Role of sialic acid residues in crossed immuno-affinoelectrophoresis of alpha 1-proteinase inhibitor

We have investigated the importance of the degree of sialylation when an acute phase glycoprotein, alpha 1-proteinase inhibitor (alpha 1-Pi), was analysed both by affinity chromatography on concanavalin A (Con A)-Sepharose and by crossed immuno-affinoelectrophoresis (CIAE) using Con A in the first dimension. Human alpha 1-Pi was isolated by immunosorption chromatography and then more or less desialylated. On Con A-Sepharose chromatography no significant difference was observed in the percentage of the two fractions (retained or not retained) whatever the degree of desialylation. In contrast by CIAE this degree was largely involved in the separation of the different isoforms obtained in the first dimension.     

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.     

Qualitative studies of lung lavage alpha 1-proteinase inhibitor

A method is described which enables identification of the molecular size of alpha 1-proteinase inhibitor (alpha 1-PI) in biological fluids. This technique when applied to bronchoalveolar lavage fluids clearly demonstrates alpha 1-PI in three molecular forms; the native molecule (Mr approximately equal to ++54 000), a partially proteolysed form (Mr approximately equal to 49 000) and in a form suggestive of a complex with enzyme (Mr approximately equal to 82 000). Samples showing the presence of native alpha 1-PI inhibited more porcine pancreatic elastase than samples where no native alpha 1-PI was seen or where the predominant form was partially proteolysed alpha 1-PI (p less than 0.01). Although the predominant band of alpha 1-PI was more frequently the partially proteolysed form in current smokers (p less than 0.01), there was no clear difference in the inhibitory function of alpha 1-PI between current smokers and non-smokers and those with and without airflow obstruction.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, JM, Warth, R. and Mu?oz, E. (1978) FEBS Letter, 86, 1-5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (alpha, beta, gamma, delta and epsilon) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants.

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg, Met358----Arg]alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu [( Met351----Glu, Met358----Arg]alpha 1-PI) does not alter activity. [Thr345----Arg, Met358----Arg]alpha 1-PI is rapidly cleaved by thrombin, while [Met358----Arg]alpha 1-PI and [Met351----Glu, Met358----Arg]alpha 1-PI form stable proteinase-inhibitor complexes. The stability of [Thr345----Arg, Met358----Arg]alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived. [Met351----Glu, Met358----Arg]alpha 1-PI and [Met358----Arg]alpha 1-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.     

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.     

Cell surface alpha 1-protease inhibitor on human peripheral mononuclear cells in culture

We have studied expression of alpha 1-protease inhibitor (alpha 1-PI) by human mononuclear cells. alpha 1-PI was detected on 50% of freshly isolated peripheral mononuclear cells. Unless a proliferative stimulus was provided, alpha 1-PI subsequently disappeared from the cell surfaces. Plant mitogens, periodate, neuraminidase-galactose oxidase, or allogeneic cells all were effective stimuli of alpha 1-PI expression. Concanavalin A stimulated de novo synthesis of alpha 1-PI in cell cultures containing both lymphocytes and mononuclear phagocytes, and alpha 1-PI simultaneously appeared on surfaces of activated lymphocytes. Inhibition of protein synthesis by cycloheximide or monocyte depletion abolished de novo alpha 1-PI synthesis, but only monocyte depletion inhibited alpha 1-PI expression. Lymphocytes, but not monocytes, displayed saturable binding of radioiodinated alpha 1-PI. The data are consistent with the interpretation that human mononuclear phagocytes synthesize and secrete alpha 1-PI. When protein synthesis is inhibited, mitogenic stimuli may provoke release of previously synthesized alpha 1-PI from mononuclear phagocytes. Secreted alpha 1-PI then may bind to specific lymphocyte cell surface receptors. This pattern of alpha 1-PI synthesis, secretion, binding, and expression on lymphoid cell surfaces appears to be a common characteristic of many immunologic reactions in vitro.     

A phenyl-beta-galactoside-specific monoclonal antibody reactive with murine and rat NK cells

The phenyl-beta-galactoside (phi-beta-gal)-specific monoclonal antibody (mAb) 49H.8 cross-reacts with the terminal disaccharide structure of the asialo GM1 (AGM1) molecule. It was found to react with phi-beta-gal determinants on murine and rat splenic natural killer (NK) cells, as measured by complement depletion studies. Flow cytometric analysis identified the antigen on two IL 2-dependent cloned murine NK cell lines and the rat large granular lymphocyte leukemia RNK. We have compared the 49H.8 reactivity to that of anti-AGM1 antisera (alpha-AGM1) on NK cells and a panel of NK related killer cells, including bone marrow-derived killer cells, lymphokine-activated killer cells (LAK), and anomalous killer cells (AK). We found that the 49H.8 specificity closely paralleled that of alpha-AGM1. When tested against Con A-reactive T cells, the 49H.8 mAb was less reactive than the alpha-AGM1, indicating that it may be a more specific marker for splenic NK populations than the alpha-AGM1.     

The murine alpha(1)-proteinase inhibitor gene family: polymorphism,chromosomal location,and structure

alpha(1)-Proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors, which function in maintaining homeostasis through regulation of numerous proteolytic processes. In laboratory mice (Mus musculus domesticus), alpha(1)-PI occurs in multiple isoforms encoded by a family of three to five genes that are polymorphic among inbred strains and that are located at the Serpina1 locus on chromosome 12. In the present study, we have characterized the alpha(1)-PI gene family of inbred mice in more detail. We show that mice express seven isoforms, all of which are encoded by genes that map to the Serpina1 locus. In addition, polymorphism at the locus is defined by three haplotypes (Serpina1(b), Serpina1(c), and Serpina1(l)) that differ with regard to both the number and identity of alpha(1)-PI genes. Finally, we present the complete sequence of an 84-kb region of Serpina1 containing a tandem repeat of two alpha(1)-PI genes.     

Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products

Previously we demonstrated that in vivo exposure of humans to NO2 resulted in significant inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the bronchoalveolar lavage fluid. However, alpha 1-PI retains its elastase inhibitory activity in vitro when exposed to 10 times the concentration of NO2 used in vivo. We suggested exogenous oxidants such as O2 and NO2 exert their effect in vivo in part through lipid peroxidation. We investigated the mechanism of inactivation of alpha 1-PI in the presence or absence of lipids under oxidant atmosphere. alpha 1-PI in solutions containing phosphate buffer (control), 0.1 mM stearic acid (saturated fatty acid, 18:0), or 0.1 mM linoleic acid (polyunsaturated fatty acid, 18:2) was exposed to either N2 or NO2 (50 ppm for 4 h). Elastase inhibitory capacity of alpha 1-PI was significantly diminished in the presence of 0.1 mM linoleic acid and under NO2 atmosphere (75 +/- 8% of control, P less than 0.01), whereas there was no change in elastase inhibitory capacity of alpha 1-PI in the presence or absence (buffer only) of 0.1 mM stearic acid under a similar condition (109 +/- 11 and 94 +/- 6%, respectively). The inactivated alpha 1-PI as the result of peroxidized lipid could be reactivated by dithiothreitol and methionine sulfoxide peptide reductase, suggesting oxidation of methionine residue at the elastase inhibitory site. Furthermore the inhibitory effect of peroxidized lipid on alpha 1-PI could be prevented by glutathione and glutathione peroxidase and to some extent by alpha-tocopherol.     

Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking

The transmembrane collagenase MT1-MMP (membrane-type 1 matrix metalloproteinase), also known as MMP-14, has a critical function both in normal development and in cancer progression, and is subject to extensive controls at the post-translational level which affect proteinase activity. As zymogen activation is crucial for MT1-MMP activity, an alpha1-PI (alpha1-proteinase inhibitor)-based inhibitor was designed by incorporating the MT1-MMP propeptide cleavage sequence into the alpha1-PI reactive-site loop (designated alpha1-PI(MT1)) and this was compared with wild-type alpha1-PI (alpha1-PI(WT)) and the furin inhibitory mutant alpha1-PI(PDX). Alpha1-PI(MT1) formed an SDS-stable complex with furin and inhibited proMT1-MMP activation. A consequence of the loss of MT1-MMP activity was the activation of proMMP-2 and the inhibition of MT1-MMP-mediated collagen invasion. alpha1-PI(MT1) expression also resulted in the intracellular accumulation of a glycosylated species of proMT1-MMP that was retained in the perinuclear region, leading to significantly decreased cell-surface accumulation of proMT1-MMP. These observations suggest that both the subcellular localization and the activity of MT1-MMP are regulated in a coordinated fashion, such that proMT1-MMP is retained intracellularly until activation of its zymogen, then proMT1-MMP traffics to the cell surface in order to cleave extracellular substrates.     

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.     

Purification and partial characterization of feline alpha1-proteinase inhibitor (falpha1-PI) and the development and validation of a radioimmunoassay for the measurement of falpha1-PI in serum

Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(1)-PI was estimated at 57,000 and the relative molecular mass (M(r)) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(1)-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline alpha(1)-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline alpha(1)-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of alpha(1)-PI in cats.     

Development of a sensitive peptidase assay: in search of cell associated proteases responsible for the cleavage of preproTGF alpha.

A radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGF alpha) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N-terminal to an octapeptide which is cognate to the N-terminal cleavage sequence of TGF alpha. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell-associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane-bound prepro TGF alpha. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo- and endo-peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1-2% of total cell activity of these enzyme classes.     

Recombinant alpha 1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a natural serine protease inhibitor. Although mainly thought to protect the airways from neutrophil elastase, alpha(1)-PI may also regulate the development of airway hyperresponsiveness (AHR), as indicated by our previous findings of an inverse relationship between lung alpha(1)-PI activity and the severity of antigen-induced AHR. Because allergic stimulation of the airways causes release of elastase, tissue kallikrein, and reactive oxygen species (ROS), all of which can reduce alpha(1)-PI activity and contribute to AHR, we hypothesized that administration of exogenous alpha(1)-PI should protect against pathophysiological airway responses caused by these agents. In untreated allergic sheep, airway challenge with elastase, xanthine/xanthine oxidase (which generates ROS), high-molecular-weight kininogen, the substrate for tissue kallikrein, and antigen resulted in bronchoconstriction. ROS and antigen also induced AHR to inhaled carbachol. Treatment with 10 mg of recombinant alpha(1)-PI (ralpha(1)-PI) blocked the bronchoconstriction caused by elastase, high-molecular-weight kininogen, and ROS, and the AHR induced by ROS and antigen. One milligram of ralpha(1)-PI was ineffective. These are the first in vivo data demonstrating the effects of ralpha(1)-PI. Our results are consistent with and extend findings obtained with human plasma-derived alpha(1)-PI and suggest that alpha(1)-PI may be important in the regulation of airway responsiveness.     

Affinity differences between some commercially available immobilized Con A with rat alpha-fetoprotein

Rat alpha-fetoprotein contains three Con A-affinity molecular variants evidenced by affino-immuno-electrophoresis, (a) Con A-non reactive (52 %), (b) Con A-weakly reactive (31 %), (c) Con A-reactive (17 %). Affinity chromatography on several commercially available Con A-linked agarose displays different alpha-fetoprotein affinity patterns. The Con A-weakly reactive variant can be either bound and eluted with the Con A-reactive fraction or unbound and eluted with the Con A-non reactive one. These differences of chromatographic behaviour should be taken into account in structural, biochemical and biological studies dealing with the Con A-affinity molecular variants of alpha-fetoprotein or of other glycoproteins.     

Glycan uniformity within molecular variants of transferrin with distinct affinity for concanavalin A

Human serum Transferrin has been fractionated on concanavalin A-Sepharose column into three fractions: Con A-non reactive (5 %), Con A-weakly reactive (30 %) and Con A-reactive (65 %). Each Con A-affinity transferrin variant contains a pair of identical oligosaccharide units either tri-branched in the Con A-non reactive variant or two-branched in the Con A-weakly reactive and Con A-reactive transferrin. This is an additional support to our proposal that glycosylation occurs uniformly along each polypeptide chain yielding identical oligosaccharide units at each glycosylated site.     

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.     

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.