SERRA HORACIO MARCELO
Congresos y reuniones científicas
Título:
SHEAR STRESS INDUCED MULTIMERIZATION OF COCHLIN AND ITS INTERACTION WITH ANNEXIN 2
Autor/es:
RENATA PICCIANI; SANJOY K. BHATTACHARYA; HORACIO M. SERRA
Lugar:
MIAMI
Reunión:
Congreso; ARVO 2012; 2012
Resumen:
Purpose: To characterize cochlin multimerization in response to shear stress and determine the interaction of native and multimerized cochlin with Annexin A2.
Methods: Native cochlin and recombinant cochlin domains were purified from HEK cells and E.coli. The proteins were subjected to purification using standard methods. Purified proteins were incubated with trabecular meshwork extract with or without being subjected to shear stress and their interaction with Annexin A2 was probed using immunoprecipitation and Western analyses. Cochlin-Annexin A2 interactions were also probed with overlay assay and mass spectrometry using a LCQ Deca XP instrument.
Results: We found native cochlin to interact with Annexin A2. In our kinetic assays, multimerized cochlin interacted with slightly higher binding strength with Annexin A2. The recombinant cochlin domains (LC and vWFA domains) poorly expressed either in E.coli or in HEK cells, they also failed to fold properly upon expression.
Conclusions: Multimeric cochlin formed in response to shear stress interacts with higher binding strength with Annexin A2 compared to native cochlin.
Methods: Native cochlin and recombinant cochlin domains were purified from HEK cells and E.coli. The proteins were subjected to purification using standard methods. Purified proteins were incubated with trabecular meshwork extract with or without being subjected to shear stress and their interaction with Annexin A2 was probed using immunoprecipitation and Western analyses. Cochlin-Annexin A2 interactions were also probed with overlay assay and mass spectrometry using a LCQ Deca XP instrument.
Results: We found native cochlin to interact with Annexin A2. In our kinetic assays, multimerized cochlin interacted with slightly higher binding strength with Annexin A2. The recombinant cochlin domains (LC and vWFA domains) poorly expressed either in E.coli or in HEK cells, they also failed to fold properly upon expression.
Conclusions: Multimeric cochlin formed in response to shear stress interacts with higher binding strength with Annexin A2 compared to native cochlin.
