Genetic polymorphism and close linkage of two α1-protease inhibitors in horse serum

Two-dimensional electrophoretic analysis of horse serum proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α-globulins. Two groups of aj-globulins, designated Pi1 and Pi2, were found to be protease inhibitors. Preliminary studies indicated that Pi1 and Pi2 proteins differed from each other in molecular weight and in protease inhibiting spectra. Extensive polymorphism was observed for both these proteins. Family data supported the hypothesis that Pi1 and Pi2 types were controlled by autosomal codominant alleles. For both Pi1 and Pi2 systems, most of the homozygous types showed two fractions each while the heterozygous types had 4 fractions. Six Pi1 and five Pi2 alleles were observed in two breeds of Swedish horses. Complete genetic linkage was observed for Pi1 and Pi2 loci as no recombinant type was observed in 40 informative matings studied.     

Genetic polymorphism and close linkage of two plasma protein loci in dogs

By using a simple method of two-dimensional horizontal electrophoresis, phenotypes of an unidentified plasma protein (PA4) were determined in 967 dogs belonging to 43 different breeds. Two codominant, autosomal alleles (F and S) of PA4 were reported. While many of the breeds of middle and north-eastern Asia (akita inu, Alaskan malamute, chow chow, samoyed, Siberian husky and Tibetan terrier) showed a substantial frequency (0.1 to 0.6) of the S allele, a majority of the European breeds had only the F allele. Evidence was provided that the PA4 locus is closely linked to the plasma pretransferrin 1 locus (PRT1). No recombinant was observed in 45 informative offspring studied. In nearly all breeds, the PA4 S allele was almost always in coupling phase with the PRT1 F allele.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Role of disulfide exchange in alpha 1-protease inhibitor.

The major endogenous inhibitor of neutrophil elastase in the plasma, alpha 1-protease inhibitor (alpha 1-PI), has a single cysteine residue which has been shown to form mixed disulfides with a number of thiols in vitro. Under normal physiological conditions, the plasma concentrations of reduced and oxidized thiols are such that a major fraction of alpha 1-PI in the circulation in vivo is in the form of mixed disulfides [Laurell, C.-B. (1979) in The Chemistry and Physiology of Human Plasma Proteins (Bing, D. H., Ed.) pp 329-341, Pergamon, New York]. We show here that the mixed disulfide between glutathione or cysteine and alpha 1-PI (alpha 1-PI-SSG or alpha 1-PI-SScys) has an intrinsic fluorescence which distinguishes it from the reduced form of alpha 1-PI. By employing the fluorescence difference, we have measured the ratio of alpha 1-PI-SH to mixed disulfide alpha 1-PI in redox buffers of different ratios of reduced to oxidized glutathione (GSH to GSSG) or reduced to oxidized cysteine (cys to cysSScys) and have calculated an equilibrium constant and redox potential of 0.74 +/- 0.08 and 8 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SSG couple and of 0.32 +/- 0.02 and 29 +/- 2 mV, respectively, for the alpha 1-PI-SH/alpha 1-PI-SScys couple. We are unable to detect any change in Trp fluorescence in the complex of alpha 1-PI and elastase when the preformed complex is added to the same GSH/GSSG or cys/cysSScys redox buffers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles ( D,F,I,S ). Evidence was provided that the F allele can be further divided into two alleles ( F ₁ and F ₂); the mobilities of F₁ and F₂ variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Genetic polymorphism of horse serum protein 3 (SP3)

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles (D, F, I, S). Evidence was provided that the F allele can be further divided into two alleles (F1 and F2); the mobilities of F1 and F2 variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum.

Horizontal polyacrylamide gel electrophoreses, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, GcF and GcS, and three Pa alleles, Pa D, Pa F and Pa S, were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. GcF and PaF, ranged from 0.72-0.93 and from 0.58-0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the GcS fraction and thus were found unsuitable for Gc typing.     

Plasma protein and liver mRNA levels of two closely related murine alpha 1-protease inhibitors during the acute phase reaction

Plasma levels of alpha 1-PI(T) and alpha 1-PI(E), two closely related murine alpha 1-protease inhibitors, having affinities for trypsin and elastase, respectively, were compared to changes in specific liver mRNA levels after induction of the acute-phase reaction by subcutaneous injection of turpentine. In earlier, qualitative experiments an increase in plasma levels of alpha 1-PI(E), but not alpha 1-PI(T), during the acute-phase reaction had been shown. It is now shown that stimulation of plasma alpha 1-PI(E) levels reaches a maximum of 35-50% above baseline 12 h after induction of the acute-phase response using either a functional or immunological assay to measure protease inhibitor activity. Consistent with earlier observations, little or no change in plasma levels of alpha 1-PI(T) is seen. Determination of mRNA levels in the mouse liver specific for alpha 1-PI(E) and alpha 1-PI(T) was accomplished using a cell-free translation system followed by immunoprecipitation of the 35S-labeled protease inhibitors. The apparent Mr's of alpha 1-PI(E) and alpha 1-PI(T) synthesized in vitro are 42K and 46K, respectively. Apparent Mr's of the native proteins in plasma are 55K and 65K. Unexpectedly, mRNA levels for both alpha 1-PI(E) and alpha 1-PI(T) were found to increase after induction of the acute-phase reaction. Maximal stimulation for both mRNAs was approximately 300% and occurred 9 h after turpentine administration. Under these conditions, levels of translatable albumin mRNA in the mouse liver decreased to 40% of baseline in 6-9 h.     

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.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.     

Structure of the oligosaccharide chains in human alpha 1-protease inhibitor.

Two glycopeptides were obtained from alpha 1-protease inhibitor after extensive pronase digestion and chromatography on Bio-Gel P-10 and concanavalin A-Sepharose. these glycopeptides were characterized by compositional analysis and sequential exoglycosidase digestion followed at each step by methylation analysis. The partially methylated alditol acetates obtained were resolved by gas chromatography and identified by mass spectrometry. The proposes structures of the oligosaccharide moieties of the glycopeptides are given below. (formula: see text) The relative amounts of the two glycopeptides isolated from concanavalin A-Sepharose suggest that each protein molecule contains four carbohydrate chains; one large chain (A) and three small chains (B).     

Two-dimensional electrophoresis of horse serum proteins: genetic polymorphism of ceruloplasmin and two other serum proteins

Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum proteins revealed genetic polymorphism of ceruloplasmin (Cp) and two unidentified serum proteins tentatively designated serum protein 1 (SP1) and serum protein 2 (SP2). Family data were consistent with the hypothesis that the observed Cp and SP1 phenotypes were each controlled by two co-dominant, autosornal alleles. The three common SP2 phenotypes were shown to be controlled by two codominant, autosomal alleles. Population data and limited family data indicated the occurrence of two additional SP2 alleles. Altogether more than 600 horses representing 13 different breeds were typed for Cp, SP1 and SP2, and allele frequency estimates were calculated. SP2 was highly polymorphic in all breeds studied whereas SP1 and Cp showed quite low degrees of polymorphism. SP1 polymorphism was observed in seven breeds while Cp polymorphism was observed only in the Icelandic toelter horse breed.     

Genetic polymorphism of alpha 2HS-glycoprotein.

A genetic polymorphism of the human serum glycoprotein, alpha 2HS-glycoprotein, can be recognized using isoelectric focusing in polyacrylamide, followed by silver-stain immunofixation. In a North American Caucasian population, two common alleles and one rare allele have been recognized, with frequencies as follows: AHSG*1: .6419, AHSG*2: .3535, and AHSG*3: .0046; polymorphism information content (PIC): .36. A black population from various islands of the Caribbean has the two most common alleles, plus a variant (B) not found in the white population. Allele frequencies in the blacks were: AHSG*1: .6901, AHSG*2: .2606, AHSG*B: .0493; PIC: .396. Family studies confirmed the allele designations. Alleles in both populations were in Hardy-Weinberg equilibrium. This polymorphism will be useful as a marker on chromosome 3q and for forensic studies. The serum concentration associated with AHSG*1 may be somewhat greater than that associated with AHSG*2. Differences between the allele products remained after removal of sialic acid from the glycoprotein with neuraminidase. The silver-stain immunofixation technique used for this polymorphism has wide application for the study of polymorphisms where the protein is present in low concentration or where only low titer antiserum is available.     

Divergent expression of alpha1-protease inhibitor genes in mouse and human.

The alpha1-protease inhibitor proteins of laboratory mice are homologous in sequence and function to human alpha1-antitrypsin and are encoded by a highly conserved multigene family comprised of five members. In humans, the inhibitor is expressed in liver and in macrophages and decreased expression or inhibitory activity is associated with a deficiency syndrome which can result in emphysema and liver disease in affected individuals. It has been proposed that macrophage expression may be an important component of the function of human alpha1-antitrypsin. Clearly, it is desirable to develop a mouse model of this deficiency syndrome, however, efforts to do this have been largely unsuccessful. In this paper, we report that aside from the issues of potentially redundant gene function, the mouse may not be a suitable animal for such studies, because there is no significant expression of murine alpha1-protease inhibitor in the macrophages of mice. This difference between the species appears to result from an absence of a functional macrophage-specific promoter in mice.     

Interaction of chymotrypsinogens with alpha 1-protease inhibitor

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.     

Concentration of some proteinase inhibitors: alpha 1-antitrypsin and alpha 2-macroglobulin in rabbit blood serum in two models of experimental atherosclerosis.

In rabbits with experimental atherosclerosis induced by a cholesterol-rich diet, alpha 1-antitrypsin concentration was decreased as compared with control, by 34%, whereas alpha 2-macroglobulin concentration was increased by 86%. In animals fed a methionine-rich diet changes in concentration of both inhibitors involved in elastase metabolism were but slight, if any.     

Characterisation of the alpha 1-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 alpha 1-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 method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.

Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.