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Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

¹ Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan 650223, China (PRC)
² Graduate School of the Chinese Academy of Sciences, Beijing 100039, China (PRC)

(RECEIVED April 27, 2005; FINAL REVISION June 14, 2005; ACCEPTED June 15, 2005)

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumindomains. B.maximaserum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (K_D) obtained for the skin and the serum proteins were 1.92 x 10^–9 M and 1.55 x 10^–9 M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys⁵³–Cys⁶²), which comprises the scissile bond Arg⁵⁸(P₁)–His⁵⁹(P₁'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Keywords: albumin; skin; trypsin; haem; amphibian; frog

Abbreviations: BmA-skin, B. maxima albumin from skin • BmA-serum, B. maxima albumin from serum • BSA, bovine serum albumin • HSA, human serum albumin • pNA, p-nitroanilide • RACE-RT-PCR, rapid amplification of cDNA ends-reverse transcription PCR

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051551105.

Reprint requests to: Yun Zhang, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32, East Jiao Chang Road, Kunming, Yunnan 650223, China (PRC); e-mail: zhangy{at}mail.kiz.ac.cn; fax: +86-871-5191823.

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