Amino acid sequence analysis of fragments generated by partial proteolysis from large simian virus 40 tumor antigen

Large simian virus 40 tumor antigen was bound as immune complex to protein A-Sepharose and then subjected to limited proteolysis which yielded several discrete fragments. Primary structures near the cleavage sites were determined by radiosequencing techniques. Experimental data for five fragments matched an amino acid sequence predicted from a nucleotide sequence at 0.51 map unit of the viral genome. We have thus identified the reading frame of translation beyond the intervening sequence at 0.60 to 0.53 map units. A cleavage map of tumor antigen was established on the basis of the sequence data and of the apparent molecular weights of the fragments. The bond most susceptible to cleavage by trypsin was between arginine-130 and lysine-131 in a cluster of five basis amino acids. Other cleavage sites were located in the COOH-terminal half of tumor antigen. Each fragment was analyzed by complete tryptic proteolysis and peptide mapping on an ion exchange column. Peaks occurring in the peptide map of large tumor antigen could thus be assigned to different segments of the protein. Two specific regions of tumor antigen were shown to be phosphorylated.     

Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis

The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV) by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4-17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes.     

Botulinum protease‐cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal‐associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA‐transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations.

     

Structures of oligosaccharides cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein

Oligosaccharides were cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein and purified by Bio-Gel P-6 and paper chromatography. The structures of five oligosaccharides, determined by compositional analyses, quantitative periodate oxidation, chronic acid oxidation, methylation analyses and enzymatic degradations, were as follows: oligosaccharide I, beta DGal1----3DGalNAc-ol; II, beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; III, alpha LFuc1----2 beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; IV, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGal1NAcol; and V, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----4 beta DGlcNAc1----6[beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----3 beta DGal1----3]DGalNAc-ol. Of the oligosaccharides 60% had a molecular size of a decasaccharide or smaller, the tetra- and pentasaccharides II and III predominating. Oligosaccharides I through IV have been previously isolated from several glycoproteins by other laboratories; the decasaccharide, V, is a new structure.     

Isolation and characterization of fragments of ATP-dependent protease Lon from Escherichia coli obtained by limited proteolysis

Conditions of limited proteolysis of the protease Lon from Escherichia coli that provided the formation of fragments approximately corresponding to the enzyme domains were found for studying the domain functioning. A method of isolation of the domains was developed, and their functional characteristics were compared. The isolated proteolytic domain (LonP fragment) of the enzyme was shown to exhibit both peptidase and proteolytic activities; however, it cleaved large protein substrates at a significantly lower rate than the full-size protease Lon. On the other hand, the LonAP fragment, containing both the ATPase and the proteolytic domains, retained almost all of the enzymatic properties of the full-size protein. Both LonP and LonAP predominantly form dimers unlike the native protease Lon functioning as a tetramer. These results suggest that the N-terminal domain of protease Lon plays a considerable role in the process of the enzyme oligomerization.     

Limited proteolysis of tumor cells increases their plasmin-binding ability

Mild proteolytic treatment of SW1116 tumor cells with trypsin or plasmin increases their plasmin-binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C-terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.     

The formation of iron-binding fragments of hen ovotransferrin by limited proteolysis

1. Iron was added to hen ovotransferrin to 30% saturation and the protein was digested with trypsin or chymotrypsin. 2. Iron-binding fragments were isolated. They carried one atom of iron/mol (mol.wt. 35000) and consisted of a single polypeptide chain derived from the N-terminal half of the protein. Carbohydrate was not present. 3. The fragments were able to bind a variety of metals and to donate iron to reticulocytes.     

Comparison of protein fragments identified by limited proteolysis and by computational cutting of proteins

Here we present a comparison between protein fragments produced by limited proteolysis and those identified by computational cutting based on the building block folding model. The principles upon which the two methods are based are different. Limited proteolysis of natively folded proteins occurs at flexible sites and never at the level of chain segments of regular secondary structure such as alpha-helices. Therefore, the targets for limited proteolysis are locally unfolded regions. In contrast, the computational cutting algorithm considers the compactness of the fragments, their nonpolar buried surface area, and their isolatedness, that is, the surface area which was buried prior to the cutting and becomes exposed subsequently. Despite the different criteria, there is an overall correspondence between sites or regions of limited proteolysis with those identified by computational cutting. The computational cutting method has been applied to several model proteins for which detailed limited proteolysis data are available, namely apomyoglobin, cytochrome c, ribonuclease A, alpha-lactalbumin, and thermolysin. As expected, more cuts are obtained computationally than experimentally and the agreement is better when a number of proteolytic enzymes are used. For example, cytochrome c is cleaved by thermolysin at 56-57, 45-46, and at 80-81, and by proteinase K at 48-49 and 50-51. Incubation of the noncovalent and native-like complex of cytochrome c fragments 1-56 and 57-104 with proteinase K yielded the gapped protein species 1-48/57-104 and finally 1-40/57-104. Computational cutting of cytochrome c reproduced the major experimental observations, with cuts at 47, 64-65 or 65-66 and 80-81 and an unstable 32-47 region not assigned to any building block. The next step, not addressed in this work, is to probe the ability of the generated fragments to fold independently. Since both the computational algorithm and limited proteolysis attempt to dissect the protein folding problem, the general agreement between the two procedures is gratifying. This consistency allows us to propose the use of limited proteolysis to produce protein fragments that can adopt an independent folding and, therefore, to study folding intermediates. The results of the present study appear to validate the building block folding model and are in line with the proposal that protein folding is a hierarchical process, where parts constituting local minima of energy fold first, with their subsequent association and mutual stabilization to finally yield the global fold.     

Size fractionation of DNA fragments by liquid-liquid chromatography

A method for the fractionation of double-stranded DNA fragments from 150 to 22000 b.p. in size by liquid-liquid chromatography is described. The procedure makes use of the fact that the partitioning of DNA in a polyethylene glycol-dextran system is size dependent and can be altered by alkali metal cations. Cellulose or celite are used as supports for the stationary, dextran-rich phase. Examples show the fractionation of digests of T7 DNA produced by Dpn II and Hind II restriction endonulceases as well as lambda DNA digests produced by Hind III and Eco RI restriction endonucleases.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Regulation by proteolysis in bacterial cells

Regulation by proteolysis plays a major role in bacterial stress responses, the cell cycle and development. Key regulators of these processes are subject to conditional proteolysis that depends on complex cellular information processing. This information includes temporal and spatial cues, and recent research has revealed a striking potential for multiple signal integration.     

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.     

Ultrastructure of C4b-binding protein fragments formed by limited proteolysis using chymotrypsin

C4b-binding protein is a regulator of the classical pathway of the complement system, acting as a cofactor to the serine protease factor I in the degradation of C4b. Its molecular weight is approximately 570,000 and it is composed of multiple, disulfide-linked 70-kDa subunits. Visualized by electron microscopy (Dahlb?ck, B., Smith, C. A., and Muller-Eberhard, H. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3641-3645), it has an unusual spider-like structure with multiple thin (30 A), elongated (330 A) tentacles. The number of tentacles was estimated to be seven. Limited proteolysis by chymotrypsin produces fragments of approximately 50- and 160-kDa, the latter composed of multiple, disulfide-linked, 25-kDa polypeptides. We now have isolated the undenatured C4b-binding protein fragments formed by treatment of the protein with chymotrypsin and have visualized them by electron microscopy. The 160-kDa fragment comprises the central portion of the C4b-binding protein, which appears as a ringlike structure with an inner diameter of 13 A and an outer diameter of 60 A and having attached an approximately 40-A long piece of each tentacle. The liberated 50-kDa fragment constitutes the major part (290-A long) of the tentacles. Chymotrypsin digestion of C4b-binding protein was also monitored as a function of time by polyacrylamide gel electrophoresis and the number of subunits cleaved was found to be seven, supporting our previous ultrastructural data which suggested that C4b-binding protein contains seven identical tentacle-like subunits.     

T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides

The antigenic structure of the rabies virus glycoprotein has been studied. A limited number of fragments were obtained by cyanogen bromide (CNBr) cleavage of viral glycoprotein, and eight large peptides were isolated by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These were tested for their capacity to stimulate the proliferation of nylon wool-purified T cells obtained from spleens of rabies-immune A/J mice. Three peptides (Cr1, Cr2 plus Cr2A, and Cr3) stimulated antigen-specific proliferation, indicating that at least three T cell determinants of the native molecule are sequential or continuous in nature. Stimulation was also obtained with 27-residue and 13-residue synthetic peptides (designated R21 and R20, respectively) that included sequences towards the carboxy terminal end of Cr1, but not with synthetic peptides that included sequences of Cr2 and Cr3 (which are both glycosylated in virus-derived material). The intact viral glycoprotein and synthetic peptide R21 stimulated T lymphocytes with surface characteristics of helper cells, and induced the production of interleukin 2 by these lymphocytes. Synthetic peptides R20 and R21 also stimulated a minor population of Lyt-2-positive cells, which were not yet identified as either suppressor or cytotoxic T lymphocytes.     

Production of prostaglandins by dispersed cells and fragments from bovine parathyroid glands

We studied the production of prostaglandins by fragments and dispersed cells from bovine parathyroid glands. Fragments released 138 +/- 19 (SE), 132 +/- 21, 4.3 +/- 0.5, and 13 +/- 6.6 pg/mg/h of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and thromboxane B2, respectively (n = 7 - 26), while dispersed cells released 414 +/- 110, 22 +/- 7.3, 27 +/- 3.8, and 29 +/- 11 pg/10(6) cells/h, respectively, of the same compounds (n = 6 - 25). Indomethacin (1 microgram/ml) inhibited the release of 6-keto-PGF1 alpha by 80-90% in fragments and cells, while mellitin stimulated release of this prostaglandin, suggesting de novo synthesis of prostaglandins in these preparations. Calcium stimulated production of 6-keto-PGF1 alpha by 1.3-fold in cells and 2.6-fold in fragments and also enhanced production of PGF2 alpha by 1.9-fold in fragments. Isoproterenol, on the other hand, had no effect on production of 6-keto-PGF1 alpha in either preparation. These results demonstrate that parathyroid tissue as well as parathyroid cells per se produce a variety of prostaglandins. We have previously shown that PGE2 and PGF2 alpha modulate cAMP accumulation and PTH release in dispersed bovine parathyroid cells. The role of the endogenous production of prostaglandins by the parathyroid gland in the acute or chronic regulation of parathyroid function, however, remains to be determined.     

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.