Two-step processing of in vivo synthesized rice lectin

The synthesis and processing of rice lectin was followed in vivo in developing rice embryos. Using labelling and pulse-chase labelling experiments, the sequence of events in the synthesis and post-translational modifications of this protein could be determined. The primary lectin product observed in vivo is a high molecular weight precursor (28 K), which is post-translationally converted to a 23 K lectin protein, and in a further step cleaved into two smaller 12 K and 10 K polypeptides. The first step of the processing of the rice lectin is a rather slow process (the precursor has a half-life of about 3 h) and resembles the so-called vectorial processing of cytoplasmically made organellar proteins. The second modification consists of a (slow) proteolytic cleavage of the basic lectin subunit into two smaller polypeptides and resembles somewhat the cleavage of some legume (storage) proteins in their protein bodies.     

Biosynthesis of Storage Proteins in Ripening Agrostemma githago L. Seeds

The synthesis of storage proteins in ripening Agrostemma githago seeds was studied by in vivo pulse and pulse-chase experiments with labeled amino acids and labeled glucosamine. It was found that storage proteins were not synthesized directly, but via cleavage of several large precursor proteins. Two disulfide-linked proteins of 38 and 25 kilodaltons were synthesized via a single large precursor protein. This precursor protein contained internal disulfide bridges, at least one of which is involved in holding the subunit structure together following cleavage of the precursor. A similar mode of biosynthesis was noted for two other disulfide-linked proteins of 36 and 22 kilodaltons. The half-life of the precursors was about 2 hours. This mode of processing is analogous to the synthesis of legumin in legumes and globulin in oats. A third pair of disulfide-bonded proteins (41 and 23 kilodaltons) was synthesized from a precursor protein in several steps. These included a legumin-like cleavage, whereafter the subunits remained disulfide-bonded. Then, from the largest subunit, a part was cleaved off, probably a storage protein of 17 kilodaltons. This 17-kilodalton protein was not disulfide-bonded to the 41 and 23-kilodalton complex. The first processing step was fast, the second slow: The half-lives of the precursors were about 3 and 10 hours, respectively. Finally, a group of 16- and 17-kilodalton proteins was synthesized by cleavage of large precursor proteins, likely in two steps. After cleavage, the proteins were not disulfide-bonded. The half-life of the precursors was short, less than 1 hour. In addition, for the 38-, 23-, and one of the 17-kilodalton proteins, a small decrease of relative molecular weight was observed as a last processing step. This was likely due to deglycosylation.     

Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.     

Structure of the head-tail connector of bacteriophage φ29

The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube.Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre.     

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.     

Characterization of spore appendages from Bacillus cereus strains

AIMS: Further characterization and comparison of spore appendages from Bacillus cereus strains. METHODS AND RESULTS: Appendages were isolated from 10 B. cereus strains from the food industry and food-borne outbreaks. The appendage proteins were dissolved in sample buffer containing 2% SDS and 5% mercaptoethanol at 100 degrees C, and subjected to SDS-PAGE. None of the appendages showed identical protein patterns. Western blots, using antibodies raised against a 3.5 kDa appendage protein, showed that the majority of the appendage proteins reacted with the antibody. Removal of the appendages by sonic treatment of the spores did not alter their heat resistance. The appendages were digested by proteinase K, pepsin, and the enzymes in the detergent Paradigm 10, but not by trypsin or chymotrypsin. Spore adhesion to stainless steel was scarcely affected by removal of the appendages. Digestion of adhered intact spores (with appendages) with Paradigm 10 showed a high degree of variation. CONCLUSIONS: Spore appendages from B. cereus are complex proteinaceous structures that differ among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Information about spore appendages and their involvement in spore adhesion is crucial for improving cleaning methods used for control of bacterial spores in the food industry.     

Formation of Influenza Virus Proteins

Eight virus-specific proteins have been found in chicken embryo fibroblasts infected with fowl plague virus. Among them are two glycoproteins which are the constituents of the hemagglutinin on the virus particle. They are derived from a large precursor glycoprotein by cleavage of a covalent linkage. The reaction can be blocked by the protease inhibitor diisopropylfluorophosphate and the amino acid analogue fluorophenylalanine. This indicates that a peptide bond is cleaved. If infected cells are kept at 25 C, a temperature at which virus maturation is inhibited, the precursor glycoprotein is cleaved at a significantly slower rate than at 37 C. It appears, however, that a reduced synthesis of the carbohydrate-free envelope protein is responsible for the block of virus maturation at 25 C rather than the lower cleavage rate of the precursor.     

Gene expression of D-beta-hydroxybutyrate dehydrogenase. II. Incorporation of pre-BDH into mitochondria

beta-hydroxybutyrate dehydrogenase (BDH), a major protein located in the inner mitochondrial membrane is encoded, as most of mitochondrial proteins, in the nuclear genome. It is synthetized on the free polysomes and post-translationally imported into the mitochondria. The neosynthesized protein is a higher molecular weight precursor. The presequence is cleaved by the matrix protease to give the mature protein. The translocation across the mitochondrial membranes needs energy. The results also indicate that cytosolic factors with low molecular weight are essential in the recognition of precursor by mitochondria and to sort out newly synthetized nuclear encoded mitochondrial proteins from others nuclear encoded proteins.     

Calcium-Dependent Cleavage of Striatal Enriched Tyrosine Phosphatase (STEP)

Striatal enriched phosphatase (STEP) is a family of protein tyrosine phosphatases enriched within the CNS. A member of this family, STEP61, is a membrane-associated protein located in postsynaptic densities of striatal neurons. In this study, we demonstrate that STEP61, is cleaved into smaller isoforms. To clarify the mechanism of cleavage, STEP61 was transiently expressed in NT2/D1 neuronal precursor cells. Exposure of transfected cells to the calcium ionophore, A23187, or to thapsigargin resulted in the rapid cleavage of STEP61. Pretreatment with the calpain inhibitor, calpeptin, or EGTA prevented proteolysis. One of the cleavage products has a relative molecular mass of 33 kDa (STEP33). A protein with the identical mobility is detected following calpain treatment of STEP61 fusion protein or postsynaptic densities purified from rat striatum. Exposure of primary neuronal cultures to glutamate also led to a significant increase in the concentration of a low molecular weight form of STEP. Taken together, these results suggest that in response to a rapid influx of calcium, STEP61, is proteolytically cleaved by calpain, leading to the release of a smaller isoform. This model may explain the rapid appearance of STEP33 in response to transient hypoxia-ischemia in the brain as cells attempt to counter the increase in tyrosine phosphorylation levels following neuronal insults.     

Variability of the Left Atrial Appendage in Human Hearts

Atrial fibrillation increases the risk of thrombus formation. It is commonly responsible for cerebral stroke whereas less frequently for pulmonary embolism. The aim of the study was to describe the morphology of the left atrial appendage in the human heart with respect to sex, age and weight. Macroscopic examination was carried out on 100 left appendages taken from the hearts of the patients aged 18–77, both sexes. All hearts preserved in 4% water solution of formaldehyde carried neither marks of coronary artery disease nor congenital abnormalities. Three axes of appendage orientation were performed. After the appendage had been cut off, morphological examination was performed in long and perpendicular axes. Measurements of the appendages were taken from anatomical specimens and their silicone casts. We classified the left atrial appendage into 4 morphological groups according to the number of lobes. Most left atrial appendages in female population were composed of 2 lobes. In the male group typically 2 or 3-lobed appendages were observed. The mean left atrial appendage orifice ranged from 12.0 to 16.0 mm and the most significant difference in the orifices between males and females was observed in LAA type 2 (about 3.3 mm). A smaller orifice and narrower, tubular shape of the LAA lobes could explain a higher risk of thrombus formation during nonvalvular atrial fibrillation in women. Knowledge of anatomical variability of the LAA helps diagnose some undefined echoes in the appendage during transesophageal echocardiographic examination.     

Properties of a Chloroplast Enzyme that Cleaves the Chlorophyll a/b Binding Protein Precursor : Optimization of an Organelle-Free Reaction

The major light-harvesting chlorophyll a/b binding protein (LHCP) of higher plant chloroplasts is nuclear-encoded, synthesized as a precursor, and processed upon import. We have previously (GK Lamppa, M Abad [1987] J Cell Biol 105: 2641-2648) identified a soluble enzyme that cleaves the LHCP precursor (pLHCP). In this study, we describe the conditions for optimal recovery of the processing activity and provide evidence that the N terminus of pLHCP is indeed cleaved, removing the transit peptide. Two pLHCP deletions were made from a cloned pLHCP gene removing 13 and 21 amino acids, respectively, from the carboxy terminus of the protein. After organelle-free processing, the cleavage products showed a shift in mobility during SDS-PAGE proportional to the size of the precursor truncations, as predicted for N-terminal processing. Unexpectedly, a third truncated precursor lacking 91 residues of the C-terminus was not cleaved although the transit peptide domain was intact, suggesting that this deletion disrupted conformational features of the precursor necessary for processing. The pLHCP processing enzyme is inhibited by 2 millimolar EDTA and the metal chelator 1, 10 phenanthroline at 0.4 millimolar, while being inhibited by EGTA only at high concentrations and insensitive to iodoacetate. Optimal processing occurs at pH 8 to 9, and 26°C. Gel filtration chromatography shows that the pLHCP processing enzyme has an apparent molecular weight of about 240,000. The identical column fractions that process pLHCP also convert the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase to its mature form.     

Amyloid-beta precursor protein processing in neurodegeneration

The amyloid-beta precursor protein is proteolytically cleaved by secretases, resulting in a series of fragments, including the amyloid-beta peptide of Alzheimer's disease. The amyloid precursor protein, when membrane anchored, could operate as a receptor. After cleavage, the soluble ectodomain exerts a trophic function in the subventricular zone. The amyloid-beta peptide itself has a depressant role in synaptic transmission, with both physiological and pathological implications. During the past two years, much time has been invested in determining the molecular pathways that regulate the processing and the signal transduction of the amyloid precursor protein. However, the absence of consistent and informative phenotypes in different loss of function animal models make elucidating the molecular actions of the amyloid-beta precursor protein an ongoing challenge.     

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.     

The role of envelope glycoprotein processing in murine leukemia virus infection.

The murine leukemia virus envelope protein is synthesized as a precursor molecule, Pr85env, which is proteolytically cleaved at an arginine residue to produce two mature envelope proteins, gp70 and p15(E). The results presented here indicate that mutation to lysine of the arginine found at the envelope precursor cleavage site results in a precursor which is cleaved with an efficiency at least 10-fold lower than the efficiency with which the wild-type protein is cleaved. This mutation has been used to investigate the requirement for envelope protein processing in various aspects of retroviral infection. Viruses produced by cells transfected with mutant proviral clones are approximately 10-fold less infectious than wild-type viruses. Mutant viruses are incapable of inducing XC cell syncytium formation and are 100-fold less efficient than wild-type viruses at rendering cells resistant to superinfection. Envelope glycoproteins bearing the lysine mutation are found in reduced amounts on the surface of infected cells, and as a result mutant virions contain significantly less envelope protein than do wild-type virions. The phenotypic effects of the processing mutation described here are most likely the result of this paucity of envelope glycoproteins in virions carrying the mutation.     

Patterning mechanisms and morphological diversity of spider appendages and their importance for spider evolution

The prosoma of spiders bears different gnathal (labrum, chelicerae, pedipalps) and locomotory appendages (legs). In most species these appendages are also used for additional functions, e.g. sensing, mating, and courtship. The opisthosoma is equipped with four pairs of highly specialized appendages. Two pairs of spinnerets are used for silk production and manipulation. The other two pairs of appendages are internalized during development and give rise to a complex respiratory system of book lungs and tracheae. Thus spiders have a number of different appendage types with radically different adult morphologies. Furthermore, all these appendage types display significant additional species specific diversity correlating with a large spectrum of functions of the appendages. Despite this importance of appendage diversity for the evolution of the spiders we know relatively little about the genetic patterning mechanisms producing this diversity of morphology. We review recent advances concerning the developmental genetics of spider appendage diversification, mainly concentrating on open questions and future directions of research. We conclude that the deeper understanding of appendage development and diversity in spiders can contribute significantly not only to evolutionary developmental biology, but also to behavioral biology, speciation research and population genetics, and the study of sexually dimorphic traits.     

Synthesis and processing of cellulase from ripening avocado fruit

The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.     

Properties and origin of filamentous appendages on spores of Bacillus cereus

Some physical, chemical, and immunological properties of filamentous appendages and the exosporium on the spores of Bacillus cereus were examined for the purpose of elucidating the origin of filamentous appendages. The main components of both filamentous appendages and the exosporium were protein and their amino acid compositions were similar in point of a high content of glycine, alanine, threonine, valine, and acidic amino acids and a low content of basic and sulphur-containing amino acids. Treatment with 1 N NaOH at 50 C solubilized the isolated appendages completely and the isolated exosporia partially. In both preparations the solubilized proteins consisted of highly acidic monomeric subunits with molecular weights between 2,000 and 5,000. Treatment of the spores with 2% 2-mercaptoethanol at 37 C resulted in the isolation of long filamentous appendages without segmentation. When the spores were treated with 10% 2-mercaptoethanol, there was partial destruction of the exosporium as well as detachment of the filamentous appendages. There was a common antigenic component in the exosporium and the tips of the filamentous appendages. Five strains of B. cereus having a common appendage antigen also had a common exosporium antigen, whereas six other strains had neither a common appendage antigen nor a common exosporium antigen. From these facts it was concluded that the filamentous appendages arose from the exosporium.     

Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli.

The 3C region of human rhinovirus 14 was expressed in Escherichia coli. The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity. Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules. Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site. This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease. The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly.     

The effect of proteolytic cleavage of potato virus X coat protein on its ability to self-assemble with RNA and viral infectivity

The process of proteolytic cleavage of potato virus X coat protein molecules inside the virions and in the dissociated state in the course of their purification and storage has been studied. In agreement with the previous reports, the intact form (Ps) of the coat protein in the viral particles was found to be gradually cleaved to three discrete lower molecular forms (Pi, Pf, Pu). During the storage of the dissociated coat protein preparations further cleavage was observed with formation of at least three additional lower molecular weight forms (Ppa, Ppb, Ppc). The location of proteolytic cleavage sites leading to formation of Ppa form was determined. The shortened forms Pi, Pf, Pu and Ppa (and possibly Ppc) were found to be incorporated into the viral particles in the course of reconstitution in vitro with the viral RNA. Infectivity of the virus containing only intact (Ps) form of the protein was found to be two to three folds higher than that of the virus containing only Pf form of the coat protein.