Yeast two-hybrid analysis of a human trabecular meshwork cDNA library identified EFEMP2 as a novel PITX2 interacting protein.
OBJECTIVE
Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld-Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs).


METHODS
A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as "bait" to identify novel PIPs. A total of 1.25×10⁶ clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays.


RESULTS
EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2.


CONCLUSIONS
We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.
Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada.
Moulinath
M
Acharya
Michael W
MW
Sharp
Farideh
F
Mirzayans
Tim
T
Footz
Lijia
L
Huang
Chanchal
C
Birdi
Michael A
MA
Walter
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005124:Eye Abnormalities / Q000175:diagnosis / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D005901:Glaucoma / Q000175:diagnosis / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D061251:Primary Cell Culture; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D000097577:Homeobox Protein PITX2
D016326:Extracellular Matrix Proteins; D018398:Homeodomain Proteins; D020033:Protein Isoforms; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C403672:EFEMP2 protein, human