Limits...
Olfactory marker protein expression is an indicator of olfactory receptor-associated events in non-olfactory tissues.

Kang N, Kim H, Jae Y, Lee N, Ku CR, Margolis F, Lee EJ, Bahk YY, Kim MS, Koo J - PLoS ONE (2015)

Bottom Line: The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events.Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues.The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain Science, DGIST, Daegu, Korea.

ABSTRACT
Olfactory receptor (OR)-associated events are mediated by well-conserved components in the olfactory epithelium, including olfactory G-protein (Golf), adenylate cyclase III (ACIII), and olfactory marker protein (OMP). The expression of ORs has recently been observed in non-olfactory tissues where they are involved in monitoring extracellular chemical cues. The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events. In addition, expression profiles and physiological functions of ORs in non-olfactory tissues are largely unknown. To overcome limitations associated with using OR as a target protein, this study used OMP with Golf and ACIII as targets to screen for potential OR-mediated sensing systems in non-olfactory tissues. Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues. The results of immunohistochemical analyses showed OMP (+) cells in mouse heart and in the following cells using the corresponding marker proteins c-kit, keratin 14, calcitonin, and GFAP in mouse tissues: interstitial cells of Cajal of the bladder, medullary thymic epithelial cells of the thymus, parafollicular cells of the thyroid, and Leydig cells of the testis. The expression of ORs in OMP (+) tissues was analyzed using a refined microarray analysis and validated with RT-PCR and real-time PCR. Three ORs (olfr544, olfr558, and olfr1386) were expressed in the OMP (+) cells of the bladder and thyroid as shown using a co-immunostaining method. Together, these results suggest that OMP is involved in the OR-mediated signal transduction cascade with olfactory canonical signaling components between the nervous and endocrine systems. The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.

Show MeSH

Related in: MedlinePlus

Protein expression profiles of OMP in various mouse tissues.Double immunoassay for olfactory marker protein (OMP). (A) In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. (B) Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4311928&req=5

pone.0116097.g001: Protein expression profiles of OMP in various mouse tissues.Double immunoassay for olfactory marker protein (OMP). (A) In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. (B) Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.

Mentions: We next performed immunological experiments using a double immunoassay technique to support the results of the mRNA studies and determine whether the OMP is expressed at the protein level. As expected, we could not detect the OMP expression with conventional western blot analysis using specific antibodies against the OMP because of the low level of the OMP expression (data not shown). To overcome this challenge, we first tested the specificity of our double immunoassay system. As shown in Fig. 1A, skeletal muscle and thymus except for kidney expressed the OMP [Fig. 1A, OMP (-)]. For this assay, OMP was immunoprecipitated with an anti-OMP antibody from goat conjugated with Protein G-Sepharose using 20 mg of total tissue lysates obtained from each tissue. The isolated immunocomplexes were subjected to western blot analysis with an anti-OMP polyclonal antibody from rabbit. For the positive control, we used the same double immunoassay with 100 μg of tissue lysate obtained from the OE (Fig. 1A, lane OE). The 19 kDa immunoreactive protein band did not appear when tissue was preincubated with the purified recombinant OMP used as a blocking reagent, indicating the specific detection of the OMP in our system [Fig. 1A, OMP (+)]. As shown in Fig. 1B, OMP protein was widely expressed in all but one tested tissue. Skeletal muscle, heart, thymus, and thyroid showed high OMP expression, whereas liver, bladder, pancreas, stomach, duodenum, testis, spleen, and lung were at relatively lower levels. OMP expression was undetectable in the kidney. The endogenous OMP was immunoprecipitated from 20 mg of total tissue lysate obtained from various tissues and from 3 mg of total cell lysate obtained from the thyroid. This OMP protein expression pattern differs from the OMP transcript pattern, in which OMP mRNA is predominantly expressed in the bladder and stomach. In addition, although the OMP protein expression level is low, it was detectable by a double immunoassay, resulting in different expression pattern at various tissues. There are two plausible explanations for low and differential expression of the OMP. The first is that OMP may be broadly expressed at low levels throughout the entire tissue. The second is that OMP may be locally expressed either in a small portion of the tissue or within a specific cell type of each tissue. Nevertheless, OMP expression is only detectable with high resolution methods using whole tissue lysates rather than by conventional methods.


Olfactory marker protein expression is an indicator of olfactory receptor-associated events in non-olfactory tissues.

Kang N, Kim H, Jae Y, Lee N, Ku CR, Margolis F, Lee EJ, Bahk YY, Kim MS, Koo J - PLoS ONE (2015)

Protein expression profiles of OMP in various mouse tissues.Double immunoassay for olfactory marker protein (OMP). (A) In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. (B) Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4311928&req=5

pone.0116097.g001: Protein expression profiles of OMP in various mouse tissues.Double immunoassay for olfactory marker protein (OMP). (A) In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. (B) Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.
Mentions: We next performed immunological experiments using a double immunoassay technique to support the results of the mRNA studies and determine whether the OMP is expressed at the protein level. As expected, we could not detect the OMP expression with conventional western blot analysis using specific antibodies against the OMP because of the low level of the OMP expression (data not shown). To overcome this challenge, we first tested the specificity of our double immunoassay system. As shown in Fig. 1A, skeletal muscle and thymus except for kidney expressed the OMP [Fig. 1A, OMP (-)]. For this assay, OMP was immunoprecipitated with an anti-OMP antibody from goat conjugated with Protein G-Sepharose using 20 mg of total tissue lysates obtained from each tissue. The isolated immunocomplexes were subjected to western blot analysis with an anti-OMP polyclonal antibody from rabbit. For the positive control, we used the same double immunoassay with 100 μg of tissue lysate obtained from the OE (Fig. 1A, lane OE). The 19 kDa immunoreactive protein band did not appear when tissue was preincubated with the purified recombinant OMP used as a blocking reagent, indicating the specific detection of the OMP in our system [Fig. 1A, OMP (+)]. As shown in Fig. 1B, OMP protein was widely expressed in all but one tested tissue. Skeletal muscle, heart, thymus, and thyroid showed high OMP expression, whereas liver, bladder, pancreas, stomach, duodenum, testis, spleen, and lung were at relatively lower levels. OMP expression was undetectable in the kidney. The endogenous OMP was immunoprecipitated from 20 mg of total tissue lysate obtained from various tissues and from 3 mg of total cell lysate obtained from the thyroid. This OMP protein expression pattern differs from the OMP transcript pattern, in which OMP mRNA is predominantly expressed in the bladder and stomach. In addition, although the OMP protein expression level is low, it was detectable by a double immunoassay, resulting in different expression pattern at various tissues. There are two plausible explanations for low and differential expression of the OMP. The first is that OMP may be broadly expressed at low levels throughout the entire tissue. The second is that OMP may be locally expressed either in a small portion of the tissue or within a specific cell type of each tissue. Nevertheless, OMP expression is only detectable with high resolution methods using whole tissue lysates rather than by conventional methods.

Bottom Line: The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events.Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues.The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain Science, DGIST, Daegu, Korea.

ABSTRACT
Olfactory receptor (OR)-associated events are mediated by well-conserved components in the olfactory epithelium, including olfactory G-protein (Golf), adenylate cyclase III (ACIII), and olfactory marker protein (OMP). The expression of ORs has recently been observed in non-olfactory tissues where they are involved in monitoring extracellular chemical cues. The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events. In addition, expression profiles and physiological functions of ORs in non-olfactory tissues are largely unknown. To overcome limitations associated with using OR as a target protein, this study used OMP with Golf and ACIII as targets to screen for potential OR-mediated sensing systems in non-olfactory tissues. Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues. The results of immunohistochemical analyses showed OMP (+) cells in mouse heart and in the following cells using the corresponding marker proteins c-kit, keratin 14, calcitonin, and GFAP in mouse tissues: interstitial cells of Cajal of the bladder, medullary thymic epithelial cells of the thymus, parafollicular cells of the thyroid, and Leydig cells of the testis. The expression of ORs in OMP (+) tissues was analyzed using a refined microarray analysis and validated with RT-PCR and real-time PCR. Three ORs (olfr544, olfr558, and olfr1386) were expressed in the OMP (+) cells of the bladder and thyroid as shown using a co-immunostaining method. Together, these results suggest that OMP is involved in the OR-mediated signal transduction cascade with olfactory canonical signaling components between the nervous and endocrine systems. The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.

Show MeSH
Related in: MedlinePlus