Calcium-dependent stability studies of domains 1 and 2 of epithelial cadherin.
Epithelial cadherin is important in establishing and maintaining cell to cell interactions in epithelial cells, thereby playing an important role during morphogenesis. The epithelial cadherin molecules have three main regions: the N-terminal extracellular region, the transmembrane region that spans the cell membrane once, and the C-terminal cytoplasmic region that communicates with the cytoskeletal actin filaments through catenins. We report studies of the calcium-dependent stability of extracellular domains 1 and 2 of epithelial cadherin as a two-domain construct (MECAD12). Circular dichroism (CD) spectra of MECAD12 indicated a typical beta-sheet conformation in all solution conditions. Thermal- and denaturant-induced unfolding was monitored by CD. Distinct calcium stabilization was observed as a shift in T(m) from 40 (apo) to 65 degrees C (10 mM Ca2+). Spectroscopic experiments agreed well with calorimetric (DSC). In the absence of calcium, the unfolding transition was shallow (deltaH(m) = 40 kcal/mol) but not obviously three state. Model-dependent analysis indicated that a second transition could be assigned to the unfolding of domain 2. A calcium-binding constant was derived from the calcium-dependent shift in temperature denaturation profiles. The Kd that was obtained (55 microM) was consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calcium-bound states. This cooperativity between the modules is consistent with the physiological role of epithelial cadherin in signal transduction through cell-adhesive contacts.
Prasad
Alka
A
Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, USA.
Pedigo
Susan
S
D000595:Amino Acid Sequence; D000818:Animals; D015820:Cadherins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004847:Epithelial Cells / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D015820:Cadherins; D014508:Urea; D002118:Calcium