Cystatin B

Cystatin B is a member of the cysteine proteinase inhibitor superfamily (PROSITE PDOC00259; reviewed in Turk and Bode 1991; Turk et al. 1997, 2002b). This name was first used to describe an inhibitor of papain and related endopeptidases isolated from chicken egg white (Barrett 1981).

Cysteine proteinases constitute a group of proteolytic enzymes that cleave peptide bonds using a catalytic cysteine residue. Their inhibitors are classified into three distinct groups:

1. Type 1 cystatins (also named stefins) are molecules of approximately 100 amino acid residues and molecular size of 11 kDa, with no disulfide bonds or carbohydrate groups. Cystatins A and B belong in this category. They are stable at neutral and alkaline pH and resist heat. These proteins are potent and reversible competitive inhibitors of cysteine proteinases, with highest inhibition constants for papain, and cathepsins B, H, and L. In addition to animal cystatins, two cystatins have been described in rice that were found to inhibit insect digestive cysteine proteinases, therefore acting as plant-resistance mechanisms (Liang et al. 1991).

2. Type 2 cystatins are slightly larger molecules of 115 amino acids and a molecular size of 13 kDa. In contrast to type 1 cystatins, proteins of this group contain one or two disulfide loops near their C-terminus. Cystatin C is the best-characterized member of this category. It was isolated from serum of patients with autoimmune diseases (Brzin et al. 1984). A mutant cystatin C, with a Q68L substitution is a major constituent of amyloid fibrils in patients with hereditary cerebral hemorrhage with amyloidosis, described in Ireland (Ghiso et al. 1986).

3. Type 3 cystatins or kininogens are much larger plasma glycoproteins of 68-120 kDa.

Residues 46-50 of cystatin B constitute the so-called QVVAG domain, which is highly conserved in type 1 and plant cystatins. This region is deleted in some EPM1 patients with splice site mutations as discussed later. In type 2 cystatins only Q46 and G50 are consistently present. Kininogens contain three QVVAG domains and are predicted to have arisen from type 1 cystatins by gene triplication (Muller-Esterl et al. 1985; Rawlings and Barrett 1990).

The other highly conserved residue among all cystatins (except rice II) is Gly4. One EPM1 patient was found to be homozygous for a G4R mutation (vide infra).

Human cystatin B (NM_000100) is a small gene of three exons and a total genomic size of 3 kb (Fig. 1a). The coding region which is 297-bp long encodes a protein of 98 amino acids (Swiss-Prot P04080). In human and mouse, cystatin B is expressed in all tissues tested (Pennacchio et al. 1996; Pennacchio and Myers 1997). In the brain, cystatin B is present in neural stem cells and in mature neurons and glial cells. However, there are some differences in the subcellular localization: in stem cells it is localized in the nucleus and in astrocytes in nucleus and cytoplasm (Riccio et al. 2001; Brannvall et al. 2003). In the cytoplasm, cystatin B is present in lysosomes. The Purkinje cells of the cerebellum, which are affected in EPM1, express cystatin B and its distribution is developmentally regulated (Riccio et al. 2005). Cystatin B is present in the nucleus of proliferating primary myoblasts and COS-1 cells (Alakurtti et al. 2005). In differentiated myotubes cystatin B is excluded from the nucleus and is detected in punctate cytoplasmic structures, some of which are lyso-somes. In embryonic liver cells, cystatin B is diffusely distributed throughout the cytoplasm (Calkins et al. 1998). Although mainly an intracellular protein, cystatin B has also been isolated from extracellular fluids (Abrahamson et al. 1986).

Human cystatin B forms inactive disulfide-linked dimers of 25 kDa. Under reducing conditions, these dimers are converted into active monomers (Wakamatsu et al. 1984). The crystal structure of human cystatin B in stoichiometric complex with papain has been determined to 2.4-Â resolution (Stubbs et al. 1990), (Fig. 2a). X-ray crystallography revealed that the molecule consists of five-stranded ^-sheets wrapped around a five-turn a-helix. Crystallography also verified, as is the case in general with cystatins, that the main interactions with papain are provided by the amino terminal

-174(CCCCGCCCCGCG^_3;12_17

-174(CCCCGCCCCGCG^_3;12_17

G4R IVS1-1G->C c168G>C

-174(CCCCGCCCCGCG)30

CA390G

G4R IVS1-1G->C c168G>C

CA390G

R68)^ c214-215delTC Q71P

-174(CCCCGCCCCGCG)30

R68)^ c214-215delTC Q71P

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