The new enzymology of precursor processing endoproteases.
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Animals↗
Biomedical subjects
S J Chan
Publications and source records attributed to S J Chan.
Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation.
Expression of the cysteine proteinase cathepsin B and its physiological inhibitor cystatin C was analyzed in vitro in 1 human fibrosarcoma and 4 human colon carcinoma cell lines. Cystatin C antigen as well as cathepsin B activity were detected in the conditioned media of the 5 cell lines. The corresponding cell extracts expressed high levels of cathepsin B activity, whereas only trace amounts of cystatin C antigen could be found. Northern-blot analysis revealed the presence in the 5 cell lines of a 0.8-kb cystatin C mRNA transcript and 2 cathepsin B transcripts of 2.3 and 4.3 kb. Pepsin treatment of tumor-cell-released cathepsin B induced an average 7.3-fold increase in activity, indicating that the enzyme was mainly present as a latent form in conditioned medium. The pepsin-activated cathepsin B from one colon carcinoma cell line was further characterized using the cysteine proteinase inhibitors E-64, recombinant cystatin C, a cystatin-C-derived peptidyl inhibitor (Z-LVG-CHN2), and cathepsin-B-specific diazomethyl ketone inhibitors (Z-FT(OBzl)-CHN2, Z-FS(OBzl)-CHN2). This activity was totally neutralized by recombinant cystatin C, suggesting a potential for interaction between released extracellular cathepsin B and cystatin C. In vitro assays of degradation of extracellular matrix showed that cysteine proteinase inhibitors could decrease matrix degradation induced by pepsin-activated conditioned media. With colon cells, this inhibition was not observed, indicating a requirement for an extracellular activation of latent cathepsin B. Our data provide evidence that cystatin C and latent cathepsin B are both released extracellularly by colon carcinoma cells in vitro. They suggest that cystatin C and cathepsin B interactions may participate, in an as yet unelucidated way, in the modulation of the invasive phenotype of human colonic tumors.
Carcinoma↗
Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.
A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.
Amino Acid Sequence↗
Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.
In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.
Amino Acid Sequence↗
Structure and evolution of insulin and insulin-like growth factors in chordates.
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Amino Acid Sequence↗
The biosynthesis and processing of neuroendocrine peptides: identification of proprotein convertases involved in intravesicular processing.
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Amino Acid Sequence↗
Evolution of the insulin gene superfamily. Sequence of a preproinsulin-like growth factor cDNA from the Atlantic hagfish.
Complementary DNAs encoding a preproinsulin-like growth factor (prepro-IGF) have been cloned from a primitive vertebrate species, the Atlantic hagfish, by using a DNA amplification strategy based on the polymerase chain reaction. A composite sequence containing a 414-nucleotide open reading frame encoding 138 amino acids and 164 nucleotides in the 3'-untranslated region was obtained. The deduced partial sequence of hagfish prepro-IGF reveals that it is organized like the mammalian prepro-IGFs with an unusually large (greater than 39-amino acid) signal peptide (initiator methionine residue is missing), 29-amino acid B, 15-amino acid C, 21-amino acid A, 10-amino acid D, and 26-amino acid E domains. All the invariant residues necessary to form the correct tertiary fold of an insulin-like molecule have been conserved in hagfish IGF. Sequence comparisons revealed that the A and B domains of hagfish IGF are equally similar to those of human IGF-I (35 out of 50 amino acids) or IGF-II (37 out of 53 amino acids). In contrast, the similarity between hagfish and mammalian pro-IGFs in the C, D, and E domains is relatively low. Northern blot analysis of RNA isolated from hagfish brain, heart, liver, skeletal muscle, and islet organ, however, indicated that hagfish IGF, like mammalian IGF-I, is expressed predominantly in the liver as a 4.2-kilobase transcript. DNA blot analysis revealed that hagfish IGF is a single copy gene. The predicted sequence of hagfish prepro-IGF thus demonstrates that the divergence of the IGF and insulin genes occurred prior to the separation of the Agnatha and that the organization and tertiary structure of IGF have been well maintained throughout 550 million years of vertebrate evolution.
Amino Acid Sequence↗
Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.
PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.
Adrenal Gland Neoplasms↗
The structure of the mouse cathepsin B gene and its putative promoter.
The mouse cathepsin B gene and its flanking regions were cloned and characterized. The gene contains 10 exons and 9 introns spanning about 20 kb. Although the exon-intron organization of the mouse cathepsin B gene showed some similarity to the rat cathepsin H and L genes, significant differences were found. In particular, the highly conserved sequence that contains the catalytically active cysteine in these genes is split at different sites by an intron. As with other thiol proteinases, there is no obvious correspondence between the coding exons and structural or functional units within preprocathepsin B. These results suggest that the lysosomal thiol proteinase genes are evolutionarily ancient and that intron shifting has occurred subsequent to their divergence from a common ancestral form. The 5'-flanking region and exon 1 sequences in the mouse cathepsin B gene have a high GC content of approximately 72%. The 5'-flanking region also contains several potential Sp1 binding sites, but lacks TATA and CAAT motifs. These characteristics suggest that cathepsin B is a "housekeeping" gene and its transcription may be controlled by multiple transcription factors, including Sp1.
Amino Acid Sequence↗
The expression of cathepsin B and other lysosomal proteinases in normal tissues and in tumors.
The mRNA for the lysosomal proteinases cathepsins B, D, H, L, and S are broadly distributed in normal rodent tissues. Although total cathepsin mRNA levels generally parallel the protein catabolic activity of the tissues, the expressions of the individual enzymes do not appear to be linked. Thus, the relative proportions of the individual messages are found to vary from tissue to tissue. Further evidence for the independent regulation of lysosomal proteinase expression is derived from observations of selective increases in mRNA levels for individual proteinases in rodent tumors. Only cathepsin B mRNA is elevated in a highly metastatic murine B16a melanoma and in a Walker-256 rat carcinosarcoma, while Moloney murine sarcoma virus-transformed fibroblasts express increased mRNA for cathepsins B, D, and L and normal levels for H and S. To address the regulation of cathepsin B expression, the mouse cathepsin B gene and its 5'-upstream region were cloned. The gene has 10 exons and 9 introns spanning about 20 kilobases. The 5'-upstream region and exon 1 are GC-rich with several potential Sp1 binding sites. TATA and CAAT motifs adjacent to the transcription start site are not evident. These properties are characteristic of mammalian "housekeeping" genes. B16 melanoma cells contain three cathepsin B transcripts of 2.2, 4.0 and 5.0 kilobases. The two larger messages, which were not found in normal tissues, contain unusually long 3'-untranslated regions resulting from the alternative cleavage and polyadenylation of the 3' end of the cathepsin B pre-mRNA in B16 melanomas. As all three messages encoded normal preprocathepsin B, cathepsin B secretion by melanoma cells is probably due to posttranslational mechanisms and not to alternative splicing or gene mutation.
Animals↗
Changes in the expression of elastase and cathepsin B with differentiation of U937 promonocytes by GMCSF.
The human promonocytic cell line, U937, when treated for up to 72h with 12,O,tetradecanoyl-phorbol-13-acetate or granulocyte-macrophage colony-stimulating factor, exhibited increased phagocytic activity and expression of the marker p150/95. There was an associated increase in the monocyte proteinase cathepsin B and its mRNA but decreased cellular levels of neutrophil elastase and elastase mRNA. Granulocyte-macrophage colony-stimulating factor therefore causes differentiation of U937 cells, with appropriate effects on the synthesis of leukocyte proteinases.
Cathepsin B↗
Molecular cloning of rat precursor cathepsin H and the expression of five lysosomal cathepsins in normal tissues and in a rat carcinosarcoma.
1. A rat cathepsin H cDNA was isolated from a rat liver cDNA library with synthetic oligonucleotide probes. 2. DNA sequence analysis indicated that it codes for rat preprocathepsin H. 3. Using this clone together with the cDNA for cathepsins B, D, L and S as probes, the expression of five major lysosomal proteinases was investigated in ten different normal rat tissues and in a rat carcinoma. 4. The common feature of their expression is that the five cathepsins have relatively high mRNA levels in lung and kidney, suggesting that they all play important roles in organs engaged in active protein metabolism. 5. In other tissues, the concentrations of the five cathepsin mRNAs are significantly different. This may indicate that their expressions are differentially regulated and that they may have specialized functions in specific tissues. 6. The cathepsin B mRNA level is at least 2.5-fold higher in the rat W256-carcinoma than in any of the normal rat tissues surveyed. 7. In contrast, the mRNA levels for the other four cathepsins show no comparable elevations. 8. This finding is consistent with previous observations reporting a correlation between cathepsins B expression and malignant tumors.
Amino Acid Sequence↗
Evolution of the insulin superfamily: cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus.
Although insulin and the insulin-like growth factors (IGFs) share marked similarities in amino acid sequence and biological activity, their evolutionary origins have not been resolved. To investigate this issue, we recently cloned a cDNA encoding an insulin-like peptide (ILP) from a primitive chordate species, amphioxus (Branchiostoma californiensis). The deduced sequence of amphioxus preproILP indicates that it is a hybrid molecule containing features characteristic of both insulin and IGF. Like proinsulin, amphioxus proILP contains a C-peptide, which is flanked by paired basic residues and is probably removed by proteolysis. However, proILP also contains an extended carboxyl-terminal peptide region that can be divided into D and E domains similar to those of proIGF. Sequence comparisons show that the amphioxus ILP A and B domains are equally homologous to those of human insulin and IGF-I and -II. Based on these results and the exon-intron organization of the amphioxus ILP gene, we propose that IGF emerged at a very early stage in vertebrate evolution from an ancestral insulin-type gene.
Amino Acid Sequence↗
Lessons learned from molecular biology of insulin-gene mutations.
Studies on naturally occurring and man-made mutations in the insulin gene have provided new insights into insulin biosynthesis, action, and metabolism. Ten families have been identified in which one or more members have single-point mutations in their insulin genes that result in amino acid substitutions within the proinsulin molecule. Six of these cause the secretion of biologically defective insulin molecules due to changes within the A or B chains. Replacing A3-Val with Leu, B24-Phe with Ser, or B25-Phe with Leu results in molecules that have essentially normal immunoreactivity but greatly reduced insulin-receptor-binding potency. Individuals with these mutations have a syndrome of mild diabetes or glucose intolerance, which is inherited in an autosomal-dominant mode and is associated with hyperinsulinemia and altered insulin-C-peptide ratios. Although affected individuals are heterozygous and coexpress both normal and abnormal molecules, the elevated circulating insulin consists mainly of the biologically defective form, which accumulates because it fails to be rapidly metabolized via receptor-mediated endocytosis. Four additional families have mutations that are associated with relatively asymptomatic hyperproinsulinemia. A point mutation affecting proinsulin occurs in 3 of the 4 families, leading to replacement of Arg-65 by His, which prevents recognition of the C-peptide-A-chain dibasic cleavage site by the appropriate beta-cell processing protease and results in the circulation of a type II proinsulin intermediate form (des 64, 65 HPI). Members of a fourth family with hyperproinsulinemia have a substitution of B10-His with Asp, resulting in a proinsulin that exhibits markedly altered subcellular sorting behavior.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat insulins I and II.
We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.
Amino Acid Sequence↗
Immunological and chemical characterization of bovine preproinsulin.
Fetal bovine pancreatic poly(A)-containing RNA directs the synthesis of an insulin immunoreactive polypeptide that is larger than proinsulin, preproinsulin, in the wheat germ cell-free translation system. We have characterized this peptide in detail both immunologically and chemically and have shown that it is 2500 daltons larger than bovine proinsulin (8700 daltons), possesses both insulin and bovine C-peptide-specific antigenic determinants, and contains all the tryptic peptides found in bovine proinsulin. Preproinsulin synthesized in the wheat germ cell-free system was precipitated with approximately 4-fold greater efficiency by bovine proinsulin antiserum than by insulin antiserum. Additional evidence was obtained which indicated that the preprotein folds and undergoes correct sulfhydryl oxidation less efficiently than proinsulin, perhaps due to the presence of the hydrophobic NH2-terminal extension. Automated sequential Edman degradation of bovine preproinsulin revealed the presence of an additional NH2-terminal sequence of 23 residues, preceding the B chain segment of proinsulin. The positions of 6 of the 7 leucine residues found in the bovine preproinsulin extension were identical to those reported previously for the rat preproinsulins. This close sequence similarity between the extensions of the bovine and rat preproinsulins supports the hypothesis that these molecules fulfill similar biosynthetic functions in vivo.
Amino Acid Sequence↗
Insulin as a cellular growth regulator.
Previous studies from this laboratory have shown that insulin treatment greatly stimulates liver cell growth in chronically diabetic rats. The effect is striking, being preceded by massive glycogen deposition and water imbibition as well as enchanced RNA and protein synthesis. More recently we have directly confirmed the stimulatory effects of insulin on liver RNA and glycogen synthesis in intact hepatocytes isolated from normal and diabetic rats. The hepatocytes specifically bind insulin (KD = 3.5X10(-9) M) and the bound hormone is degraded systematically, indicating the possibility of receptor-mediated uptake of the hormone. Recent studies in other laboratories of the growth factors, non-suppresible insulin-like activity (NSILA) and nerve growth factor (NGF), as well as of relaxin, have disclosed structural relationships between these peptides and pro-insulin, indicating the existence of families of closely related anabolic effectors. A new hypothesis regarding the role of the insulin-receptor complex in initiating the metabolic and mitogenic effects of the hormone is briefly discussed.
Amino Acid Sequence↗
Biosynthesis of insulin and glucagon: a view of the current state of the art.
It is now well established that insulin biosynthesis proceeds through a precursor molecule, proinsulin. This single polypeptide chain form has been identified as a ribosomal product in the microsomal fraction from islet tissues. The newly synthesized peptide chain, after folding and thiol oxidation, is transferred to the Golgi apparatus where it begins to undergo proteolytic processing to insulin and packaging into secretory granules. The secretion from the cells of significant amounts of newly synthesized material by exocytosis begins only one hour or more after biosynthesis and this process is regulated by several factors, including glucose. Foci of current attention discussed in this paper include (1) the possible existence of larger precursor forms than proinsulin, especially short-lived biosynthetic transients with extended NH2-termini analogous to the recently described immunoglobulin L chain and proparathyroid hormone precursors; (2) the large-scale production of insulin by chemical or genetic engineering approaches; (3) isolation of beta-cell plasma membranes; (4) regulatory mechanisms for the biosynthesis and secretion of insulin, the possible role of mRNA modification in this process, and effects of somatostatin on insulin biosynthesis and secretion; (5) studies on the secretion, metabolism and clinical usefulness of the proinsulin C-peptide; (6) finally, the biosynthesis of glucagon and other peptide hormones and the general significance of precursor forms.
Adenylyl Cyclases↗