The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II.

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

Vaccinia virus encodes a family of genes with homology to serine proteinase inhibitors.

Nucleotide sequencing of a region of the vaccinia virus genome proximal to the right inverted terminal repeat (ITR) identified two open reading frames (ORFs) encoding proteins of 39K and 40K with amino acid homology to each other, to another vaccinia virus gene near the opposite end of the virus genome and to the superfamily of serine proteinase inhibitors (serpins). Serpins have now been found in poxviruses from the genera orthopox (cowpox and vaccinia viruses), leporipox (myxoma virus) and avipox (fowlpox virus). One of the vaccinia virus serpins identified here (B13R) shares 92% amino acid identity with the serpin from cowpox virus and 46% and 19% identity with vaccinia serpins B24R and K2L, respectively. The amino acid sequence of B13R reported here differs at 11 positions from a recently reported sequence and contains an additional three internal residues. The serpin genes near the right ITR are separated by 8 kb of DNA. Both genes contain early transcriptional termination signals just downstream of the ORFs and are transcribed in a rightward direction towards the end of the genome. Analysis of mRNAs from virus-infected cells demonstrated that all three vaccinia virus serpin genes are transcribed early during infection. The amino acid sequences at the active sites of these serpins suggest that they may inhibit serine proteinases of differing biochemical specificities. The possible functions of these genes are discussed.

Alpha 1-PI for emphysema due to alpha 1-antitrypsin deficiency.

Alpha 1-antitrypsin deficiency is a genetic disorder that may result in premature pulmonary emphysema. Affected persons are deficient in the protective protein, alpha 1-proteinase inhibitor (alpha 1-PI). The diagnosis should be considered in patients with signs of emphysema by age 40 (especially nonsmokers), those with predominantly lower lobe disease and those with a family history of premature emphysema. Recently, human alpha 1-PI has been purified and is available to prevent the development of disabling emphysema in affected individuals. Weekly infusion of the concentrate is effective in raising serum levels and is very safe, with only rare minor adverse effects.

Tumor-associated expression of a serum protein, termed aX protein (alpha 1-inhibitor III), and its mRNA in rat liver.

A cDNA clone bearing the mRNA sequence for rat alpha X protein (alpha X) was isolated from a cDNA library constructed from rat liver mRNA. The nucleotide sequence of alpha X protein cDNA showed 97% homology with that of the 3'-proximal domain of alpha 1-inhibitor III cDNA. The amino acid sequence deduced from that of alpha X cDNA also exhibited high homology with the primary sequences of alpha 1-inhibitor III and alpha 2-macroglobulin. K231 ascites hepatoma cells were transplanted into male ACI rats, and the level of alpha X mRNA in the liver of the tumor-bearing rats was determined by RNA blot hybridization with the cDNA probe. The serum concentration of alpha X decreased to about 30% of the control value with time after transplantation. The amount of alpha X mRNA in the liver of tumor-bearing rats was proportional to the serum concentration of alpha X. The serum concentrations of transferrin and albumin in the tumor-bearing rats also decreased to about 30 and 60% of the normal levels, respectively. However, the amounts of mRNAs for transferrin and albumin in the liver of tumor-bearing rats did not decrease. These findings indicate that the mechanisms of tumor-associated decrease in the concentrations of different serum proteins in tumor-bearing rats may differ.

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Genetic markers and inflammatory bowel disease: immunoglobulin allotypes (GM, KM) and protease inhibitor.

We have studied immunoglobulin allotypes (GM and KM) in 101 patients with Crohn's disease, 51 patients with ulcerative colitis, and 99 healthy local blood donor controls. In addition, protease inhibitor (PI) types were examined in a random subset of patients and in all controls. No significant differences were found between Crohn's disease patients and controls, or between ulcerative colitis patients and controls, in the frequencies of GM phenotypes, GM haplotypes, KM phenotypes, or PI phenotypes.

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20.

Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a full-length cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.

Widespread expression of amyloid beta-protein precursor gene in rat brain.

The neuritic plaque is a characteristic finding in Alzheimer's disease. A major component of the plaque core is a 4.2 kd polypeptide, amyloid beta-protein (ABP), which is derived from the C-terminus of a larger precursor protein (ABPP). The authors have studied the transcription of ABPP mRNA in the adult rat brain by Northern analysis and in situ hybridization, and report that the ABPP gene gives rise to essentially the same multitranscript family of mRNAs as in the human, and that differential transcription patterns exist between brain and kidney. Morphologically, ABPP mRNA is expressed ubiquitously in neurons of the fore and hindbrain. ABPP transcripts also are present less frequently in occasional glial cells and at moderate to low frequency in nonneural cell types, namely, the choroid plexus epithelium, ependymal cells, and leptomeningeal membranes. Neuronal transcripts are most abundant in cerebral cortical layers II and V, the pyramidal cell layer of the hippocampus, the olfactory cortex, nucleus basis pontis, cranial nerve nuclei, and, significantly, in Purkinje cells and cerebellar granule cells. Because the cerebellum is relatively uninvolved in Alzheimer's disease, these findings suggest that high intraneuronal expression of ABPP may be a necessary but not sufficient requirement for plaque formation.

A male accessory gland peptide with protease inhibitory activity in Drosophila funebris.

Male accessory glands of Drosophila funebris synthesize and secrete a peptide that shows a protease-inhibiting activity. Amino acid sequencing of the purified peptide revealed that the peptide consists of 63 amino acid residues. It is a serine protease inhibitor belonging to the pancreatic trypsin inhibitor (Kunitz) family. The inhibitory function and the kinetic characteristics of the inhibition have been examined with various substrates. The peptide possibly plays a role as an acrosin inhibitor involved in Drosophila reproduction.

[The protease inhibitor system of macaques. The identification of alleles using isoelectric focusing and family analysis]. / Sistema ingibitora proteaz makak. Identifikatsiia allelei s pomoshch'iu izoélektrofokusirovaniia i semeinogo analiza.

Five alpha-1-antitrypsin (alpha-1-AT) phenotypes have been revealed by isoelectrofocusing (IEF) in sera of 215 crab-eating macaques. Alpha-1-AT was monomorphic in sera of 250 Rhesus monkeys. A new allele of macaque Pi-system, designated as B' was postulated in addition to existing two (B and C) on the basis of IEF data. The above conclusion was supported by family analysis, based on 35 monkey birth cases. Alpha-1-AT phenotype frequencies were in agreement with Hardy-Weinberg equation both in wild and capture born crab-eating macaques. Alpha-1-AT was found to be microheterogeneous: several zones of the protein were revealed by IEF and Western blotting with anti human alpha-1-AT serum. The pregnancy caused a sharp increase of one band. This may lead to false identification of alpha-1-AT phenotypes, particularly when acid starch gel electrophoresis is used for alpha-1-AT identification. Such misinterpretation during alpha-1-AT phenotyping may explain the disagreement with Hardy-Weinberg equation described earlier for crab-eating macaques.

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.

Structure and expression of the alternatively-spliced forms of mRNA for the mouse homolog of Alzheimer's disease amyloid beta protein precursor.

The human amyloid beta protein (BP) is a major constituent of the amyloid deposited in the brain of patients with Alzheimer's disease and is derived from a larger precursor protein (BPP). In human three alternatively-spliced forms of BPP mRNA were found and two of them were shown to encode a protease inhibitory activity. We have isolated the corresponding species of cDNA in mice and found that the inhibitor domain is highly conserved through mammalian evolution. The homology between human and mouse was 94.6%. Northern blot using specific probes showed that the mRNA for BPP with inhibitor domain was present in every tissue, particularly at a higher level in the kidney. On the other hand, that without inhibitor domain was found most abundantly in the brain but much less in the kidney and the intestine. These data suggest that the individual BPP mRNA species were produced in a tissue-specific manner in mouse as in the case of human.

Homologous rat hepatic protease inhibitor genes show divergent functional responses to inflammation.

The genes encoding three distinct serine protease inhibitors (Spi) have been cloned from rat liver. These inhibitors are highly homologous with each other and are similar to alpha 1-antitrypsin at the nucleic and amino acid sequence level. Although previous investigators have examined the regulation of the Spi 2 locus by inflammation, the use of various techniques and the complexity of this genetic locus have led to incomplete and somewhat confusing results. Oligonucleotide probes specific for Spi 2.1, Spi 2.2, Spi 2.3, and a 3' mouse cDNA probe for alpha 1-antitrypsin mRNA were used to measure these mRNA after induction of inflammation with subcutaneous turpentine in Fischer rats. alpha 1-Antitrypsin mRNA increased 1.8-fold, and Spi 2.2 increased 7-fold. In contrast, Spi 2.1 and 2.3 mRNA sequences decreased fourfold. The maximal changes occurred between 24 and 48 h after inflammation, with a gradual return toward normal over the next 4 days. Since Spi 2.1, Spi 2.3, and alpha 1-antitrypsin mRNA sequences are responsive to growth hormone, two other growth hormone-responsive mRNA sequences, alpha 2u-globulin and insulin-like growth factor I, were measured, and they also decreased after induction of inflammation. The results of this study show that, despite a marked similarity of nucleotide sequence, Spi 2.1 and 2.3 genes respond very differently from Spi 2.2 and alpha 1-antitrypsin to both growth hormone and inflammation. We speculate that the functions of Spi 2.1 and 2.3 products are different from those of Spi 2.2 and alpha 1-antitrypsin and may involve the regulation of growth.

Vaccinia virus encodes two proteins that are structurally related to members of the plasma serine protease inhibitor superfamily.

Nucleotide sequencing adjacent to the right inverted terminal repetition of the vaccinia virus genome revealed two genes encoding polypeptides that are structurally related to members of the plasma serine protease inhibitor superfamily (SPI). Inclusion in the superfamily is based on extensive amino acid sequence similarities as well as a consensus sequence adjacent to the active-site region near the carboxyl ends of the proteins. The genes designated SPI-1 and SPI-2 are located 10,000 and 17,000 base pairs from the right end of the genome, respectively. The predicted SPI-1 polypeptide is 11 amino acids longer than that of SPI-2, and the deduced masses are 40,471 and 38,125 daltons, respectively. Similarities between SPI-1 and SPI-2 are indicated by the percentage of identical amino acids (44%) and corresponding hydrophobicity plots. The maximum amino acid sequence diversity occurs precisely in the putative active-site region, suggesting that SPI-1 and SPI-2 may inhibit different proteases. SPI-2 is homologous to a previously described cowpox virus gene (D. J. Pickup, B. S. Ink, W. Hu, C. A. Ray, and W. K. Joklik, Proc. Natl. Acad. Sci. USA 83:7698-7702, 1986). Evidence for a cowpox virus homolog of SPI-1 was obtained by DNA hybridization. Thus, the presence of two genes that belong to the plasma serine protease inhibitor superfamily may be characteristic of orthopoxviruses.

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.

Molecular cloning of cDNA for sarcocystatin A and analysis of the expression of the sarcocystatin A gene during development of Sarcophaga peregrina.

Sarcocystatin A is a cysteine proteinase inhibitor purified from the hemolymph of Sarcophaga peregrina larvae [Suzuki, T., & Natori, S. (1985) J. Biol. Chem. 260, 5115-5120]. We isolated a cDNA clone for sarcocystatin A and analyzed the structure and expression of the sarcocystatin A gene. Sarcocystatin A consists of 102 amino acid residues. Significant homology was found between amino acid sequences of sarcocystatin A and other mammalian cystatins, and highly conserved sequences among mammalian cystatins were also found in sarcocystatin A. Using cloned cDNA as a probe, we investigated expression of the sarcocystatin A gene during the development of Sarcophaga. Results showed that this gene was transiently activated in the very early embryonic stage and in the pupal stage, suggesting that sarcocystatin A participates in morphogenesis of larval and adult structures of Sarcophaga.

Structure and negative transcriptional regulation by glucocorticoids of the acute-phase rat alpha 1-inhibitor III gene.

DNA clones representing the negative acute-phase gene coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from a rat genomic library. Structural analysis established the existence of at least four different members of the alpha 1-inhibitor III gene family. Partial DNA sequence analysis of the 5'-terminal regions was performed for the alpha 1-inhibitor III gene and the related alpha 1-inhibitor IV gene. The transcription start site of the alpha 1-inhibitor III gene was located by S1 mapping and primer extension. No stable alpha 1-inhibitor IV mRNA was detected in rat liver. In an experimentally induced acute-phase reaction, the transcription rate of the alpha 1-inhibitor III gene was reduced 12.7-fold at 6 h after stimulation. Four hours after injection of a high dose of dexamethasone into rats, the transcription rate of this gene was reduced 9-fold. Thus, glucocorticoids alone are capable of causing a strong transient down-regulation of the transcription of this gene in rats, independent of other inflammatory mediators. An inverted consensus glucocorticoid responsive element (5'GGACACAATAT3') shared with the glucocorticoid-regulated alpha 1-fetoprotein, alpha 2u-globulin, and alpha 1-acid glycoprotein genes was detected by computer-assisted sequence analysis in the promoter proximal 5'-flanking region of the alpha 1-inhibitor III gene.