Polymorphism of pig serum alpha-protease inhibitor-3 (PI3) and assignment of the locus to the Pi1, Po1A, Po1B, Pi2, Igh linkage group

Polymorphism of an alpha-protease inhibitor, PI3, in pig serum samples was detected using 2D agarose gel (pH 5.4)--polyacrylamide gel (pH 9.0) electrophoresis. Evidence was obtained that the five variants observed (A, B1, B2, C and D) are under genetic control by codominant alleles (Pi3A, Pi3B1, Pi3B2, Pi3C and Pi3D) at one autosomal locus. Variants A, B1, B2 and C inhibited chymotrypsin; there was no appreciable inhibition of trypsin and papain. Variant D did not inhibit chymotrypsin, and therefore its classification as a PI3 variant was put in question. PI3 typing was not possible in about 50% of the studied pigs since in those cases the PI3 variants were either too weak or absent. On the basis of backcross matings and haplotyping in complete families for protease inhibitor loci Pi1, Po1A, Pi2 and Pi3 it was proved that the Pi3 locus belongs to the protease inhibitor gene cluster, and the position of the locus in the linkage group was proposed as being Pi1-Po1A-(Po1B)-Pi3-Pi2-(Igh1, Igh2, Igh3, Igh4).     

The serum protease inhibitor linkage group in Belgian pig breeds

Genetic variants of pig serum alpha-protease inhibitors (protease inhibitors-1 and -2, PI1 and PI2; postalbumin-1A and -1B, PO1A and PO1B) were studied by 2-D electrophoresis of serum samples. The inheritance data confirmed the close linkage between the loci of these inhibitors. The order between these loci was indicated as Pi1-Po1A-Po1B-Pi2 and these were spread over a distance of about 1 cM. Very strong linkage disequilibrium was observed between the alleles at these loci. The two breeds studied (Belgian Landrace and Piétrain) showed very different allele and haplotype frequencies. Both breeds showed extensive polymorphism at Po1A and Pi2 loci.     

Extensive genetic polymorphism of four plasma alpha-protease inhibitors in pigs and evidence for tight linkage between the structural loci of these inhibitors

Two-dimensional horizontal gel electrophoresis of pig plasma samples (under non-denaturing conditions) using Immobiline pH gradient gels 4.0-6.0 for the first dimension separation, resulted in clear resolution of the variants of four different alpha-protease inhibitors (protease inhibitor -1 and -2, PI1 and PI2; post-albumin -1A and -1B, PO1A and PO1B). All these variants were readily visualized by general protein staining. About 900 families each of Swedish Landrace (SL) and Yorkshire (SY) breeds were studied. The extensive inheritance data, including the recombinants encountered, indicated that each of these four inhibitors is controlled by a separate, autosomal locus and that the four loci are tightly linked (spread over a distance of 1-1.5 cM) with the order as Pi1-Po1A-Po1B-Pi2. The alleles observed were two of Pi1, 14 of Po1A, 11 of Po1B and 8 of Pi2. About 40 haplotypes were observed in each of the two breeds. The allele frequencies at Po1A, Po1B and Pi2 loci were remarkably different in the two breeds; the alleles at these three loci showed a very strong linkage disequilibrium (0.8-1.0). The females showed much higher recombination frequencies than the males in the Po1A-Pi2 interval, suggesting that gene conversion-like events may be occurring at these loci. This linkage in pigs and similar ones comprising some plasma alpha-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.     

Some new variants of serum protease inhibitors in Meishan pigs

Serum samples of Meishan (13 animals) and Meishan x Wild Boar crosses (361 animals) were analysed by means of two-dimensional electrophoresis. Some new variants in protease inhibitor systems PO1A, PO1B and PI2 are reported.     

Simultaneous phenotyping of pig plasma α-protease inhibitors (PI1, PO1A, PO1B, PI2) and four other proteins (PO2, TF, CP, HPX) by a simple method of 2D horizontal electrophoresis

A simple and rapid method of 2D agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis was developed for the simultaneous phenotyping of pig plasma alpha-protease inhibitors (protease inhibitor-1 and -2; postalbumin-1A and -1B), postalbumin-2, transferrin, ceruloplasmin and haemopexin. These eight plasma proteins were clearly visible on gels stained with Coomassie Brilliant Blue G250. The 2D patterns and mobilities of several variants of alpha-protease inhibitors were described. By using two agarose gels and 10 polyacrylamide gels, 120 samples were easily analysed in a day. Since alpha-protease inhibitors show extensive polymorphism and as the gene for postalbumin-2 is closely linked to the halothane sensitivity locus Hal, this method is a useful tool for conducting parentage control and for predicting Hal genotypes of individual pigs.     

Hepatitis C virus protease gene diversity in patients coinfected with human immunodeficiency virus

The clonal variability of the hepatitis C virus (HCV) protease gene in 24 individuals with HCV genotypes 1a, 1b, 2b, and 3a who were coinfected with the human immunodeficiency virus was evaluated. Within-genotype variability at the nucleotide and amino acid levels ranged from 6.5 to 8.6% and 2.2 to 3.8%, respectively. After adjustments were made for correlation of intrapatient clonal variation, mixed-model analysis indicated that nucleotide and amino acid variability among patients with different genotypes did not differ significantly. However, within individual patients, clonal variability differed by up to 5.3% and 5.8% at the nucleotide and amino acid levels, respectively, and genotype 1a had significantly greater nucleotide variability than other genotypes (P = 0.01). Significant variability exists within HCV protease gene variants at the patient level and could affect the effectiveness of HCV protease inhibitors.     

Pig plasma protease inhibitor gene complex: isolation and partial characterization of three inhibitors

1. Pig plasma alpha-protease inhibitors (protease inhibitor-1, PI1; protease inhibitor-2, PI2; postalbumin-1A, PO1A; postalbumin-1B, PO1B), all encoded by one gene complex (gene cluster), were isolated by rivanol-ammonium sulphate fractionation and double-one dimensional IPG-PAGE. The proteins were recovered from the polyacrylamide gel by a combination of electrophoresis and isoelectric focusing. 2. Molecular wt estimated by SDS-PAGE under reducing conditions was 63,000 each for PI1 and PI2 and 60,000 each for PO1A and PO1B. The two main components of a genetic variant of PI2 differed in mol. wt by approx. 1000. 3. PO1A, PO1B and PI2 were shown to be glycoproteins. The major component of both PO1A and PO1B contained about 15% carbohydrate and the two components of PI2 had about 24 per cent and 21 per cent carbohydrate, respectively. 4. Neuraminidase treatment showed that the main component of PO1A had 8 sialic acid residues and fast and slow components of PI2 had respectively 11 and 10 residues. 5. Amino acid compositions of PO1A, PO1B and PI2 were very similar to one another, indicating that the genes for these proteins have evolved by regional duplications of a common ancestral gene. 6. The results (mol. wt, amino acid and carbohydrate compositions) confirm that pig PI2 is homologous to human plasma alpha 1-antichymotrypsin.     

Association of the X-linked lymphoproliferative disease gene product SAP/SH2D1A with 2B4, a natural killer cell-activating molecule, is dependent on phosphoinositide 3-kinase

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.     

Genetic variants of protease inhibitors against fungal protease and α-chymotrypsin from hemolymph of the silkworm,Bombyx mori

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus melleus and pancreatic alpha-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotrypsin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.     

Effect of maturation on activities of various proteases and protease inhibitors in the muscle of Ayu (Plecoglossus altivelis).

1. Increases in activities of muscle muticatalytic proteinase, modori-inducing proteinase (latent trypsin-like proteinase), cathepsin B and L-like proteases and cathepsin D were observed more markedly for male fish than female fish, in the spawning stage. 2. Decreases in inhibitory activities of muscle serine and cysteine protease inhibitors were observed more markedly for male fish than female fish in the spawning stage.     

Computational characterization of structural role of the non-active site mutation M36I of human immunodeficiency virus type 1 protease

A prominent characteristic of human immunodeficiency virus type 1 (HIV-1) is its high genetic variability, which generates diversity of the virus and often causes a serious problem of the emergence of drug-resistant mutants. Subtype B HIV-1 is dominant in advanced countries, and the mortality rate due to subtype B HIV-1 has been decreased during the past decade. In contrast, the number of patients with non-subtype B viruses is still increasing in developing countries. One of the reasons for the prevalence of non-subtype B viruses is lack of information about the biological and therapeutic differences between subtype B and non-subtype B viruses. M36I is the most frequently observed polymorphism in non-subtype B HIV-1 proteases. However, since the 36th residue is located at a non-active site of the protease and has no direct interaction with any ligands, the structural role of M36I remains unclear. Here, we performed molecular dynamics (MD) simulations of M36I protease in complex with nelfinavir and revealed the influence of the M36I mutation. The results show that M36I regulates the size of the binding cavity of the protease. The reason for the rare emergence of D30N variants in non-subtype B HIV-1 proteases was also clarified from our computational analysis.     

Extensive genetic polymorphism of four plasma α-protease inhibitors in pigs and evidence for tight linkage between the structural loci of these inhibitors

Summary. Two-dimensional horizontal gel electrophoresis of pig plasma samples (under non-denaturing conditions) using Immobiline pH gradient gels 4.0–6.0 for the first dimension separation, resulted in clear resolution of the variants of four different α-protease inhibitors (protease inhibitor -1 and -2, P11 and P12; postalbumin -1A and -1B, PO1A and PO1B). All these variants were readily visualized by general protein staining. About 900 families each of Swedish Landrace (SL) and Yorkshire (SY) breeds were studied. The extensive inheritance data, including the recombinants encountered, indicated that each of these four inhibitors is controlled by a separate, autosomal locus and that the four loci are tightly linked (spread over a distance of 1–1.5 cM) with the order as Pil-Po1A-Po1B-Pi2 . The alleles observed were two of Pil, 14 of Po1A , 11 of Po1B and 8 of Pi2 . About 40 haplotypes were observed in each of the two breeds. The allele frequencies at Po1A, Po1B and Pi2 loci were remarkably different in the two breeds; the alleles at these three loci showed a very strong linkage disequilibrium (0.8–1.0). The females showed much higher recombination frequencies than the males in the Po1A-Pi2 interval, suggesting that gene conversion-like events may be occurring at these loci. This linkage in pigs and similar ones comprising some plasma α-protease inhibitor genes in humans and in rodents, reported recently in the literature, indicate evolutionary conservation of a homologous linkage group in these species.     

Novel HIV-1 protease inhibitors active against multiple PI-resistant viral strains: coadministration with indinavir

HIV-1 protease inhibitors (PI) with an N-arylpyrrole moiety in the P(3) position afforded excellent antiviral potency and substantially improved aqueous solubility over previously reported variants. The rapid in vitro clearance of these compounds in human liver microsomes prompted oral coadministration with indinavir to hinder their metabolism by the cyctochrome P450 3A4 isozyme and allow for in vivo PK assessment.     

Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds

Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.     

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.     

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.     

Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.