Human cystatin D: cDNA cloning, characterization of the E. coli expressed inhibitor, and identification of the native protein in saliva

José P Freije, Milagros Balbin, Magnus Abrahamson, Gloria Velasco, Henrik Dalboge, Anders Grubb, Carlos Lopez-Otin

Research output: Contribution to journalArticlepeer-review

Abstract

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse- transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity.
Original languageEnglish
Pages (from-to)15737-15744
JournalJournal of Biological Chemistry
Volume268
Issue number21
Publication statusPublished - 1993

Subject classification (UKÄ)

  • Medicinal Chemistry
  • Pharmacology and Toxicology

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