Protection of phenylalanine ammonia-lyase from proteolytic attack

Phenylalanine ammonia-lyase contained within permeabilized cells of Rhodosporidium toruloides was protected from proteolytic attack by trypsin, chymotrypsin and duodenal juice. The inactivation by the proteases was biphasic. The enzyme contained within the yeast cells had a similar Km for phenylalanine and Ki for cinnamic acid to the protein in free solution. Phenylalanine ammonia-lyase present in the yeast depleted duodenal juice of free phenylalanine, while the enzyme in free solution did not. The possibility of using permeabilized cells of R. toruloides as a vehicle for protecting orally ingested therapeutic enzymes from proteolytic inactivation is discussed.     

The resistance of cellulases and xylanases to proteolytic inactivation

The sensitivity of a range of cellulases and xylanases to proteolytic inactivation was investigated. The xylanases, all the Clostridium thermocellum cellulases and cellulase E from Pseudomonas fluorescens subsp. cellulosa exhibited no decrease in catalytic activity during a 3-h incubation with proteinases of the small intestine. Under these conditions, the control Escherichia coli enzymes analysed had half-lives of 4.3–13.5 min. The addition of substrate significantly decreased the sensitivity of proteinase-labile enzymes to inactivation. The significance of these data in relation to the use of cellulases and xylanases for improving animal nutrition is discussed.     

Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.     

Proline versus charge concept for protein stabilization against proteolytic attack

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.Key words: antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, autoantibodies     

Enzyme stabilization towards thermal,chemical and proteolytic deactivation

An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21), β-d-fructofuranosidase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23).     

Entrapment of phenylalanine ammonia-lyase in silk fibroin for protection from proteolytic attack

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.     

Protein engineering of bacterial alpha-amylases

alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas.     

Lysosomal beta-hexosaminidase is highly resistant towards proteolytic degradation in vitro.

1. A partially purified enzyme preparation of beta-hexosaminidase from human fibroblasts was treated with proteases and the effect on its molecular weight and enzymatic activity was studied. 2. Both the forms A and B of the enzyme appeared to be resistant to a protease treatment that degraded the majority of the contaminating proteins to a large extent. 3. The same result was obtained with enzyme preparations from cells treated with tunicamycin. 4. Also the molecular weights of the individual polypeptide chains of the enzyme were not decreased, as was shown by SDS-PAGE, followed by immuno-blotting.     

Concerted loss of cyclooxygenase and peroxidase activities from prostaglandin H synthase upon proteolytic attack

Prostaglandin H synthase has two distinct enzymatic activities: a cyclooxygenase that forms PGG2 from arachidonate and a peroxidase that can reduce hydroperoxides, such as PGG2, to the corresponding alcohols. The relative sensitivities of the two synthase activities to proteolytic attack have been examined, using trypsin, chymotrypsin, and proteinase K, all known to attack the native apoprotein in the arg 253 region. The relation between the specific activity of the synthase and the loss of the two activities and the cleavage of the synthase subunit during trypsin digestion was also examined. The cyclooxygenase and peroxidase activities declined in concert throughout room temperature digestions with each of the three proteases. There was no indication of a selective loss of either activity in any of the digestions. In separate digestions with the same preparation of synthase, 3.3% (w/w) proteinase K resulted in more extensive loss of activity (90% decrease after 90 min) than did 3% (w/w) trypsin (70% decrease after 120 min) or 5% (w/w) chymotrypsin (60% decrease after 135 min). In tryptic digestions of synthase preparations with cyclooxygenase specific activity between 16 and 125 k units/mg protein, the fractional loss of cyclooxygenase activity was, within experimental error, the same as that of peroxidase activity. The extent of cleavage of the 70 kDa synthase subunit was greater than the loss of enzymatic activity, with the discrepancy being larger for synthase preparations with lower specific activity. The presence of a variable amount of catalytically-inactive, protease-sensitive, synthase protein could account for the difference between surviving activity and intact subunit in six out of the seven synthase preparations examined. Thus, it is likely that the cyclooxygenase and peroxidase activities are destroyed together during proteolytic attack on the arg 253 region of the native synthase apoprotein.     

The chemical basis of resistance of Sorghum bicolor to attack by Locusta migratoria

The resistance of young Sorghum bicolor to attack by Locusta migratoria (L.) has been investigated from a chemical standpoint. Absence of phagostimulants is not important. The release on biting of HCN from the non-deterrent cyanogenic glucoside, dhurrin, has been shown quantitatively to play an important part in the unpalatability. Correlation of feeding data with release rates of HCN showed that the increase in palatability of Sorghum did not closely correspond to the rapid fall in release of HCN as the plants aged, and when different concentrations of HCN were directly introduced into the mouthparts of Locusta using a cannula, the concentration of HCN released after the first growth stage was insufficient to account for the continued unpalatability. The effects on Locusta of other chemicals in Sorghum were monitored using a series of crude plant extracts. A lipid-soluble extract from plants at growth stage 1 and an extract of phenolic acids naturally released from the plants at stage 2 both showed deterrent effects. The individual phenolic acids were identified and all were found to occur naturally as esters with no deterrent properties. Estimates were made of the concentrations of free acids released by disruption of the plant tissues in several ways: maximum concentration occurs at growth stage 2. Bioassays showed that the deterrent effects of HCN and the phenolic acid mixture were additive and the change in palatability of the plant with increasing maturity is thus completely accounted for.

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Résumé On montre que Sorghum bicolor, au stade de plantule, est très peu apprécié par Locusta migratoria, mais qu'il devient de plus en plus appétissant au cours de sa croissance et de sa maturation. Ce changement n'est pas dû uniquement aux quantites croissantes de phagostimulants nutritifs.On a quantifié les changements du nivéau de la cyanogénèse chez Sorghum au fur et à mesure de sa croissance et on a mesuré le taux de dégagement de HCN par hydrolyse enzymatique après lésion des tissus. Par une technique nouvelle de biotitrage utilisant une canule implantée en permanence à travers le labium, on a montré que les concentrations de HCN dégagées naturellement par les jeunes plantes ont un effet répulsif sur Locusta. On a montré que la cyanogénése est la cause du caractère inappétissant des très jeunes plantes mais son niveau n'est pas suffisamment élevé pour expliquer le maintien d'une appétence réduite.Au second stade de croissance étudié, les acides phénoliques produits par la plante accroissent la répulsion causée par la cyanogénèse. On a trouvé que ces acides étaient présents dans la plante sous forme d'esters et qu'ils étaient dégagés enzymatiquement lors de lésions causées aux tissus par l'insecte s'alimentant, de la même façon que HCN. Aucun des acides phénoliques n'est dégagé à une concentration suffisante pour être répulsif, mais le mélange est répulsif à cause de l'effet cumulé des différentes substances chimiques.Le changement du caractère appétissant de Sorghum bicolor (var. 65D) pour Locusta migratoria est attribuable aux effects combinés de la cyanogénése et du dégagement d'acide phénolique.

     

Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.