Identification and cloning of a new calmodulin-like protein from human epidermis.
After separating by two-dimensional gel electrophoresis an extract of total proteins from human stratum corneum, two spots were extracted and analyzed for their peptide sequence. The resulting internal protein sequences provided evidence for the identification of a new calcium-binding protein. Cloning of the corresponding full-length cDNA was achieved by reverse transcriptase-polymerase chain reaction using two keratinocyte libraries, one from proliferating cultured keratinocytes and one from differentiated keratinocytes of reconstructed human epidermis. The cDNA had an open reading frame encoding a new calcium-binding protein of 146 amino acids, a member of the calmodulin family. We named this new protein calmodulin-like skin protein (CLSP), since reverse transcriptase-polymerase chain reaction studies of CLSP expression in 10 different human tissues revealed that this protein was particularly abundant in the epidermis where its expression is directly related to keratinocyte differentiation. Expression of the cloned cDNA in Escherichia coli yielded a recombinant protein which allowed its further characterization. rCLSP is able to bind calcium, and similarly to calmodulin, exposes thereafter hydrophobic parts which most likely interact with target proteins. Epidermal proteins retained by CaM affinity column are quantitatively and qualitatively distinct from those of the rCLSP column. Sequencing of a rCLSP affinity purified protein revealed 100% identity with transglutaminase 3, a key enzyme in terminal differentiation, indicating an important role of CLSP in this process.
Méhul
B
B
Life Sciences Research, L'Oréal, Centre Charles Zviak, 92583 Clichy, France. Bmehul@recherche.loreal.com
Bernard
D
D
Simonetti
L
L
Bernard
M A
MA
Schmidt
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D002147:Calmodulin / Q000737:chemistry; D002454:Cell Differentiation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004817:Epidermis / Q000737:chemistry*; D015723:Gene Library; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism; D014018:Tissue Distribution; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D018076:DNA, Complementary; C498712:TGM3 protein, human; D011503:Transglutaminases; D002118:Calcium