CYSTATIN-E IS A NOVEL HUMAN CYSTEINE PROTEINASE-INHIBITOR WITH STRUCTURAL RESEMBLANCE TO FAMILY-2 CYSTATINS

A new member of the human cystatin superfamily, called cystatin E, has been found by expressed sequence tag (EST) sequencing in amniotic cel l and fetal skin epithelial cell cDNA libraries. The sequence of a ful l-length amniotic cell cDNA clone contained an open reading frame enco ding 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. Re combinant cystatin E was produced in a baculovirus expression system a nd isolated. An antiserum against the recombinant protein could be use d for affinity purification of cystatin E hom human urine, as confirme d by N-terminal sequencing. The mature recombinant protein processed b y insect cells started at amino acid 4 (cystatin C numbering), and dis played reversible inhibition of papain and cathepsin B (K-i values of 0.39 and 32 nM, respectively), in competition with substrate. Cystatin E is thus a functional cysteine proteinase inhibitor despite relative ly low amino acid sequence similarities with human cystatins (26-34% i dentity 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 M-r cystatins, cystatin E is a glycoprotein, carrying an N-linked carbohydrate chain at position 108. Northern blot analysis revealed that the cystatin E g ene is expressed in most human tissues, with the highest mRNA amounts found in uterus and liver. A strikingly high incidence of cystatin E c lones in cDNA libraries from fetal skin epithelium and amniotic membra ne cells (>0.5% of clones sequenced) indicates a protective role of cy statin E during fetal development.