| 1. |
- Balbin, Milagros, et al.
(författare)
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A sequence variation in the human cystatin D gene resulting in an amino acid (Cys/Arg) polymorphism at the protein level
- 1993
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Ingår i: Human Genetics. - 1432-1203. ; 90:6, s. 668-669
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Tidskriftsartikel (refereegranskat)abstract
- A polymorphism in the coding region of the human cystatin D gene has been detected by direct sequencing of amplified DNA from different individuals. The variation, resulting from a T/C transition in exon 1 of the gene, causes an amino acid variation, Cys/Arg, at the protein level. An allele-specific oligonucleotide hybridization assay was developed and used to demonstrate this polymorphism in the population. The deduced frequencies were 0.55 and 0.45 for the Cys and Arg variant-encoding alleles, respectively.
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| 2. |
- Balbin, Milagros, et al.
(författare)
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Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases
- 1994
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 269:37, s. 23156-23162
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin D is a novel member of the cystatin superfamily of cysteine proteinase inhibitors present in saliva and tears. Two alleles of the cystatin D gene (CST5), encoding protein variants with either Cys or Arg as residue 26 in their 122-residue polypeptide chains, are present in the population. Expression of the two alleles was investigated by immunochemical analyses of the secreted cystatin D in saliva from individuals homozygous for each of the two alleles, with results demonstrating that both are expressed at similar levels. The inhibitory characteristics of the two cystatin D variants were studied, by determination of dissociation equilibrium constants (Ki) for their complexes with papain and with the mammalian cysteine proteinases, cathepsins B, H, L, and S. The results demonstrate that 1) cystatin D has a characteristic inhibition profile since it does not inhibit cathepsin B (Ki > 1 microM), and when compared to cystatin C and all other known cystatins it is a much poorer inhibitor of cathepsin L (mean Ki 25 nM) but binds cathepsin H and S relatively tightly (mean Ki values of 8.5 and 0.24 nM, respectively); and 2) the inhibitory activities of the two cystatin D variants are not significantly different, demonstrating that the presence of an extra cysteine residue in the cystatin D molecule affects neither the stability nor the functional activity of the inhibitor, thus explaining the widespread distribution of the Cys26-cystatin D encoding allele in the population. The inhibitory properties displayed by cystatin D suggest that it has a function in saliva as inhibitor of either endogenous or exogenous enzymes with cathepsin S- or H-like properties.
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| 3. |
- Freije, José P, et al.
(författare)
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Human cystatin D: cDNA cloning, characterization of the E. coli expressed inhibitor, and identification of the native protein in saliva
- 1993
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 268:21, s. 15737-15744
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Tidskriftsartikel (refereegranskat)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.
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| 4. |
- Freije, José P, et al.
(författare)
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Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor
- 1991
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 266:30, s. 20538-20543
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Tidskriftsartikel (refereegranskat)abstract
- A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.
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