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 provi ded evidence for the identification of a new calcium-binding protein. Cloni ng of the corresponding full-length cDNA was achieved by reverse transcript ase-polymerase chain reaction using two keratinocyte libraries, one from pr oliferating cultured keratinocytes and one from differentiated keratinocyte s of reconstructed human epidermis. The cDNA had an open reading frame enco ding a new calcium-binding protein of 146 amino acids, a member of the calm odulin family. We named this: new protein calmodulin-like skin protein (CLS P), since reverse transcriptase-polymerase chain reaction studies of CLSP e xpression in 10 different human tissues revealed that this protein was part icularly abundant in the epidermis where its expression is directly related to keratinocyte differentiation. Expression of the cloned cDNA in Escheric hia coli yielded a recombinant protein which allowed its further characteri zation. rCLSP is able to bind calcium, and similarly to calmodulin, exposes thereafter hydrophobic parts which most likely interact with target protei ns. Epidermal proteins retained by CaM affinity column are quantitatively a nd qualitatively distinct from those of the rCLSP column. Sequencing of a r CLSP affinity purified protein revealed 100% identity with transglutaminase 3, a hey enzyme in terminal differentiation, indicating an important role of CLSP in this process.