Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom.

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.

The primary structure of Vipera ammodytes venom trypsin inhibitor I.

The primary structure of Vipera ammodytes venom trypsin inhibitor I consists of 61 amino acid residues [sequence in text]. The N-terminal group of the inhibitor is pyrrolidonecarboxylic acid. The sequential data were obtained by analysis of peptides isolated from tryptic and chymotryptic digests and by analysis of peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The primary structure of trypsin inhibitor I presented shows approximately 80% sequence homology with chymotrypsin inhibitor isolated from the venom of the same snake, and nearly 50% homology with bovine basic pancreatic trypsin inhibitor. It belongs to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Primary structure of ammodytoxin C further reveals the toxic site of ammodytoxin.

The sequence of ammodytoxin C, a presynaptically toxic, basic phospholipase A2 of Vipera ammodytes ammodytes venom was determined. The toxin differs only in two amino acid residues from the most toxic isotoxin ammodytoxin A and is 18-times less lethal. Ammodytoxin B which is 30-times less lethal than ammodytoxin A differs from it only in three amino acid residues. From the three-dimensional model of ammodytoxin A, it can be seen that mutated regions of ammodytoxin B and ammodytoxin C are on the surface, and relatively distant from each other. The observed decrease in toxicity of ammodytoxin C could be a consequence of changed charge in position 128 where a Lys is exchanged for Glu. The resulting change in electrostatic properties of the molecule which influences the orientation of the molecule during the approach to the charged nerve-terminal membrane might be responsible for the observed decrease in toxicity.

Primary structure of a new cysteine proteinase inhibitor from pig leucocytes.

The primary structure of a pig leucocyte cysteine proteinase inhibitor, also called cathelin, was determined. The sequence was obtained from analyses of peptides isolated from the chymotryptic, endoproteinase Lys-C and protease V8 digests, and by analysis of the peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The inhibitor consists of 96 residues. The N-terminal residue of the inhibitor is pyrrolidone-carboxylic acid. The amino acid sequence of cathelin suggests the appearance of a new family of cysteine proteinase inhibitors.

Purification of the complex of cathepsin L and the MHC class II-associated invariant chain fragment from human kidney.

The complex of cathepsin L and the fragment of the MHC class II-associated invariant chain was purified from human kidney. M(r) of the complex, as determined by gel filtration, is about 40,000. Both components were identified by amino acid and sequence analyses. The bound invariant chain fragment is almost identical to the additional segment found in p41, but not in the p31 form of the invariant chain. The complex has significantly enhanced stability at neutral and slightly alkaline pH, and reduced proteolytic activity against the synthetic substrate Z-Phe-Arg-MCA compared to free cathepsin L. The complex exhibits no enzymatic activity against the protein substrate azocasein. For the first time, the invariant chain was found in a complex with a protein, which was not an MHC molecule.

Amino acid sequence of human liver cathepsin B.

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.

Amino acid sequences of the human kidney cathepsins H and L.

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.

Inactivation of human cystatin C and kininogen by human cathepsin D.

A papain inhibitor of 22 kDa was isolated from human placenta and shown to be identical to residues Cys246-Leu373 of the third domain of human kininogen. This kininogen domain and recombinant human cystatin C were inactivated by peptide bond cleavages at hydrophobic amino acid residues due to the action of cathepsin D. These results further support the proposed role of cathepsin D in the regulation of cysteine proteinase activity.

Cathepsin L is capable of truncating cystatin C of 11 N-terminal amino acids.

Cystatin C with the 11 N-terminal amino acids truncated shows a much lower affinity for cysteine proteinases than the intact inhibitor. Such truncation of cystatin C is recorded after action of glycyl endopeptidase and cathepsin L. Incubation of cystatin C with papain, cathepsin B or cathepsin H led to no changes in the cystatin C molecule. Isoelectric focusing of the cathepsin L and cystatin C mixture showed the formation of two new bands. One of them appeared whether E-64 or PMSF was added or not, evidently representing a cystatin C/cathepsin L complex. The other band is the truncated cystatin C molecule. N-terminal sequencing after separation by HPLC showed that cystatin C is cleaved by cathepsin L at the Gly11-Gly12 bond. The action of cathepsin L on cystatin C may be explained by the cleavage of the scissile bond in an inappropriate complex.

Papaya proteinase IV amino acid sequence.

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.

The complete amino acid sequence of bovine cathepsin S and a partial sequence of bovine cathepsin L.

The complete amino acid sequence of bovine spleen cathepsin S has been determined. The single-chain protein contains 217 residues and has a Mr of 23,682. The primary structure was determined by sequencing of native protein and the peptides obtained by proteolytic cleavage with beta-trypsin, papaya proteinase IV and by chemical cleavage with cyanogen bromide. Comparison of the amino terminal sequences of the heavy and the light chain of bovine cathepsin L with that of bovine cathepsin S clearly indicates that the enzymes are structurally different.

Stem bromelain: amino acid sequence and implications for weak binding of cystatin.

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64[L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)b utane].

Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family.

A new stefin type low-M(r) cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M(r) was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k(ass) > or = 1 x 10(6) M-1 x s-1) and forms very tight complexes with these enzymes (Ki < or = 190 pM). The affinity for cathepsins B and H (Ki > or = 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

Selective cleavage of glycyl bonds by papaya proteinase IV.

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO2 is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in beta-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases.

For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M(r) of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (kass = 9.6 x 10(6) M-1.s-1, Ki = 29 pM). The binding to cathepsin H was also rapid (kass = 2.1 x 10(6) M-1.s-1), but weaker (Ki = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (kass = 1.4 x 10(5) M-1.s-1), but still tight (Ki = 1.9 nM).

Human plasma kininogens are identical with alpha-cysteine proteinase inhibitors. Evidence from immunological, enzymological and sequence data.

Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.

The amino acid sequence of a novel inhibitor of cathepsin D from potato.

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.

Mild hyperhomocysteinemia and fibrinolytic factors in patients with history of venous thromboembolism.

Mild hyperhomocysteinemia is recognized as a risk factor for venous thromboembolism (VTE), though its role in the thrombogenic processes is not understood. Its possible association with impaired fibrinolysis was investigated in 157 patients (61 women, 96 men) below the age of 60 years (43+/-11, mean+/-SD) with a history of objectively confirmed VTE. Patients had significantly higher fasting total plasma homocysteine (tHcy) levels than 138 apparently healthy subjects (8.0, 6.6-9.9 micromol/L vs. 7.2, 5.9-8.6 micromol/L, P=0. 001; median, range between first and third quartile). In 17 of 157 patients (12%) hyperhomocysteinemia (tHcy>11.4 micromol/L for women and tHcy>12.6 micromol/L for men) was established. The adjusted odds ratio as an estimate of relative risk for VTE was 2.3 (0.8-7.0; 95% confidence interval). When patients with hyperhomocysteinemia were compared to patients without hyperhomocysteinemia, no significant differences in t-PA (antigen 9.2+/-5.5 microg/L and 9.7+/-4.7 microg/L, respectively; activity 1.3+/-0.5 IU/mL and 1.3+/-0.7 IU/mL, respectively) and PAI-1 (antigen 19.3+/-17.5 microg/L and 22.6+/-20. 4 microg/L, respectively; activity 15.0+/-12.6 and 15.8+/-13.3 IU/mL, respectively) were observed. In conclusion, this study showed an association between mild hyperhomocysteinemia and VTE, but provided no evidence for an independent association between hyperhomocysteinemia and alterations in fibrinolytic proteins.

Chelidocystatin, a novel phytocystatin from Chelidonium majus.

Greater celandine (Chelidonium majus L.) has traditional uses in European and Chinese herbal medicine. In the plant sap significant inhibitory activity against papain was observed. A cysteine proteinase inhibitor, named chelidocystatin, was isolated from the plant using papain Sepharose affinity chromatography followed by gel filtration and ion-exchange chromatography. Chelidocystatin showed a M(r) of 10,000 on SDS-PAGE with the pI of 9.3, and was a strong inhibitor of cathepsin L (Ki = 5.6 x 10(-11) M), papain (Ki = 1.1 x 10(-10) M) and cathepsin H (Ki = 7.5 x 10(-9) M). The complete amino acid sequence of the protein was obtained with N-terminal sequencing and sequencing of the peptides after digestion of the protein. Moreover, a major part of the sequence was verified by molecular cloning. The conserved glycine residue at the N-terminal region and the QVVAG motif, which are both believed to be involved in the inhibitory activity, indicate that it is a member of the cystatin superfamily. The amino acid sequence of chelidocystatin shows a high degree of homology with cysteine proteinase inhibitors belonging to the phytocystatin group, especially with the recently described carrot and sunflower phytocystatins with which it shares 57% and 54% homology, respectively.