Analyses of the expression and function of the aspartic protease napsin

Two-dimensional gel electrophoresis (2 DE) is one of the powerful tools for the expression profiling of proteins. Using 2 DE, two polypeptides which are expressed exclusively in primary lung adenocarcinomas have been described. Mass spectrometry of tryptic digests of these proteins revealed that they were identical to napsin A. Napsin A is a member of the aspartic proteinase family, which is expressed in the lung and kidney. The aims of this study were to examine the clinical significance of napsin A in lung cancer and to investigate its functions in the normal lung and in cancer development.

Northern blot analysis showed a strong expression of napsin in lung and kidney tissues, and weak expression in spleen and peripheral blood leukocytes. In situ hybridization showed its expression to be localized to the type II pneumocytes of the lung, from which primary lung adenocarcinoma arises. Napsin mRNA expression was examined by in situ hybridization using 118 lung tissues including 39 primary lung adenocarcinomas. Surfactant protein-A (SP-A), SP-B and thyroid transcription factor-1 (TTF-1) are commonly-used markers for lung adenocarcinoma and their expressions were examined by immunohistochemistry. Napsin had the highest sensitivity (84.6%), together with TTF-1, and the highest specificity (94.3%), for adenocarcinoma in non-small cell lung carcinoma. Napsin expression was associated with the differentiation grade of adenocarcinoma: the expression rate decreased with the extent of dedifferentiation of adenocarcioma. These findings suggest that napsin is a promising marker for the identification of primary lung adenocarcinoma.

SP-B is a hydrophobic protein, which is produced in type II pneumocytes and secreted into the air space to reduce the surface tension of the lung. SP-B is synthesized as a precursor protein, which is then processed into the mature protein by multiple steps. The processing of SP-B has been shown to be mediated by an aspartic proteinase, raising the hypothesis that napsin is the proteinase involved in SP-B processing. Cryo-electron microscopy in isolated mouse type II pneumocytes showed that napsin was expressed in the lamellar body, where SP-B is stored, and in the multivesicular body, supporting the hypothesis. Incubation of recombinant pro SP-B with napsin A resulted in cleavage of pro SP-B, which was inhibited by pepstatin A, an inhibitor of aspartic proteinases. A cleavage site was identified as being 22 amino acids upstream of the NH2terminus of mature SP-B. Down-regulation of napsin expression by siRNA resulted in decreased levels of mature SP-B, suggesting that napsin is essential for SP-B processing. Cathepsin H, another enzyme implicated in SP-B processing, cleaved recombinant SP-B proproteins at a site 13 amino acids upstream of the N-terminus of mature SP-B and at the boundary between the mature protein and the C-terminal propeptide. These data suggest that napsin A and cathepsin H are involved in different steps of SP-B processing.

In situ hybridization of kidney tissues demonstrated that only 1 of 29 renal cell carcinomas expressed napsin mRNA although cells of the proximal convoluted tubules, from which renal cell carcinoma originates, did express this transcript. HEK293 cells transfected with napsin A cDNA showed reductions in anchorage-independent growth and tumor growth in SCID mice, compared to control cells transfected with an empty vector. Napsin A possesses an RGD-motif, a motif for integrin binding, in its C-terminus. Expression vectors carrying mutations in the catalytic domain and in the RGD motif were constructed. Mutation of the RGD motif abolished the ability of napsin A to inhibit anchorage-independent growth and tumor growth whereas mutation of the catalytic site had no effect. These data show that napsin A inhibits tumor growth in an RGDmotif dependent manner.

These results support the proposal that napsin may be an enzyme involved in SP-B processing in the lung. In addition, napsin expression appears to be inhibitory for cancer growth in kidney cells.

List of scientific papers

I. Chuman Y, Bergman A, Ueno T, Saito S, Sakaguchi K, Alaiya AA, Franzen B, Bergman T, Arnott D, Auer G, Appella E, Jornvall H, Linder S (1999). Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas. FEBS Lett. 462(1-2): 129-34.
https://pubmed.ncbi.nlm.nih.gov/10580105

II. Ueno T, Linder S, Elmberger G (2003). Aspartic proteinase napsin is a useful marker for diagnosis of primary lung adenocarcinoma. Br J Cancer. 88(8): 1229-33.
https://pubmed.ncbi.nlm.nih.gov/12698189

III. Ueno T, Linder S, Na CL, Rice WR, Johansson J, Weaver TE (2004). Processing of pulmonary surfactant protein B by napsin and cathepsin H. J Biol Chem. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/14766755

IV. Ueno T, Elmberger G, Weaver TE, Linder S (2004). Expression of the aspartic proteinase napsin A leads to suppression of tumor growth in an RGD-motif dependent manner. [Manuscript]