Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris

Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha(2)beta(2) E1. Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1). The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation.     

Changes in serum level and affinity for concanavalin A of human alpha 1-proteinase inhibitor in severe burn patients: relationship to natural killer cell activity

In serum from five patients with severe burns, alpha 1-proteinase inhibitor (alpha 1-PI) was analyzed and then isolated by immunosorption chromatography. By Con A-Sepharose chromatography alpha 1-PI was separated into two types of fractions: the first containing the Con A-non-reactive isoforms and the second containing the Con A-reactive isoforms. The increase of alpha 1-PI serum level in burn patients is associated on the fifth day after the burn with a significant shift toward species enriched in bi-antennary oligosaccharides (Con A-reactive isoforms). This latter change passed very quickly and ten days after the burn, whereas the alpha 1-PI serum level was still high, the difference in proportions of Con A-reactive and non-reactive isoforms was not statistically significant. With respect to the difference in oligosaccharide structure, it appeared that the glycan moiety was involved in the inhibitory effect on natural killer cell activity. At the same concentration, purified alpha 1-PI and retained alpha 1-PI isoforms had an equal effect, whereas the non-retained alpha 1-PI isoforms were more efficient (P less than or equal to 0.01). Purified alpha 1-PI and its isoforms inhibited the natural killer cell activity in a dose-dependent manner.     

Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.     

Distinct and additive effects of elastase and endotoxin on expression of alpha 1 proteinase inhibitor in mononuclear phagocytes

Expression of alpha 1 proteinase inhibitor (alpha 1-PI) in human mononuclear phagocytes may provide a local mechanism for inactivation of serine proteases at sites of tissue injury, thereby preventing incidental damage to surrounding tissue and allowing for orderly initiation of repair. We have previously shown that serine (neutrophilic or pancreatic) elastase and lipopolysaccharide (LPS) each mediate an increase in the expression of alpha 1-PI in human peripheral blood monocytes and bronchoalveolar macrophages. In this study we demonstrate that elastase and LPS have an additive positive regulatory effect on alpha 1-PI expression. Distinct pretranslational and translational mechanisms of action for elastase and LPS, respectively, account for the additive effect. The possibility that translational regulation of alpha 1-PI by LPS involves a mechanism analogous to that of the yeast gene GCN4 during amino acid starvation and that of the human ferritin gene in response to iron is discussed.     

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.     

Hydroxyl radical production by H2O2 plus Cu,Zn-superoxide dismutase reflects the activity of free copper released from the oxidatively damaged enzyme.

To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD.     

Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris.

Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a [1.7 micromol.min-1.(mg protein)-1] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.     

Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1 (Yps1p) endoprotease

Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family. A chimeric gene encoding polyhistidine-tagged human pre-elafin fused to the yeast alpha-factor precursor was expressed in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature alpha-factor peptide. This enabled the use of a simple bioassay (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule. We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endoprotease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues. Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin. The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography. Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin.     

Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants

Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.     

The effect of reducing agents on proteolytic enzymes and oxidation of alpha 1-proteinase inhibitor

We have studied the effect of the mucolytic agent N-acetylcysteine and dithiothreitol on the oxidation of alpha 1-PI by hydrogen peroxide, and their effect on porcine pancreatic elastase and leukocyte elastase. In addition, the effect of S-(carboxymethyl)cysteine (= carbocisteine, a mucolytic agent which does not have reducing properties) was studied in vitro and in patients with chronic obstructive bronchitis. Following addition of 59.6mM N-acetylcysteine, the amidolytic activity of leukocyte elastase was decreased by 55.3% and that of porcine pancreatic elastase by 57.0%. Dithiothreitol (5.7 mM) caused the loss of 97.4% and 67.6% of amidolytic activity of leukocyte elastase and porcine pancreatic elastase respectively whereas S-(carboxymethyl)cysteine had no effect. Similar results were found for the effect on elastolytic activity. Oxidation of alpha 1-PI by 8.6mM H2O2 resulted in partial loss of inhibitory function (mean 68.7% activity of native alpha 1-PI). N-Acetylcysteine and dithiothreitol prevented oxidation of alpha 1-PI when pre-incubated with H2O2 or incubated with alpha 1-PI and H2O2 simultaneously (94.5% and 94.4% activity of native alpha 1-PI for N-acetylcysteine; 78.3% and 87.6% activity for dithiothreitol - p less than 0.025). S-(Carboxymethyl)cysteine, when pre-incubated with H2O2 or incubated concurrently with alpha 1-PI and H2O2, caused a further decrease in the porcine pancreatic elastase inhibitory capacity of alpha 1-PI (53.1% and 63.0% respectively - p less than 0.025). None of the agents reversed oxidative inactivation once it had occurred. S-(Carboxymethyl)cysteine had no effect on alpha 1-PI function in sputum at the dose used.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyll and Pichia stipitis gene xyl2,encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively,were amplified by PCR.The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-PI2.Subsequently the vector pYES2-P12 was transformed into S.cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12.The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%.     

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.     

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.     

The effect of catalase and methionine-S-oxide reductase on oxidised alpha 1-proteinase inhibitor

Oxidative damage to alpha 1-proteinase inhibitor (alpha 1-PI) may be important in the pathogenesis of emphysema. We have studied the ability of 2 enzymes (catalase and methionine-S-oxide reductase) to prevent and reverse oxidation of alpha 1-PI by hydrogen peroxide. Pre-incubation of catalase with H2O2 protected alpha 1-PI from oxidation, but the enzyme could not reverse prior oxidation of alpha 1-PI. In contrast, methionine-S-oxide reductase fully restored activity to H2O2-oxidised alpha 1-PI. Sputum sol-phase from smokers and non-smokers contained alpha 1-PI that was only about 30% active. Functional activity increased in both smokers (p less than 0.025) and non-smokers (p less than 0.05) approximately 2-fold following incubation with the reductase. Western blotting of the samples showed that about 20% of the alpha 1-PI was present as an enzyme-inhibitor complex and 20% was proteolytically cleaved. These observations suggest proteolysis, complexing with enzyme and oxidation are mechanisms of inactivation of alpha 1-PI in lung secretions.     

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.     

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.     

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.     

A novel mode of polymerization of alpha1-proteinase inhibitor

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.