Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor, and identification of the native protein in saliva.
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.
Freije
J P
JP
Departamento de Biología Funcional, Universidad de Oviedo, Spain.
Balbín
M
M
Abrahamson
M
M
Velasco
G
G
Dalbøge
H
H
Grubb
A
A
López-Otín
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D002401:Cathepsin B / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D015891:Cystatins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C071378:CST5 protein, human; D004247:DNA; D002401:Cathepsin B; D010206:Papain