Characterization of cystatin N, a novel cysteine proteinase inhibitor

A novel cysteine proteinase inhibitor, cystatin N, was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the Family 2 cystatins, cystatin N is secreted, glycosylated and developmentally regulated. The highest expression of cystatin N mRNA is in skin, and in situ hybridization reveals significant cystatin N expression in the outer root sheath of the hair follicle and the suprabasal layer of rat skin. Cystatin N is also expressed in brain, and is induced during differentiation of a conditionally-immortalized E17 rat hippocampal cell line (H19-7) by bFGF or activated Raf via MEK-dependent and -independent signaling pathways. Cystatin N protein is increased during PC12 cell differentiation, and is secreted into the medium of primary embryonic hippocampal cultures. Analysis of the Ki of recombinant His-tagged cystatin N toward cathepsins B and H revealed that cystatin N has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between cystatin N and cystatin C, a well-characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Overexpression or suppression of cystatin N in H19-7 cells demonstrated that cystatin N is neither sufficient nor necessary for the morphological differentiation of H19-7 cells. In addition, we generated K14 promoter driven cystatin N transgenic mice to study the function of cystatin in skin. Our results suggest that misexpression of cystatin N in the basal layer of skin does not significantly alter the skin development. Thus, the physiological function of cystatin N is still unidentified.