Limits...
Secreted osteopontin is highly polymerized in human airways and fragmented in asthmatic airway secretions.

Arjomandi M, Frelinger J, Donde A, Wong H, Yellamilli A, Raymond W - PLoS ONE (2011)

Bottom Line: Furthermore, we examined the relationship between airway sOPN and cellular inflammation.We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form.Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America. mehrdad.arjomandi@ucsf.edu

ABSTRACT

Background: Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and a cytokine with diverse biologic roles. OPN undergoes extensive post-translational modifications, including polymerization and proteolytic fragmentation, which alters its biologic activity. Recent studies suggest that OPN may contribute to the pathogenesis of asthma.

Methodology: To determine whether secreted OPN (sOPN) is polymerized in human airways and whether it is qualitatively different in asthma, we used immunoblotting to examine sOPN in bronchoalveolar lavage (BAL) fluid samples from 12 healthy and 21 asthmatic subjects (and in sputum samples from 27 healthy and 21 asthmatic subjects). All asthmatic subjects had mild to moderate asthma and abstained from corticosteroids during the study. Furthermore, we examined the relationship between airway sOPN and cellular inflammation.

Principal findings: We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form. Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN. Polymeric sOPN in BAL fluid was associated with increased alveolar macrophage counts in airways in all subjects.

Conclusions: These results suggest that sOPN in human airways (1) undergoes extensive post-translational modification by polymerization and proteolytic fragmentation, (2) is more fragmented and less polymerized in subjects with mild to moderate asthma, and (3) may contribute to recruitment or survival of alveolar macrophages.

Show MeSH

Related in: MedlinePlus

Monomeric, cleaved, and polymeric rOPN, BAL, and sputum sOPN.1A- Size exclusion chromatogram of rOPN and TGM2-treated (polymerized) rOPN; V0: void volume; i: thyroglobulin (669 kD); ii: apoferritin (460 kD); iii: gamma globulin (158 kD); iv: bovine serum albumin (65.5 kD); v: human chymase (30 kD). 1B- Immunoblot of rOPN, thrombin-cleaved rOPN, and TGM2-treated (polymerized) rOPN. Lane 1: rOPN; Lane 2: rOPN treated with thrombin; Lane 3: polymeric rOPN made using TGM2. 1C- Immunoblot of BAL samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody. 1D- Immunoblot of induced sputum samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198733&req=5

pone-0025678-g001: Monomeric, cleaved, and polymeric rOPN, BAL, and sputum sOPN.1A- Size exclusion chromatogram of rOPN and TGM2-treated (polymerized) rOPN; V0: void volume; i: thyroglobulin (669 kD); ii: apoferritin (460 kD); iii: gamma globulin (158 kD); iv: bovine serum albumin (65.5 kD); v: human chymase (30 kD). 1B- Immunoblot of rOPN, thrombin-cleaved rOPN, and TGM2-treated (polymerized) rOPN. Lane 1: rOPN; Lane 2: rOPN treated with thrombin; Lane 3: polymeric rOPN made using TGM2. 1C- Immunoblot of BAL samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody. 1D- Immunoblot of induced sputum samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody.

Mentions: Human rOPN (1 µg/ml) (1433-OP/CF; R&D Systems; Minneapolis, MN) was incubated with recombinant human transglutaminase 2 (TGM2) (0.06 µg/ml) (4376-TG-050; R&D Systems) in reaction buffer consisting of 5 mM CaCl2, 1 mM DTT, and 50 mM Tris-HCl (pH 7.5) at 37°C for 2 h (Figure 1B).


Secreted osteopontin is highly polymerized in human airways and fragmented in asthmatic airway secretions.

Arjomandi M, Frelinger J, Donde A, Wong H, Yellamilli A, Raymond W - PLoS ONE (2011)

Monomeric, cleaved, and polymeric rOPN, BAL, and sputum sOPN.1A- Size exclusion chromatogram of rOPN and TGM2-treated (polymerized) rOPN; V0: void volume; i: thyroglobulin (669 kD); ii: apoferritin (460 kD); iii: gamma globulin (158 kD); iv: bovine serum albumin (65.5 kD); v: human chymase (30 kD). 1B- Immunoblot of rOPN, thrombin-cleaved rOPN, and TGM2-treated (polymerized) rOPN. Lane 1: rOPN; Lane 2: rOPN treated with thrombin; Lane 3: polymeric rOPN made using TGM2. 1C- Immunoblot of BAL samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody. 1D- Immunoblot of induced sputum samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198733&req=5

pone-0025678-g001: Monomeric, cleaved, and polymeric rOPN, BAL, and sputum sOPN.1A- Size exclusion chromatogram of rOPN and TGM2-treated (polymerized) rOPN; V0: void volume; i: thyroglobulin (669 kD); ii: apoferritin (460 kD); iii: gamma globulin (158 kD); iv: bovine serum albumin (65.5 kD); v: human chymase (30 kD). 1B- Immunoblot of rOPN, thrombin-cleaved rOPN, and TGM2-treated (polymerized) rOPN. Lane 1: rOPN; Lane 2: rOPN treated with thrombin; Lane 3: polymeric rOPN made using TGM2. 1C- Immunoblot of BAL samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody. 1D- Immunoblot of induced sputum samples from 5 healthy and 5 asthmatic subjects probed with affinity purified polyclonal anti-human sOPN antibody.
Mentions: Human rOPN (1 µg/ml) (1433-OP/CF; R&D Systems; Minneapolis, MN) was incubated with recombinant human transglutaminase 2 (TGM2) (0.06 µg/ml) (4376-TG-050; R&D Systems) in reaction buffer consisting of 5 mM CaCl2, 1 mM DTT, and 50 mM Tris-HCl (pH 7.5) at 37°C for 2 h (Figure 1B).

Bottom Line: Furthermore, we examined the relationship between airway sOPN and cellular inflammation.We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form.Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America. mehrdad.arjomandi@ucsf.edu

ABSTRACT

Background: Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and a cytokine with diverse biologic roles. OPN undergoes extensive post-translational modifications, including polymerization and proteolytic fragmentation, which alters its biologic activity. Recent studies suggest that OPN may contribute to the pathogenesis of asthma.

Methodology: To determine whether secreted OPN (sOPN) is polymerized in human airways and whether it is qualitatively different in asthma, we used immunoblotting to examine sOPN in bronchoalveolar lavage (BAL) fluid samples from 12 healthy and 21 asthmatic subjects (and in sputum samples from 27 healthy and 21 asthmatic subjects). All asthmatic subjects had mild to moderate asthma and abstained from corticosteroids during the study. Furthermore, we examined the relationship between airway sOPN and cellular inflammation.

Principal findings: We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form. Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN. Polymeric sOPN in BAL fluid was associated with increased alveolar macrophage counts in airways in all subjects.

Conclusions: These results suggest that sOPN in human airways (1) undergoes extensive post-translational modification by polymerization and proteolytic fragmentation, (2) is more fragmented and less polymerized in subjects with mild to moderate asthma, and (3) may contribute to recruitment or survival of alveolar macrophages.

Show MeSH
Related in: MedlinePlus