein disulfide isomerase. This results in a conformation that is suitable for myocilin structure and function.
Supported in part by National Institutes of Health research grant EY 07065, Research to Prevent Blindness, Inc., New York, NY and Mayo Foundation, Rochester, MN.
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