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- Ni, Jiun, et al.
(författare)
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Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins
- 1997
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 272:16, s. 10853-10858
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Tidskriftsartikel (refereegranskat)abstract
- A new member of the human cystatin superfamily, called cystatin E, has been found by expressed sequence tag (EST) sequencing in amniotic cell and fetal skin epithelial cell cDNA libraries. The sequence of a full-length amniotic cell cDNA clone contained an open reading frame encoding a putative 28-residue signal peptide and a mature protein of 121 amino acids, including four cysteine residues and motifs of importance for the inhibitory activity of Family 2 cystatins like cystatin C. Recombinant cystatin E was produced in a baculovirus expression system and isolated. An antiserum against the recombinant protein could be used for affinity purification of cystatin E from human urine, as confirmed by N-terminal sequencing. The mature recombinant protein processed by insect cells started at amino acid 4 (cystatin C numbering), and displayed reversible inhibition of papain and cathepsin B (Ki values of 0.39 and 32 nM, respectively), in competition with substrate. Cystatin E is thus a functional cysteine proteinase inhibitor despite relatively low amino acid sequence similarities with human cystatins (26-34% identity with sequences for the Family 2 cystatins C, D, S, SN, and SA; <30% with the Family 1 cystatins, A and B, and domains 2 and 3 of the Family 3 cystatin, kininogen). Unlike other human low Mr cystatins, cystatin E is a glycoprotein, carrying an N-linked carbohydrate chain at position 108. Northern blot analysis revealed that the cystatin E gene is expressed in most human tissues, with the highest mRNA amounts found in uterus and liver. A strikingly high incidence of cystatin E clones in cDNA libraries from fetal skin epithelium and amniotic membrane cells (>0.5% of clones sequenced) indicates a protective role of cystatin E during fetal development.
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| 2. |
- Ni, J, et al.
(författare)
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Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor
- 1998
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 273:38, s. 24797-24804
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Tidskriftsartikel (refereegranskat)abstract
- A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.
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