Assessment of alpha-1-antitrypsin deficiency heterozygosity as a risk factor in the etiology of emphysema. Physiological comparison of adult normal and heterozygous protease inhibitor phenotype subjects from a random population.

For plethysmographic studies of lung mechanics and measurement of pulmonary diffusing capacity, 62 subjects were drawn from a randomly selected population sample. Data obtained from the 24 subjects of heterozygous phenotype for alpha-1-antitrypsin deficiency (PiMZ) were compared by age group with data from 38 normal (PiM) subjects matched for sex, age, and smoking history. Comparison of mean values by age group for lung volumes, diffusing capacity, lung elastic recoil, maximum expiratory flow, and the occurrence of frequency dependence of dynamic compliance revealed no differences between phenotype groups. There was no evidence of an accelerated effect of aging among PiMZ subjects when compared with normal counterparts nor was there evidence of an increased effect of smoking. From these data it appears that the PiMZ phenotype per se is not a risk factor in the development of emphysema.

Regulation of the expression of tissue inhibitor of metalloproteinases and collagenase by retinoids and glucocorticoids in human fibroblasts.

The regulation of the expression of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP) was examined in response to both retinoid compounds and glucocorticoids. Effective retinoids induced a dose-dependent, specific increase in the production of TIMP of approximately two- to threefold by monolayer cultures of human fibroblasts derived from various tissues, while simultaneously causing a decrease in collagenase secretion of similar magnitude. These effects were apparent by 8-12 h in culture and disappeared within 24 h after the withdrawal of retinoid compounds. The retinoid effect on TIMP production was mediated via an increased biosynthesis of new inhibitor protein. Similarly, increased steady state levels of TIMP messenger RNA (mRNA) accompanied by decreased quantities of collagenase mRNA were demonstrated, suggesting transcriptional control of the retinoid action. The data suggest that retinoids co-regulate the expression of collagenase and TIMP, and do so in an inverse manner. Dexamethasone caused a dose-dependent, specific decrease in collagenase production without altering the biosynthesis of TIMP. These findings were paralleled by a marked reduction in collagenase mRNA, without any accompanying change in TIMP mRNA. Therefore, TIMP and collagenase expression appear to be independently modulated by glucocorticoids.

Elastase regulates the synthesis of its inhibitor, alpha 1-proteinase inhibitor, and exaggerates the defect in homozygous PiZZ alpha 1 PI deficiency.

The net balance of neutrophil elastase, an enzyme that degrades many components of the extracellular matrix, and its inhibitor, alpha-1-proteinase inhibitor (alpha 1 PI), is thought to be a critical determinant in the development of destructive lung disease, especially in individuals with homozygous alpha 1 PI deficiency. Synthesis and secretion of alpha 1 PI has been recently demonstrated in cells of mononuclear phagocyte lineage, including peripheral blood monocytes and tissue macrophages. In this study we show that alpha 1 PI gene expression in human monocytes and bronchoalveolar macrophages is affected by a novel mechanism, whereby elastase directly regulates the synthesis of its inhibitor. In nanomolar concentrations, neutrophil or pancreatic elastase mediates a dose- and time-dependent increase in steady state levels of alpha 1 PI mRNA and in the rate of synthesis of alpha 1 PI in human monocytes and bronchoalveolar macrophages. Antisera to neutrophil elastase or pretreatment of elastase with the serine proteinase inhibitor diisopropylfluorophosphate abrogates the effect of elastase on alpha 1 PI expression. Elastase also stimulates the synthesis of alpha 1 PI in monocytes from homozygous PiZZ alpha 1 PI-deficient individuals, but has no effect on the rate of secretion; hence, the enzyme mediates an effect on alpha 1 PI that increases the intracellular accumulation of inhibitor and exaggerates the intrinsic defect in secretion of alpha 1 PI that characterizes the homozygous PiZZ alpha 1 PI deficiency.

Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

A genetic locus closely linked to a protease inhibitor gene complex controls the level of multiple RNA transcripts.

The two major protease inhibitors in mouse plasma are alpha 1-protease inhibitor (alpha 1-PI), putative inhibitor of neutrophil elastase, and contrapsin, an inhibitor in vitro of trypsinlike proteases. We have shown by nucleotide sequence analysis that these two inhibitors are related (R. E. Hill, P. H. Shaw, P. A. Boyd, H. Baumann, and N. D. Hastie, Nature (London) 311:175-177, 1984). Here, we show that the contrapsin and alpha 1-PI genes are members of two different multigene families, each containing at least three genes in mice and rats. We established the chromosomal locations of these genes by analyzing the segregation of restriction fragment length polymorphisms in recombinant inbred mouse strains. These experiments show that the multiple genes in each family are clustered and that the two gene families are closely linked on chromosome 12. Thus the genes for contrapsin and alpha 1-PI are likely to have evolved by duplication of a common ancestral gene. The contrapsin multigene family codes for multiple mRNA transcripts in the liver. There is a genetic difference among inbred mouse strains in the regulation of two of these transcripts. In some inbred strains the transcripts are synthesized constitutively; in others they are induced by inflammation. We mapped in recombinant inbred strains the regulatory locus responsible for this genetic variation and found it is linked to the contrapsin multigene family, which suggests a cis-acting regulatory element. We also found that the contrapsin and the alpha 1-PI multigene families have acquired very different regulatory responses since the time of the gene duplication event.

Glucocorticoid modulation of plasminogen activators and of one of their inhibitors in the human mammary carcinoma cell line MDA-MB-231.

In cultures of the human mammary carcinoma-derived cell line MDA-MB-231, plasminogen activator (PA) activity was reduced substantially following treatment with the glucocorticoid dexamethasone. These cells produced urokinase-type PA (u-PA) and tissue-type PA (t-PA), and both enzymes were decreased in dexamethasone-treated cultures. The drop in u-PA activity was associated with a decrease in the synthesis of single-chain pro-u-PA and in the concentration of u-PA messenger RNA; however, the decrease in u-PA activity was more extensive than could be accounted for by inhibition of enzyme synthesis only, suggesting that postsynthetic events were also involved. The comparatively small dexamethasone-induced decrease in t-PA activity was not associated with a change in the concentration of t-PA messenger RNA. Hence, the two PA genes are differentially regulated by the same hormone. MDA-MB-231 cells also produced a PA-specific inhibitor related to that produced by bovine aortic endothelial cells (PAI-1). This inhibitor was present in two forms: one functionally active, and the other which required activation by sodium dodecyl sulfate; both forms were increased in cultures exposed to dexamethasone. Thus, glucocorticoid-induced inhibition of PA activity in these cells results from a decrease in u-PA synthesis and a concomitant increase in the production of a PA inhibitor.

cDNA and deduced amino acid sequence for the rat hydrophobic pulmonary surfactant-associated protein, SP-B.

Pulmonary surfactant prevents collapse of lung alveoli by lowering surface tension at the air/liquid interface. The hydrophobic surfactant associated proteins SP-B and SP-C have been shown to be important in surfactant function and metabolism. A cDNA clone for rat SP-B was isolated and sequenced. Northern analysis showed mRNA for SP-B was present in whole lung and was greatly enriched in alveolar type II cells, but was not present in brain, kidney, spleen or liver. A full length transcript of the rat SP-B cDNA clone consists of 1536 bases and encodes an open reading frame of 376 amino acids. The predicted molecular mass of the primary translation product is 42 kDa and the predicted molecular mass of the mature protein is 8 kDa. Extensive homology exists between the rat sequence for SP-B and those reported for human and canine SP-B. The position of 25 cysteine residues has been extremely well preserved across all three species. An N-linked glycosylation site in the COOH region has been conserved across all three species. A search of the NIH database revealed homology between rat SP-B and the active site for the mouse contrapsin serum proteinase inhibitor.

Sequences of the genes and polypeptide precursors for two bovine protease inhibitors.

The genes for bovine pancreatic trypsin inhibitor and a homologous protease inhibitor have been characterized using messenger RNA, clones of complementary DNA copies and genomic fragments. Both genes consist of three exons and two introns and are virtually identical in sequence, suggesting that recently they either arose by gene duplication or were subject to gene conversion. However, they differ markedly in their promoter regions and in a segment within the first intron. Perhaps as a consequence, the two genes can be expressed using different transcription start sites, and the two proteins are found in different bovine tissues. Each middle exon encodes primarily the mature protein, while the other two exons define amino- and carboxyl-terminal extensions of 33 or 35 and 7 amino acid residues, respectively. The amino-terminal extensions contain a signal peptide-like sequence, suggesting that the proteins are destined for cellular compartments. The remaining extensions at both ends may be involved in targetting of the proteins, and there are some similarities to other proteins destined for cellular compartments.

Molecular cloning of human C1 inhibitor: sequence homologies with alpha 1-antitrypsin and other members of the serpins superfamily.

Genetic and acquired diseases in man show that the proteolytic activity of the complement component C1 is crucially regulated by C1 inhibitor (C1-INH), a plasma protein whose suspected relatedness to other serine proteinase inhibitors (serpins) contrasts with its atypically large size and high degree of glycosylation. Indeed we have found that the C1-INH polypeptide precursor synthesized in a cell-free system is a 64-kDa protein, hence it exceeds the length of the precursor forms of typical serpins. Seeking more conclusive sequence information and a probe for the structural locus, we isolated C1-INH cDNA clones from a library representing size-enriched human liver mRNA. Nucleotide sequence analysis of a clone covering the carboxyterminal half of C1-INH conclusively documents the relatedness of this protein with the serpins, and reveals 27% amino acid identity with alpha 1-antitrypsin.

Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family.

Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA. Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene. The cloned genes were identified with a probe made from a salivary cystatin cDNA. The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined. The gene, which we name CST1, contains three exons and two intervening sequences. The expected CAT and ATA boxes are present in the 5'-flanking region of the gene. Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA. This gene, which we name CST2, has the same gene organization as CST1. The complete nucleotide sequence of a third gene was determined. It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene. These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene. These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members.

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C.

Recombinant cystatin C producing clones were isolated from a human placenta lambda gt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.

Cloning a synthetic gene for human stefin B and its expression in E. coli.

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.

Total synthesis of the cystatin alpha gene and its expression in E. coli.

A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E. coli. The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter. The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.

Cystatin domains in alpha-2-HS-glycoprotein and fetuin.

We have found that chain A of alpha-2-HS-glycoprotein contains two cystatin domains that show closest similarity to those of kininogen. Most likely, the two proteins diverged after the primary duplication of a single cystatin domain as the two cystatin domains of alpha-2-HS-glycoprotein are more similar, especially in disulfide bonding, to the corresponding domains of kininogen than to each other. We also propose that the carboxyl-terminal (non-cystatin) parts of kininogen and alpha-2-HS-glycoprotein contain homologous segments. We suggest that alpha-2-HS-glycoprotein may act as an inhibitor of the cysteine proteinases responsible for bone resorption. We have also found that fetuin is closely related to alpha-2-HS-glycoprotein.

Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily. Presence of two cystatin domains in the N-terminal region.

A new member of the cystatin superfamily is introduced. Human plasma histidine-rich glycoprotein (HRG) was found to contain 2 cystatin-like sequences in tandem in the N-terminal region. Domain 1 (residues 1-112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113-225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.

Efficient production of native, biologically active human cystatin C by Escherichia coli.

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

mRNA for kininogens and thiostatins (major acute phase alpha 1-proteins) in the liver of kininogen-deficient rats.

mRNAs for low and high molecular weight kininogens (1.6 and 3.0 kb in size, respectively) and for two thiostatins (1.6 kb in size) were found in the liver of kininogen-deficient Brown-Norway (BN/Mai Pfd) rats. The levels of mRNAs for thiostatins, but not those for low and high molecular weight kininogens (arising from a single kininogen gene), increased strongly during acute inflammation. The pattern of DNA restriction sites for the kininogen gene and the thiostatin genes in the mutant rat strain was identical to that in at least four other rat strains.

Pulmonary emphysema: current concepts of pathogenesis.

Pulmonary emphysema is a major public health problem and is primarily a disease of smokers. The pathogenesis of emphysema in smokers is likely to be multifactorial and may involve protease-antiprotease imbalance, abnormal host response to injury, the inactivation of antiproteases by oxidants, and direct damage of lung tissue by pulmonary phagocytes. The data regarding current concepts of pathogenesis of emphysema in smokers are reviewed in this article.

Regulation of proteinase inhibitor genes in food plants.

Proteinase inhibitor proteins in the families Solanaceae, Leguminosae and Graminae are stored in seeds and tubers and are also found to accumulate in leaves in response to pest attacks. The isolation of the proteinase Inhibitor I cDNA and its gene from tomato has provided information concerning proteinase inhibitor synthesis, processing and compartmentation in leaves under pest attacks. Strategies are now being developed using proteinase inhibitor genes, to genetically engineer the quantity and quality of these potentially defensive and highly nutritional proteins of important crop plants.