The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles (PiF, G, I, L, N, S1, S2, U), another 10 alleles, designated PiH, J, K, O, P, Q, R, V, X, Z, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pi1, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 2. Protease inhibition

The protease inhibitory spectra of the eight homozygous Thoroughbred Pi types against trypsin, elastase and chymotrypsin have been determined. The α1-protease inhibitor proteins exhibit three classes of inhibitory specificity towards these enzymes. The Pi types F, I, N and U exhibit class I (trypsin, elastase and chymotrypsin) and class II (trypsin and elastase) types of inhibition and fit Juneja et al.s (1979) classification of two separate genetic systems Pi 1 and Pi 2 based on differences in the inhibitory spectra against trypsin and chymotrypsin. The remaining four Pi types are exceptions to Juneja et al.s (1979) classification. Types G, L, S₁ and S₂ possess class I but not class II proteins. A third class of proteins (class III) which exclusively inhibit chymotrypsin was detected in all eight protease inhibitor types. Type G is well represented by class III proteins because two of the three major proteins of the ISO-DALT pattern inhibit only chymotrypsin and is thus an exception to Juneja et al.s (1979) classification.     

A new genetic variant Z2 in the Pi system of horses

A new genetic variant in the horse Pi system, designated Z2, was reported in Polish Arabs by using two-dimensional agarose polyacrylamide gel electrophoresis. The frequencies of Pi alleles F, G, L, L2, N, S, U, W, Z and Z2 were found to be 0.036, 0.005, 0.171, 0.013, 0.008, 0.237, 0.416, 0.003, 0.107 and 0.004 respectively.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Equus przewalskii plasma protease inhibitor (Pi) system

A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) were found to control the plasma Pi proteins of the E. przewalskii haplotypes. In contrast a fourth locus, Spi 4, was found in some E. caballus haplotypes. The significance of these results with respect to the complexity of the protein pattern exhibited by the equine Pi multigene family is discussed.     

Protease inhibitor system in horses: classification and detection of a new allele

A method of horizontal thin layer polyacrylamide gel electrophoresis at acid pH has been developed for the separation of the prealbumins in equine plasma. Using this method, it has been possible to split the S allele into two, S₁ and S₂, bringing the total number of prealbumin alleles in Thoroughbred horses to eight. The gene frequencies of these eight alleles in Australian Thoroughbreds are presented. All eight prealbumin types exhibit antiprotease activity and therefore, it is suggested that the name prealbumin (Pr) should be abandoned in favour of protease inhibitor (Pi) although at this stage it is not known whether this incorporates the Pi1 and Pi2 described by Juneja et al. (1979).     

Epitope topology and removal of mouse acrosomal plasma membrane by P12-targeted immunoaggregation

P12 is a Kazal-type trypsin inhibitor that has been purified from mouse seminal vesicle secretion. We observed a slight impact of P12 on sperm capacitation, and demonstrated the removal of plasma membrane overlaying the acrosome region by immunoaggregation of P12 on mouse sperm. Further, we compared the immunoreactivity of P12 antibody to ten P12 variants, including six single-site mutated mutants (R19L, Y21V, D22G, R43G, K44S, and R45T), two multisite mutated mutants (R43G/K44S/R45T and L50H/R52G/K53A), and two deletion mutants (Nd10 and Cd8) in which 10 and 8 residues were deleted from the N- and C-terminals, respectively. We found that the N-terminal region, 43RKR45, and the C-terminal region, but not R19, Y21, and D22, are involved in the three epitopes that reside on one side and are three-dimensionally distant from R19, Y21, and D22 on the P12 molecule. Based on the epitope topology, we elucidated the structural basis by which P12 antibody immunoaggregated P12 on sperm head.     

侧孢短芽胞杆菌产生线虫侵染性蛋白酶的优化

采用单因素试验确定侧孢短芽胞杆菌G4产线虫侵染性蛋白酶的最佳碳氮源,通过Placket-Burman设计筛选影响蛋白酶活力的主效因子,最陡坡试验和Box-Behnken设计获得主效因子的最佳水平,建立线虫侵染性蛋白酶的最佳生产体系:葡萄糖9.78 g/L、牛肉膏16.65 g/L、磷酸氢二钾0.75 g/L、可溶性淀粉12.5 g/L、氯化钠0.75 g/L、硫酸镁0.5 g/L、初始pH值自然、装液量50 mL,37℃摇瓶培养32 h,蛋白酶活力可达12 379.41 U/mL,较优化前的2 476.3 U/mL提高了4倍。     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles ( Pi^{F, g, i, L, N, S1, S2, u)} , another 10 alleles, designated PiH, j, k, ^o, ^p, ^q , ^r, ^v, ^{x, z}, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pil, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar K_is, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.     

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function.     

Combining mutations in HIV-1 protease to understand mechanisms of resistance

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.     

Characterization and suppression of dysfunctional human alpha1-antitrypsin variants

Human alpha1-antitrypsin-deficient variants may aggregate in the liver, with subsequent deficiency in the plasma, which can lead to emphysema. The structural and functional characteristics of 10 dysfunctional alpha1-antitrypsin variants (R39C, S53F, V55P, I92N, G115S, N158K, E264V, A336T, P369S, and P369L) were analyzed in detail. Most of them were unstable, as compared to the wild-type molecule, and many of the variants folded into an intermediate form. When five thermostable mutations (T68A, A70G, M374I, S381A, and K387R) were introduced into dysfunctional alpha1-antitrypsin variants, the stabilities and inhibitory activities of most of the variants were restored to levels comparable to those of the wild-type molecule. However, the extremely unstable S53F variant was not stabilized sufficiently by these mutations so as to exhibit function. N158K variant, which carries a mutation in the region critical for the reactive site loop insertion into beta-sheet A, exhibited a reduced level of inhibitory activity, despite conformational stabilization. These results show that aberrant folding caused by conformational destabilization due to mutations can be compensated for by increasing the overall stability of the alpha1-antitrypsin molecule, with exception of a mutation in the highly localized region critical for functional execution.     

Detection of the frequencies of GBA gene major mutations in patients with Gaucher disease in Ukraine

The molecular diagnostics of 27 from 26 Ukrainian families has been performed. The common mutations in GBA gene (N370S, L444P and 84GG) accounted for up to 58% of all cases: mutation N370S was detected in 42.3% alleles, mutation L444P was observed in 15.4% alleles and mutation 84GG was not found at all. The other mutations were: P178S, W184R and Rec Nci I (in compounds with N370S) in the patients with nonneuronopathic form of Gaucher disease, and the genotypes G377S/c 999G --> A and D409H/R120W/G202R were detected in patients with chronic neuronopathic form of Gaucher disease. The data analysis of the genotype and disease progression in the patients allows confirming the known genotype-phenotype correlation.     

Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex.

Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 A X-ray data set has been collected on crystals belonging to space group P2(1)2(1)2(1), with a = 41.66 A, b = 51.41 A, and c = 107.72 A. There is most likely one molecule per asymmetric unit.     

Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S

The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PR(L24I), PR(I50V), and PR(G73S) to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50V alters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PR(L24I) and PR(I50V), showed a 4% and 18% lower k(cat)/K(m), respectively, relative to PR. The relative k(cat)/K(m) of PR(G73S) varied from 14% to 400% when assayed using different substrates. Inhibition constants (K(i)) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR(L24I) and PR(I50V), respectively, relative to PR and PR(G73S). The dimer dissociation constant (K(d)) was estimated to be approximately 20 nM for both PR(L24I) and PR(I50V), and below 5 nM for PR(G73S) and PR. Crystal structures of the mutants PR(L24I), PR(I50V) and PR(G73S) were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10-1.50 Angstrom. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PR(L24I) and PR(I50V) had reduced intersubunit contacts, consistent with the increased K(d) for dimer dissociation. Relative to PR, PR(I50V) had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K(i). The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.     

Analysis of the structure of chemically synthesized HIV-1 protease complexed with a hexapeptide inhibitor. Part I: Crystallographic refinement of 2 Å data

The structure of a complex between a hexapeptide-based inhibitor, MVT-101, and the chemically synthesized (Aba 67,95,167,195; Aba: l-α-amino-n-butyric acid) protease from the human immunodeficiency virus (HIV-1), reported previously at 2.3 Å has now been refined to a crystallographic R factor of 15.4% at 2.0 Å resolution. Root mean square deviations from ideality are 0.18 Å for bond lengths and 2.4° for the angles. The inhibitor can be fitted to the difference electron density map in two alternative orientations. Drastic differences are observed for positions and interactions at P3/S3 and P3′/S3′ subsites of the two orientations due to different crystallographic environments. © 1997 Wiley-Liss, Inc.     

Crystal structures of five bovine chymotrypsin complexes with P1 BPTI variants

The bovine chymotrypsin-bovine pancreatic trypsin inhibitor (BPTI) interaction belongs to extensively studied models of protein-protein recognition. The accommodation of the inhibitor P1 residue in the S1 binding site of the enzyme forms the hot spot of this interaction. Mutations introduced at the P1 position of BPTI result in a more than five orders of magnitude difference of the association constant values with the protease. To elucidate the structural aspects of the discrimination between different P1 residues, crystal structures of five bovine chymotrypsin-P1 BPTI variant complexes have been determined at pH 7.8 to a resolution below 2 A. The set includes polar (Thr), ionizable (Glu, His), medium-sized aliphatic (Met) and large aromatic (Trp) P1 residues and complements our earlier studies of the interaction of different P1 side-chains with the S1 pocket of chymotrypsin. The structures have been compared to the complexes of proteases with similar and dissimilar P1 preferences, including Streptomyces griseus proteases B and E, human neutrophil elastase, crab collagenase, bovine trypsin and human thrombin. The S1 sites of these enzymes share a common general shape of significant rigidity. Large and branched P1 residues adapt in their complexes similar conformations regardless of the polarity and size differences between their S1 pockets. Conversely, long and flexible residues such as P1 Met are present in the disordered form and display a conformational diversity despite similar inhibitory properties with respect to most enzymes studied. Thus, the S1 specificity profiles of the serine proteases appear to result from the precise complementarity of the P1-S1 interface and minor conformational adjustments occurring upon the inhibitor binding.