Inter-alpha-trypsin inhibitor: emergence of a family within the Kunitz-type protease inhibitor superfamily.

Plasma inter-alpha-trypsin inhibitor (I alpha TI) is a major representative of the superfamily of Kunitz-type protease inhibitors in mammals. Formerly, I alpha TI was considered to be a single polypeptide, but recent molecular genetic studies have demonstrated an unexpected multipolypeptide chain structure. The newly discovered genes and gene products form the basis of a new family of I alpha TI-related protease inhibitors.     

Inter-alpha-trypsin inhibitor and pre-alpha-trypsin inhibitor in health and disease. Determination by immunoelectrophoresis and immunoblotting.

Inter-alpha-trypsin inhibitor (ITI) consists of 3 polypeptides cross-linked by chondroitin sulphate, which is o-glycosidically linked to the smallest of the polypeptides, designated bikunin. Pre-alpha-trypsin inhibitor (p alpha I) consists of bikunin and a fourth polypeptide, also associated by chondroitin sulphate. Crossed immunoelectrophoresis (CIE) of plasma, using immunoglobulins to ITI, revealed 3 precipitation-lines, two of which increased in size during disease. Molecular mass determination by polyacrylamide gel electrophoresis showed that the immunoprecipitates contained mixtures of proteins. Therefore CIE is unfit for quantitation of the individual proteins related to ITI. Immunoblotting suggested that the plasma concentrations of p alpha I and of bikunin was increased in uraemia, rheumatoid arthritis and after trauma. The plasma concentrations of ITI and of p alpha I were decreased in a patient with endocarditis.     

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides.

Four 11-residue peptides based on the Bowman-Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human beta-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.     

Inter-alpha-trypsin inhibitor attenuates complement activation and complement-induced lung injury

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.     

Solubilization of native and derived forms of cellulose by cell-free microbial enzymes

1. Cell-free enzymes from Myrothecium verrucaria and Trichoderma koningii hydrolyse native undegraded cellulose, as found in cotton fibres, in a random manner to short insoluble fibres and to minor amounts of soluble products. 2. Enzyme preparations from M. verrucaria fail to attack the short fibres whereas preparations from T. koningii solubilize them completely to sugars at an optimum pH4.2-4.6. 3. The mode of hydrolysis of cotton cellulose by preparations from T. koningii involves from the earliest stages the formation of reducing sugars, followed closely by the appearance of short fibres, until the insoluble and soluble products each constitute about 40-50% of the weight of the initial substrate. After this stage the quantity of sugars increases at the expense of the insoluble short fibres. 4. Depending upon the method of preparation, derived forms of cellulose may be hydrolysed more slowly, much more rapidly, or at the same rate as cotton fibres by enzyme preparations from T. koningii.     

Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components.

The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels.     

Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).     

Inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.     

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor

Bowman-Birk inhibitor proteins (BBIs), which are potent inhibitors of chymotrypsin-like proteases, do not inhibit human beta-tryptase despite this protein having a chymotrypsin-like fold. We have reported previously that, in contrast, BBI-derived peptides (whose sequences incorporate the solvent exposed reactive site loop motif) are able to inhibit human beta-tryptase. This is due to their small size, which allows them to access the restricted active site(s) of tryptase, which has an unusual tetrameric arrangement with four active sites flanking a central pore. In this paper, we have examined the possibility of creating additional interactions within this pore by adding extensions to the BBI-peptide motif. We have taken the core disulfide-bridged sequence SCTKSIPPQCY and examined a series of extensions, at both the C- and N-termini, that bear a second positively charged Lys residue at their end. The aim was to construct inhibitors that could make additional interactions in tryptase by spanning the gap between adjacent active sites in the enzyme, producing a double-headed inhibitor; a positively charged group was used as the dominant specificity of this enzyme is for a positively charged P1 residue. Both N- and C-terminal extensions are found to produce inhibitors of much increased potency, with a strong dependence of potency on chain length. Moreover, it was found that the C- and N-terminal extensions were able to synergise, with their combination on the same peptide producing an even better inhibitor with a potency 10(4)-fold greater than the original sequence. We suggest that the C- and N-terminal extensions are picking up interactions with separate additional sites on the tryptase, making the doubly extended BBI peptide a tri-functional tryptase inhibitor.     

Hydrogen exchange in native and alcohol forms of ubiquitin.

Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inter-alpha-trypsin inhibitor-related immunoreactivity in human tissues and body fluids.

The distribution of inter-alpha-trypsin inhibitor (ITI) and related inhibitors was investigated in normal human tissues and body fluids by using an enzyme-linked immunosorbent assay (ELISA) and a streptavidin-biotin-peroxidase immunohistochemical technique. ITI-related immunoreactivity was localized in different cell types of various organs, such as liver, kidney, testis, gross intestine, cutis and brain. Specific immunoreactivity was also detected in serum, urine and bronchial mucus. This widespread, but not ubiquitous pattern of localization suggests that, in addition to the well known plasmatic role, ITI and/or ITI-related inhibitors may play a number of different physiological roles in various human tissues.     

1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms

The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.     

Correlation between native and desialyzed forms of orosomucoid

The correlation between ORM phenotypes in untreated serum and ORM phenotypes after isoelectric focusing of neuraminidase-treated serum is demonstrated. ORM subtypes were determined using isoelectric focusing in immobilized pH gradients. Population genetic studies of ORM polymorphism in the Galician population were also carried out.     

Inhibition of lentil copper/TPQ amine oxidase by the mechanism-based inhibitor derived from tyramine

Copper amine oxidase from lentil (Lens esculenta) seedlings was shown to catalyze the oxidative deamination of tyramine and three similar aromatic monoamines, benzylamine, phenylethylamine and 4-methoxyphenylethylamine. Tyramine, an important plant intermediate, was found to be both a substrate and an irreversible inhibitor of the enzyme whereas the other amines were not inhibitory. In the course of tyramine oxidation the enzyme gradually became inactivated with the concomitant appearance of a new absorption at 560 nm due to the formation of a stable adduct. Inactivation took place only in the presence of oxygen and was probably due to the reaction of the enzyme with the oxidation product of tyramine, p-hydroxyphenylacetaldehyde. The kinetic data obtained in this study indicate that tyramine represents a new interesting type of physiological mechanism-based inhibitor for plant copper amine oxidases.     

Isolation and characterization of native and lower molecular weight forms of sheep plasminogen.

Two major forms of native sheep plasminogen (SPg-a) have been isolated from plasma by affinity chromatography. These forms differ in molecular weight, charge characteristics, affinity for epsilon-aminocaproic acid (epsilon-Ahx), and carbohydrate content. Upon treatment of SPg-a with plasmin, lower molecular weight plasminogens can be isolated. A plasminogen (SPg-b) of molecular weight approximately 8,000 less than native plasminogen is rapidly produced when either major plasminogen form is treated with plasmin. The molecular weight differences found in the major SPg-a forms are retained in the SPg-b forms, derived from each SPg-a. Upon protracted treatment of either major form of SPg-a or SPg-b with plasmin, a plasminogen (SPg-c) or molecular weight approximately 32,000 less than SPg-b is produced. A single peptide (P) is also produced in this step. The SPg-c species produced from each original SPg-a major form possess essentially the same molecular weights and carbohydrate compositions; but the P cleaved retains the molecular weight and carbohydrate differences found in each major SPg-a or SPg-b form. A large decrease in the S20,w of SPg-a is observed upon the binding of epsilon-Ahx to this protein. A much smaller alteration in the S20,w of SPg-b and SPg-c is observed upon binding of epsilon-Ahx to these proteins.     

Two-domain structure of the native and reactive centre cleaved forms of C1 inhibitor of human complement by neutron scattering

The C1 inhibitor component of human complement is a member of the serpin superfamily, and controls C1 activation. Carbohydrate analyses showed that there are seven O-linked oligosaccharides in C1 inhibitor. Together with six N-linked complex-type oligosaccharides, the carbohydrate content is therefore 26% by weight and the molecular weight (Mr) is calculated as 71,100. Neutron scattering gives an Mr of 76,000 (+/- 4000) and a matchpoint of 41.8 to 42.3% 2H2O, in agreement with this carbohydrate and amino acid composition. Guinier plots to determine the radius of gyration RG were biphasic. Neutron contrast variation of C1 inhibitor in H2O-2H2O mixtures gave an overall radius of gyration RG at infinite contrast of 4.85 nm, from analyses at low Q, and a cross-sectional RG of 1.43 nm. The reactive centre cleaved form of C1 inhibitor has the same Mr and structure as the native molecule. The length of C1 inhibitor, 16 to 19 nm, is far greater than that of the putative serpin domain. This is attributed to an elongated structure for the carbohydrate-rich 113-residue N-terminal domain. The radial inhomogeneity of scattering density, alpha, is large at 59 x 10(-5) from the RG data and 28 x 10(-5) from the cross-sectional analysis, and this is accounted for by the high oligosaccharide content of C1 inhibitor. The scattering data were modelled using small spheres. A two-domain structure of length 18 nm based on two distinct scattering densities accounted for all the contrast variation data. One domain is based on the crystal structure of alpha 1 antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an extended heavily glycosylated N-terminal domain of length 15 nm, whose long axis is close to the longest axis of the serpin domain. Calculation of the sedimentation coefficient s0(20),w for C1 inhibitor using the hydrodynamic sphere approach showed that a two-domain head-and-tail structure with an Mr of 71,000 and longest axis of 16 to 19 nm successfully reproduced the s0(20),w of 3.7 S. Possible roles of the N-terminal domain in the function of C1 inhibitor are discussed.     

Characterization of ancestral and derived Y-chromosome haplotypes of New World native populations.

We analyze the allelic polymorphisms in seven Y-specific microsatellite loci and a Y-specific alphoid system with 27 variants (alphah I-XXVII), in a total of 89 Y chromosomes carrying the DYS199T allele and belonging to populations representing Amerindian and Na-Dene linguistic groups. Since there are no indications of recurrence for the DYS199C-->T transition, it is assumed that all DYS199T haplotypes derive from a single individual in whom the C-->T mutation occurred for the first time. We identified both the ancestral founder haplotype, 0A, of the DYS199T lineage and seven derived haplogroups diverging from the ancestral one by one to seven mutational steps. The 0A haplotype (5.7% of Native American chromosomes) had the following constitution: DYS199T, alphah II, DYS19/13, DYS389a/10, DYS389b/27, DYS390/24, DYS391/10, DYS392/14, and DYS393/13 (microsatellite alleles are indicated as number of repeats). We analyzed the Y-specific microsatellite mutation rate in 1,743 father-son transmissions, and we pooled our data with data in the literature, to obtain an average mutation rate of.0012. We estimated that the 0A haplotype has an average age of 22,770 years (minimum 13,500 years, maximum 58,700 years). Since the DYS199T allele is found with high frequency in Native American chromosomes, we propose that 0A is one of the most prevalent founder paternal lineages of New World aborigines.