Human cystatin, a new protein inhibitor of cysteine proteinases

A new low-molecular weight protein inhibitor of cysteine proteinases, human cystatin, was isolated from sera of patients with autoimmune diseases. It inhibits papain, human cathepsin H and cathepsin B. According to its partially determined amino-acid sequence, human cystatin is highly homologous to egg white cystatin, but only distantly related to stefin, the cytosolic protein inhibitor of cysteine proteinases isolated from human polymorphonuclear granulocytes. Very probably human cystatin is identical with human gamma-trace, a microprotein of known sequence but hitherto unknown function.     

Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.     

The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases.

The crystal structure of chicken egg white cystatin has been solved by X-ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X-ray data between 6 and 2.0A. The molecule consists mainly of a straight five-turn alpha-helix, a five-stranded antiparallel beta-pleated sheet which is twisted and wrapped around the alpha-helix and an appending segment of partially alpha-helical geometry. The 'highly conserved' region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta-hairpin loop which on opposite sides is flanked by the amino-terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I - Trp104I. These loops and the amino-terminal Gly9I - Ala10I form a wedge-shaped 'edge' which is quite complementary to the 'active site cleft' of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino-terminal segment Gly9I - Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin-like inhibitors seems to be fundamentally different from the 'standard mechanism' defined for serine proteinases and most of their protein inhibitors.     

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.     

Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein

A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant. The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2₁ with unit cell parameters of 52.2 Å, 85.8 Å, 103.9 Å, and 81.8° corresponding to two molecules per asymmetric unit. The crystals diffract at least to a resolution of 2.0 Å and are suitable for an X-ray structure analysis. © 1995 Wiley-Liss, Inc.     

Affinity chromatography of a cysteine proteinase inhibitor from chicken egg protein

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.     

Crystallization and preliminary X-ray diffraction studies of dogfish C-reactive protein

Crystals of dogfish (Mustelus canis) C-reactive protein were obtained through vapor phase equilibration using the sitting drop rod technique with ammonium sulfate as the precipitating agent. The space group was determined to be P1 (triclinic lattice) with unit cell dimensions of a = 82.91, b = 92.25 and c = 103.40 Å; α = 83.36°, β = 89.76°, and γ = 81.30°. These crystals diffract to about 2.6 Å resolution and contain two hexamers in the asymmetric unit. © 1993 Wiley-Liss, Inc.     

Purification and characterization of a low molecular weight basic protein from marine turtle egg white

The egg white of marine turtle (Caretta caretta Linn.) and one species of tortoise (Geomyda trijuga trijuga Schariggar) contain a low molecular weight basic protein. It has been purified to homogeneity from the egg white of marine turtle and characterized in terms of its major physicochemical and chemical properties. The molecular weight of this protein calculated from gel filtration, sodium dodecyl sulfate-gel electrophoresis in the presence of urea, sedimentation-diffusion data, and amino acid composition is 4300. Its isoelectric point is at pH 11.1 and intrinsic viscosity is 0.038 dl g-1 in 0.2 M NaCl. It has a Stokes radius of 12.6 A and a diffusion coefficient of 16.50 x 10(-7) cm2 s-1. Analysis of the far-ultraviolet circular dichroic spectrum has shown that the basic protein contains 27% beta-pleated sheet and little or no alpha-helix. It possesses a single polypeptide chain of 40 amino acid residues with three disulfide bonds. It lacks serine, methionine, phenylalanine and carbohydrate moiety. It binds to DNA and stimulates ATPase activity due to its strong basicity. The complex of DNA-basis protein is partially resistant to the action of DNase.     

Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin

The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Crystallization and preliminary X-ray data of a phleomycin-binding protein from Streptoalloteichus hindustanus

Large single crystals of a glycopeptide resistance protein encoded by the SH-ble gene from Streptoalloteichus hindustanus have been grown using vapor diffusion techniques with ammonium sulfate as the precipitant. The diffraction pattern extends to 2.0 A resolution. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have unit cell dimensions of a = 48.8 A and c = 112.5 A.     

Crystallization and preliminary X-ray diffraction studies of colicin E3 immunity protein

Immunity protein, an inhibitor of the ribonuclease activity of the protein antibiotic colicin E₃, crystallizes in the orthorhombic space group C222 with cell dimensions $\text{a = 78·7}\text{A}\text{?}\text{, b = 54·1}\text{A}\text{?}\text{, c = 36·1}\text{A}\text{?}$ and one molecule of M_r 9800 per asymmetric unit. The crystals are suitable for high resolution X-ray analysis.     

Crystallization and preliminary X-ray data of human plasma retinol-binding protein

Crystals of human plasma retinol-binding protein have been obtained from 4.5 m-NaCl buffered at pH 6.8 with 20 mm-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system. $\text{a = b = 104.2}\text{A}\text{?}\text{and}\text{c = 74.5}\text{A}\text{?}$. The crystals diffract to a resolution of 2.0 Å.     

Crystallization of and preliminary X-ray diffraction data for TET-repressor and the TET-repressor-tetracycline complex

The TET-repressor encoded by the transposon Tn10 has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P4₃2₁2 (or P4₁2₁2) with cell dimensions $\text{a = b = 74.3(1)}\text{A}\text{?}\text{, c = 94.2(2)}\text{A}\text{?}\text{and}\text{a = b = 73.3(1)}\text{A}\text{?}\text{, c = 94.6(2)}\text{A}\text{?}$ for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,000 per asymmetric unit, and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor, subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 Å and those of the complexed repressor to 2.8 Å.     

Crystallization and preliminary X-ray diffraction data of two heparin-binding fragments of human fibronectin

Two different heparin-binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep-2A fragment, consisting of type III domains 12–14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I2₁2₁2₁ with a = 68.1 Å, b = 88.6 Å, and c = 144.9 Å. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6₁22 or P6₅22 witha a = b = 66.7 Å and c = 245.7 Å. The 40 kDa hep-2B fragment, consisting of type III domains 12–15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep-2A fragment with space group I222 or I2₁2₁2₁ and a = 67.5 Å, b = 87.0 Å, and c = 144.3 Å. All crystal forms diffract to at least 3.5 Å resolution and contain a single molecule in the asymmetric unit. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction data for the collagenase of Hypoderma lineatum larvae

Collagenase from the larvae of the arthropod Hypoderma lineatum has been crystallized. The crystals are tetragonal, space group I422, $\text{a = b = 112}\text{A}\text{?}\text{, c = 166}\text{A}\text{?}$, with two molecules in the asymmetric unit.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Isolation and complete amino acid sequence of a basic low molecular weight protein from black swan egg white

The following complete amino acid sequence of a low molecular weight basic protein (Mr 4,454) from black swan egg white has been determined: less than Glu-Val-Arg-Lys-Tyr-Cys-Pro-Lys-Val-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys-Ala-Asp-Val-Trp-Ser-Leu-Ser-Ser-Asp-Cys-Lys-Phe-Tyr-Cys-Cys-Leu-Pro-Pro-Gly-Trp-Lys. There is significant homology between this protein, provisionally designated cygnin, and the NH2-terminal region of the second domain of chicken ovotransferrin. The disulfide bonds have not been assigned; however, the arrangement of half-cystines in cygnin is sufficiently different from that of the known transferrins to suggest that cygnin is derived from another gene.     

Crystallization and preliminary X-ray crystallographic data of a histidine-binding protein from Escherichia coli

Histidine-binding protein, purified from periplasmic space of Escherichia coli K12, has been crystallized in a form suitable for X-ray analysis. Crystals of average size 0.3 mm x 0.15 mm x 0.15 mm have been grown by the hanging-drop method, with ammonium sulfate as precipitant. The space group if I4(1)22, with the unit cell dimensions a = b = 119.1 A; c = 151.8 A; Vm = 2.7 A3/dalton. There appear to be two protein subunits of molecular weight 25,000 each in the asymmetric unit.