Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.     

Wheat-germ agglutinin is synthesized as a glycosylated precursor

The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [³⁵S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with M_r 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of M_r 23 000 and the mature monomer of M_r 18000. When [³H]mannose was used in labeling studies, only the polypeptide of M_r 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of M_r 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - WGA wheat-germ agglutinin     

A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa.

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.     

Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.     

Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.     

The cytotoxin of Actinobacillus pleuropneumoniae (pleurotoxin) is distinct from the haemolysin and is associated with a 120 kDa polypeptide

Mutants of Actinobacillus pleuropneumoniae strain HK 361 (serotype 2) were isolated which were deficient in type II (Ca2(+)-dependent) haemolysin activity (Hly-). Some of the Hly- mutants secreted a potent, heat-labile extracellular cytotoxic activity against porcine alveolar macrophages. Comparison of cell-free culture supernatant from the parent strain and some Hly- mutants by SDS-PAGE and immunoblotting revealed the loss of a major extracellular polypeptide of 109 kDa. Two Hly- mutants which in addition failed to secrete a 120 kDa polypeptide produced no extracellular cytotoxic activity, suggesting that the 120 kDa protein was the cytotoxin. Antiserum raised to the culture supernatant from a Hly- mutant lacking the 109 kDa polypeptide recognized the 120 kDa band, but not the 109 kDa band, in immunoblots and neutralized the cytotoxic activity, but not the haemolytic activity, of A. pleuropneumoniae. The 120 kDa polypeptide and extracellular cytotoxic activity were widespread among A. pleuropneumoniae strains, but absent from related bacterial pathogens of the pig: Actinobacillus suis, Haemophilus parasuis and Pasteurella multocida. A clear correlation was found between the presence of the 120 kDa polypeptide and cytotoxic activity in culture supernatants. The cytotoxic activity of all the strains tested was neutralized by antibody to the Hly- extracellular material and by convalescent pig serum. It is proposed that the 120 kDa polypeptide represents the cytotoxin of A. pleuropneumoniae, that it is distinct from the haemolysin, and that it be termed pleurotoxin.     

The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes.

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.     

Cytoplasmic p53 polypeptide is associated with ribosomes.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.     

A 220 kDa polypeptide, immunolocalized to epithelial tight junctions, is associated with brain clathrin preparations

Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

On inactive precursor of cathepsin D from chicken liver

Two enzyme preparations (components 1 and 11) were isolated from chicken liver by means of DEAE-cellulose chromatography. The highest proteolytic activity was seen in the zone of comparatively low protein concentration. Storage of the isolated first component in acid medium at pH 3 resulted in considerable increase of its activity, as well as in increase of its ability to bind to diazocarboxyl inhibitor. In the course of this reaction an additional, highly active component and an inactive, low-molecular-weight fraction were formed.     

The neuroendocrine polypeptide 7B2 is a precursor protein

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.     

Association of the precursor of cathepsin D with coated membranes. Kinetics and carbohydrate processing

Specific anti-chlathrin antibodies were used to isolate clathrin-coated membranes from homogenates of metabolically labelled fibroblasts. The isolated membranes were extracted with detergents and cathepsin D was isolated from the extracts. The 53-kDa precursor of cathepsin D was transiently associated with the coated membranes with a maximum approximately 60 min after synthesis. At maximum about 4.0% of the precursor was recovered with the coated membranes and the associated precursor contained complex oligosaccharides. The proportion of complex oligosaccharides in the coated membrane-associated precursor did not differ from that in the total precursor. These data support the view that coated membranes are involved in the transport of cathepsin D between trans Golgi and a prelysosomal organelle.     

Lamin A is not synthesized as a larger precursor polypeptide

Isolation of rat liver nuclei in the presence of N-ethylmaleimide (NEM) led to the recovery in the final nuclear matrix of a higher molecular weight form of lamin A. The 2 kDa larger form was identified as lamin A by isoelectric point determination, recognition by an anti-lamin A and C monoclonal antibody and peptide mapping using V8 protease and N-chlorosuccinimide. The 2 kDa extension was tentatively localized to the carboxy-terminus of lamin A. Pulse-chase labeling and immunoprecipitation studies using baby hamster kidney cells showed that lysis of the cells in the presence of NEM allowed the recovery of a stable higher molecular weight form of lamin A. We conclude from these results that NEM prevented the degradation of the native form of lamin A previously thought to represent a higher molecular weight transient precursor form.     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

Human cathepsin H.

Cathepsin H was purified from human liver by a method involving autolysis and acetone fractionation, and chromatography on DEAE-cellulose, Ultrogel AcA 54, hydroxyapatite and concanavalin A-Sepharose. The procedure allowed for the simultaneous isolation of cathepsin B and cathepsin D. Cathepsin H was shown to consist of a single polypeptide chain of 28 000 mol.wt., and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. The enzyme existed in multiple isoelectric forms, the two major forms having pI values of 6.0 and 6.4; it hydrolysed azocasein (pH optimum 5.5), benzoylarginine 2-naphthylamide (Ba-Arg-NNap), leucyl 2-naphthylamide (Arg-NNap), (pH optimum 6.8). Arg-NNap and Arg-NMec, unlike Bz-Arg-NNap-, were not hydrolysed by human cathepsin B. Cathepsin H was similar to cathepsin B in being irreversibly inactivated by exposure to alkaline pH. Sensitivity to chemical inhibitors by 1 microM-leupeptin, which gave essentially complete inhibition of the other lysosomal cysteine proteinases, cathepsins B and L.     

Cystic fibrosis: the ciliary inhibitor is a small polypeptide associated with immunoglobulin G.

A small polypeptide within the molecular weight range of 6,000 to 11,000 was found in immunoglobulin fractions from sera of cystic fibrosis homozygotes and heterozygotes. This factor appears to be responsible for interfering with ciliary activity in oyster gills. Since it can be dissociated from IgG without reductive cleavage it must be bound in a non-covalent manner. After its dissociation the IgG fractions from cystic fibrosis sera no longer inhibited ciliary activity. This finding explains the differences previously observed in the molecular weights of the ciliary inhibitor synthesized by cultured cells and that of sera from cystic fibrosis genotypes.     

Human liver cathepsin L.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.     

Human ACE I/D polymorphism is associated with individual differences in exercise heat tolerance.

We hypothesized that there is an association between the angiotensin I-converting enzyme (ACE) insertion (I)/deletion (D) polymorphism with the variability in exercise heat tolerance in humans. Fifty-eight Caucasian men were exposed to a 2-h exercise heat-tolerance test. We analyzed the association between their heat-tolerance levels with the ACE DD (n = 25) and I+ (n = 33) genotypes and with various anthropometrical parameters and aerobic fitness. It was found that the relative changes in body core temperature, heat storage, and heart rate during the 120-min exposure to exercise heat stress was consistently lower in the I+ genotype group compared with the DD genotype group (0.8 +/- 0.2 vs. 1 +/- 0.1 degrees C, P < 0.05; 17.7 +/- 1.8 vs. 19.8 +/- 1.3 W/M(2), P < 0.05; and 33 +/- 7 vs. 44 +/- 5 beats/min, respectively, P = 0.06). No significant association was found between heat strain response and the anthropometrical measurements or aerobic fitness in the various genotype groups. We suggest that the ACE I+ polymorphism may be considered as a possible candidate marker for increased heat tolerance.     

Human pancreatic polypeptide has a marked diurnal rhythm that is affected by ageing and is associated with the gastric TFF2 circadian rhythm

Normal circadian variations in vasoactive intestinal polypeptide, somatostatin, cholecystokinin and pancreatic polypeptide were measured to determine if these alter with aging and to identify gastrointestinal regulatory hormones that might control the dramatic diurnal variation in the gastric cytoprotective trefoil protein TFF2. Plasma pancreatic polypeptide concentrations showed a marked diurnal rhythm (p < 0.0001). Basal and postprandial pancreatic polypeptide concentrations increased with age (p < 0.01). The timing of the diurnal rhythm was consistent with pancreatic polypeptide inhibiting TFF2 secretion and there was a negative association between pancreatic polypeptide and TFF2 concentrations (p < 0.002). The much higher pancreatic polypeptide concentrations in older people will induce increased satiety that may contribute to 'anorexia of ageing'. These results identify potential therapies for treatment of gastric mucosal morbidity and age-associated loss of appetite.