Characterization of two distinct calcium-binding sites in the amino-terminus of human profilaggrin.
Profilaggrin is a large phosphorylated protein (approximately 400 kDa in humans) that is expressed in the granular cells of epidermis where it forms a major component of keratohyalin. It consists of multiple copies of similar filaggrin units plus amino- and carboxy-terminal domains that differ from filaggrin. Proteolytic processing of profilaggrin during terminal differentiation results in the removal of these domains and generation of monomeric filaggrin units, which associate with keratin intermediate filaments to form macrofibrils in the stratum corneum. The amino-terminal domain contains two calcium-binding motifs similar to the EF-hands found in the S-100 family of calcium-binding proteins. In this report, we expressed the 293-residue amino-terminal pro-domain of human profilaggrin as a polyhistidine fusion protein in Escherichia coli, and characterized calcium binding by a 45Ca++ binding assay and fluorescence emission spectroscopy. Fluorescence measurements indicated that the profilaggrin polypeptide undergoes conformational changes upon the removal of Ca++ with ethylenediamine tetraacetic acid, demonstrating the presence of two calcium-binding sites with affinities for calcium that differ ninefold (1.4 x 10(-4) M and 1.2 x 10(-3) M). We suggest that this functional calcium-binding domain at the amino-terminus of human profilaggrin plays a role in profilaggrin processing and in other calcium-dependent processes during terminal differentiation of the epidermis.
Presland
R B
RB
Department of Oral Biology, University of Washington, Seattle 98195.
Bassuk
J A
JA
Kimball
J R
JR
Dale
B A
BA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D000091344:Filaggrin Proteins; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D010750:Phosphoproteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D002118:Calcium