The folding process of hen lysozyme: a perspective from the ‘new view’

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the ‘new view’ of protein folding based on energy surfaces and land scapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.     

Invertase activity in the gut of 6th instar larvae ofSpodoptera mauritia Boisd. (Noctuidae,Lepidoptera)

Summary Invertase activity has been studied in the fore-, mid- and hindgut of the 6th instar larva ofSpodoptera mauritia. The highest activity was in the midgut except during the early hours of the larval period when the foregut showed comparatively increased activity. The hindgut invertase activity may be from the voiding of enzyme along with the undigested food.Acknowledgments. The authors wish to thank the Department of Zoology, for providing the facilities for this work. The senior author gratefully acknowledges the junior research fellowship from the University of Calicut.     

Identification of chicken lysozyme <Emphasis Type="Italic">g</Emphasis>2 and its expression in the intestine

Lysozyme is an important component of the innate immune system, protecting the gastrointestinal tract from infection. The aim of the present study was to determine if lysozyme is expressed in the chicken (Gallus gallus) intestine and to characterise the molecular forms expressed. Immunohistochemical staining localised lysozyme to epithelial cells of the villous epithelium along the length of the small intestine. There was no evidence for lysozyme expression in crypt epithelium and no evidence for Paneth cells. Immunoblots of chicken intestinal protein revealed three proteins: a 14-kDa band consistent with lysozyme c, and two additional bands of approximately 21 and 23 kDa, the latter consistent with lysozyme g. RT-PCR analyses confirmed that lysozyme c mRNA is expressed in 4-day, but not older chicken intestine and lysozyme g in 4- to 35-day chicken intestine. A novel chicken lysozyme g2 gene was identified by in silico analyses and mRNA for this lysozyme g2 was identified in the intestine from chickens of all ages. Chicken lysozyme g2 shows similarity with fish lysozyme g, including the absence of a signal peptide and cysteines involved in disulphide bond formation of the mammalian and bird lysozyme g proteins. Analyses using SecretomeP predict that chicken lysozyme g2 may be secreted by the non-classical secretory pathway. We conclude that lysozyme is expressed in the chicken small intestine by villous enterocytes. Lysozyme c, lysozyme g and g2 may fulfil complimentary roles in protecting the intestine.Received 4 August 2004; received after revision 1 September 2004; accepted 7 September 2004     

Solution structure and activity of mouse lysozyme M

The three-dimensional structure of mouse lysozyme M, glycoside hydrolase, with 130 amino acids has been determined by heteronuclear NMR spectroscopy. We found that mouse lysozyme M had four alpha-helices, two 3(10)helices, and a double- and a triple-stranded anti-parallel beta-sheet, and its structure was very similar to that of hen lysozyme in solution and in the crystalline state. The pH activity profile of p-nitrophenyl penta N-acetyl-beta-D-chitopentaoside hydrolysis by mouse lysozyme M was similar to that of hen lysozyme, but the hydrolytic activity of mouse lysozyme M was lower. From analyses of binding affinities of lysozymes to a substrate analogue and internal motions of lysozymes, we suggest that the lower activity of mouse lysozyme M was due to the larger dissociation constant of its enzyme-substrate complex and the restricted internal backbone motions in the molecule.     

A new lysozyme from the eastern oyster (<Emphasis Type="Italic">Crassostrea virginica</Emphasis>) indicates adaptive evolution of <Emphasis Type="Italic">i</Emphasis>-type lysozymes

A new lysozyme (cv-lysozyme 2) with a MALDI molecular mass of 12 984.6 Da was purified from crystalline styles and digestive glands of eastern oysters (Crassostrea virginica) and its cDNA sequenced. Quantitative real time RT-PCR detected cv-lysozyme 2 gene expression primarily in digestive gland tissues, and in situ hybridization located cv-lysozyme 2 gene expression in basophil cells of digestive tubules. Cv-lysozyme 2 showed high amino acid sequence similarity to other bivalve mollusk lysozymes, including cv-lysozyme 1, a lysozyme recently purified from C. virginica plasma. Differences between cv-lysozyme 2 and cv-lysozyme 1 molecular characteristics, enzymatic properties, antibacterial activities, distribution in the oyster body and site of gene expression indicate that the main role of cv-lysozyme 2 is in digestion. While showing that a bivalve mollusk employs different lysozymes for different functions, findings in this study suggest adaptive evolution of i type lysozymes for nutrition. Received 30 August 2006; received after revision 14 October 2006; accepted 6 November 2006     

A cold-active salmon goose-type lysozyme with high heat tolerance

The Atlantic salmon (Salmo salar) goose-type lysozyme gene was isolated and revealed alternative splicing within exon 2 affecting the signal peptide-encoding region. The lysozyme was produced in Escherichia coli, and the recombinant enzyme showed a high specific lytic activity that was stimulated by low or moderate concentrations of mono- or divalent cations. Relative lytic activities of 70 and 100% were measured at 4°C and 22°C, respectively, and there was no detectable activity at 60°C. However, 30% activity was retained after heating the enzyme for 3 h at 90°C. This unique combination of thermal properties was surprising since the salmon goose-type lysozyme contains no cysteines for protein structure stabilization through disulphide bond formation. The results point to a rapid reversal of inactivation, probably due to instant protein refolding. Received 14 August 2007; received after revision 07 September 2007; accepted 12 September 2007     

A small chimerically bifunctional monomeric protein: <Emphasis Type="Italic">Tapes japonica</Emphasis> lysozyme

The lysozyme of the marine bilave Tapes japonica (13.8 kDa) is a novel protein. The protein has 46% homology with the destabilase from medicinal leech that has isopeptidase activity. Based on these data, we confirmed hydrolysis activity of T. japonica lysozyme against three substrates: L--Glu-pNA, D--Glu-pNA, and -(-Glu)-L-Lys. The optimal pH of chitinase and isopeptidase activity was 5.0 and 7.0, respectively. The isopeptidase activity was inhibited with serine protease inhibitor, but the lytic and chitinase activities were not. Moreover, only isopeptidase activity is decreased by lyophilization, but lytic and chitinase activities were not. We conclude that T. japonica lysozyme expresses isopeptidase and chitinase activity at different active sites.Received 25 February 2003; received after revision 29 May 2003; accepted 12 June 2003     

Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast.

Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.     

Lipoxygenase - a versatile biocatalyst for biotransformation of endobiotics and xenobiotics

Lipoxygenase, a member of the arachidonate cascade enzymes, dioxygenates polyenoic fatty acids to finally yield products with profound and distinct biological activity. This review summarizes the available evidence for another role played by lipoxygenases in the metabolism of endobiotics and xenobiotics. Although other mechanisms exist, a direct hydrogen abstraction by the enzyme and the peroxyl radical-dependent chemical oxidation appear to be central to the co-oxidase activity of lipoxygenases. Besides polyunsaturated fatty acids, H2O2, fatty acid hydroperoxides, and synthetic organic hydroperoxides support the lipoxygenase-catalyzed xenobiotic oxidation. The major reactions documented thus far include oxidation, epoxidation, hydroxylation, sulfoxidation, desulfuration, dearylation, and N-dealkylation. It is noteworthy that lipoxygenases are also capable of glutathione conjugation of certain xenobiotics. The enzyme system appears to be inducible following exposure to chemicals. Lipoxygenases are inhibited by a large number of chemicals, some of which also serve as co-substrates. Available data suggest that lipoxygenases contribute to in vivo metabolism of xenobiotics in mammals.     

Enzymatic basis for protection of fish embryos by the fertilization envelope.

The mechanism by which the fertilization envelope (FE) is able to protect the embryo of fish until hatching is almost unknown, except for its function as a physical barrier. FE extract from activated or fertilized eggs of the fish Salmo gairdneri was demonstrated to contain enzyme activities using an agar plate enzyme assay. The enzymes apparently active were carboxymethylcellulase (cellulase; EC 3.2.1.4), laminaranase (endo-1,3(4)-beta-glucanase; EC 3.2.1.6), carboxymethylchitinase (chitinase; EC 3.2.1.14), xylanase (endo-1,4-beta-xylanase; EC 3.2.1.8), mannanase (mannan 1,2-(1,3)-alpha-mannosidase; EC 3.2.1.77), dextranase (EC 3.2.1.11), a protease and lysozyme (EC 3.2.1.17). The FE extract exerted an antifungal or fungicidal action on the fungus Saprolegnia parasitica, whereas an extract from the vitelline envelopes (VE) has no apparent enzyme activity nor antifungal or fungicidal action. Enzymes acquired by the FE through the cortical reaction may have an important defensive role, protecting the embryo against invaders or pathogens.     

Structural basis of bacterial defense against g-type lysozyme-based innate immunity

Gram-negative bacteria can produce specific proteinaceous inhibitors to defend themselves against the lytic action of host lysozymes. So far, four different lysozyme inhibitor families have been identified. Here, we report the crystal structure of the Escherichia coli periplasmic lysozyme inhibitor of g-type lysozyme (PliG-Ec) in complex with Atlantic salmon g-type lysozyme (SalG) at a resolution of 0.95 Å, which is exceptionally high for a complex of two proteins. The structure reveals for the first time the mechanism of g-type lysozyme inhibition by the PliG family. The latter contains two specific conserved regions that are essential for its inhibitory activity. The inhibitory complex formation is based on a double ‘key-lock’ mechanism. The first key-lock element is formed by the insertion of two conserved PliG regions into the active site of the lysozyme. The second element is defined by a distinct pocket of PliG accommodating a lysozyme loop. Computational analysis indicates that this pocket represents a suitable site for small molecule binding, which opens an avenue for the development of novel antibacterial agents that suppress the inhibitory activity of PliG.     

Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme

Invertebrate (I-) type lysozymes, like all other known lysozymes, are dedicated to the hydrolysis of peptidoglycan, the major bacterial cell wall polymer, thereby contributing to the innate immune system and/or digestive system of invertebrate organisms. Bacteria on the other hand have developed several protective strategies against lysozymes, including the production of periplasmic and/or membrane-bound lysozyme inhibitors. The latter have until now only been described for chicken (C-) type lysozymes. We here report the discovery, purification, identification and characterization of the first bacterial specific I-type lysozyme inhibitor from Aeromonas hydrophila, which we designate PliI (periplasmic lysozyme inhibitor of the I-type lysozyme). PliI has homologs in several proteobacterial genera and contributes to I-type lysozyme tolerance in A. hydrophila in the presence of an outer membrane permeabilizer. These and previous findings on C-type lysozyme inhibitors suggest that bacterial lysozyme inhibitors may have an important function, for example, in bacteria-host interactions.     

Recognition of bacterial peptidoglycan by the innate immune system

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan (PGN) is a unique and essential component of the cell wall of virtually all bacteria and is not present in eukaryotes, and thus is an excellent target for the innate immune system. Indeed, higher eukaryotes, including mammals, have several PGN recognition molecules, including CD14, Toll-like receptor 2, a family of peptidoglycan recognition proteins, Nod1 and Nod2, and PGN-lytic enzymes (lysozyme and amidases). These molecules induce host responses to microorganisms or have direct antimicrobial effects.Received 15 January 2003; received after revision 28 February 2003; accepted 26 March 2003     

Differential evolution of signal-responsive RNA elements and upstream factors that control alternative splicing

Cell signal-regulated alternative splicing occurs for many genes but the evolutionary origin of the regulatory components and their relationship remain unclear. This review focuses on the alternative splicing components of several systems based on the available bioinformatics data. Eight mammalian RNA elements for signal-regulated splicing were aligned among corresponding sequences from dozens of representative vertebrate species to allow for assessment of the trends in evolutionary changes. Four distinct trends were observed. Four of the elements are highly conserved in bird, reptile and fish species examined (i); two elements can be found in fish but the sequences have been changing till in marsupials or higher mammals (ii); one element is almost exclusively found in mammals with mostly the same sequence (iii); and one element can be found in birds or lower vertebrates but expanded abruptly to have variable numbers of copies in mammals (iv). All examined prototype trans-acting factors and protein kinases emerged earlier than the RNA elements but additional (paralog) factors emerged in the same or later species. Thus, after their emergence mainly in fish or mammals with pre-existing prototype trans-acting factors/kinases, half of the elements have been highly conserved from fish to humans but the other half have evolved differentially with additional trans-acting factors. Their differential evolution likely contributes to the exon- and species/class-specific control of alternative splicing and its regulation by cell signals. The evolvement of a group of mammal-specific components would help relay signals from extracellular stimuli to the splicing machinery and thus contribute to higher proteomic diversity.     

Presence of a protein disulfide isomerase (EC 5.3.4.1) in wheat germ]

The presence of a protein disulfide isomerase (rearrangease) in wheat embryo has been demonstrated by its ability in reactivating randomly cross-linked ribonuclease. This activity requires a dialysable cofactor; after dialysis, the activity is recovered by addition of reduced glutathione. The enzyme can be precipitated by 70% saturation ammonium sulfate.     

Cardiac design in lower vertebrates: what can phylogeny reveal about ontogeny?

In very few instances can the cardiovascular systems of adult 'lower' vertebrates serve as direct models for development in 'higher' vertebrates, primarily because numerous evolutionary specializations for preferential distribution of cardiac output between systemic tissues and gas exchange organs occur in the highly derived circulation of most extant lower vertebrates. Yet, the extensive literature on the cardiovascular anatomy and physiology of aquatic and air breathing fishes, amphibians and reptiles offers important conceptual insights into both patterns and mechanisms of development in birds and mammals. The primary contribution of such studies to the student of developing bird and mammal circulations is the clear demonstration that surprisingly complex hemodynamic function can develop from supposedly 'simple' cardiovascular systems typified by incompletely divided heart chambers. Thus, the hemodynamics of embryonic bird and mammal circulations should be determined by measurement, rather than inferred from structure.     

A method for automatic recording of serum lysozyme activity with the fragiligraph.

A quick and simple method for the estimation of lysozyme activity using the Fragiligraph, was described. Diminution of turbidity in a suspension of Micrococcus lysodeikticus produced by the addition of standard lysozyme (hen egg white) or serum sample, was continuously recorded for 5 min by the Fragiligraph. The normal mean serum lysozyme activity value obtained by this method is 6,80 mug/ml +/- 1.85.     

The electrophoretic mobility of serum lysozyme.

The electrophoretic mobility of serum lysozyme in 2 patients with raised enzyme levels was identical to that of gamma-globulins. Similar mobility was observed after incubation of lysozyme and normal serum. Incubation with one hypogammaglobulinemic serum showed that lysozyme could also acquire alpha2 mobility.     

Effect of Corynebacterium parvum on serum lysozyme (muramidase) levels (author's transl)]

An i.v. injection of 548 microgram of killed Corynebacterium parvum into C57B1 mice leads to significant changes in serum lysozyme (muramidase) levels. After an initial fall at 24 h, the activity of the enzyme increased progressively, reached a peak on the 9th day and returned to control range after the 15th day.