Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

家蝇溶菌酶MDLZM2基因的克隆、序列分析及原核表达

对家蝇溶菌酶(Musca domestica lysozyme,MDLZM2)基因进行克隆、序列分析,构建原核表达载体并在大肠杆菌中表达。从Gen Bank家蝇基因组中筛选获得MDLZM2基因。以该基因的序列设计引物,进行PCR扩增,测序分析获得该基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构和保守结构域等方面进行预测和分析。构建p EASY-E1-MDLZM2重组质粒,转化到大肠杆菌BL21(DE3)p Lys S Chemically Competent Cell中进行诱导表达及纯化。结果表明MDLZM2基因ORF全长552 bp,编码183个氨基酸,理论分子量21.2 k Da;等电点为6.13,具有Lysozyme家族的蛋白保守结构域。成功构建重组原核表达p EASY-E1-MDLZM2并诱导表达、纯化重组蛋白,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut.     

The role and mechanism of the up-regulation of fibrinolytic activity in painful peripheral nerve injury

Peripheral nerve injury is followed by Wallerians degeneration (WD) and degradation and regeneration of the extracellular matrix (ECM) are very important events in these processes. We tested fibrinolytic activities and the expression level of tPA and uPA levels in chronic constriction injury (CCI) model. The presented data demonstrates that in the injury nerve of CCI model, the fibrinolytic activities is upregulated and main caused by the upregulation of tPA expression. We also find that the activities of tPA and MMP-9 are co-upregulated post-CCI, both at CCI site and proximal site. These results indicate that tPA activity may regulate the MMP-9 activity in injury nerve post-CCI.     

海刺参i型溶菌酶基因的重组表达及抑菌谱分析

将本实验室已分离到的海刺参 (Stichopus japonicus) i型溶菌酶的基因(GenBank Accession No. EF036468)亚克隆进原核表达载体pET32a(+)中, 构建重组质粒pET32a(+)-SjLys, 转化至大肠杆菌BL21(DE3)pLysS。阳性克隆子经诱导表达、亲和纯化和透析复性, 得到了酶活力为19.2 U/mg的重组SjLys (rSjLys), 并对rSjLys进行了抑菌谱测定。结果表明, rSjLys对革兰氏阳性菌和阴性菌均有抑制作用, 尤其对常见的海洋致病菌副溶血弧菌和铜绿假单胞菌具有很强的抑菌活性。更为显著的结果是, rSjLys经100oC、40 min加热处理后, 失活的rSjLys对革兰氏阳性菌和阴性菌的抑菌能力高于rSjLys的9%~25%。上述结果表明, 海刺参溶菌酶是一种具有糖苷酶活性和非酶抑菌活性的特殊的i型溶菌酶, 且具有很广的抑菌活性, 在海刺参机体先天免疫系统中是一个重要的效应分子。     

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS)

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.     

Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm

An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.     

C-terminus of TRAP in Staphylococcus can enhance the activity of lysozyme and lysostaphin

In Staphylococcus aureus , the target of RNAIII activating protein (TRAP) is a membrane-associated protein whose C-terminus can be used as a vaccine to provide protection against staphylococcal infection. Here, we show for the first time by surface plasmon resonance and enzyme-linked immunosorbent assay that TRAP can specifically bind lysozyme and lysostaphin through its C-terminus (amino acids 155–167) and enhance lysozomal activities in vitro . It was also found that the traP mutant strain is more resistant to lysostaphin than wild-type. Our previous data showed that the C-terminus of TRAP might be extracellular. So our results suggested that the C-terminus of TRAP could act as the specific targeting protein of the lysozyme/lysostaphin on the S. aureus cell wall and the biological significance of the interaction might be to facilitate lysozyme/lysostaphin-mediated cell lysis.     

Recombinant expression and characterization of a lysozyme from the midgut of Triatoma brasiliensis (Hemiptera,Reduviidae) in comparison with intestinal muramidase activity

Insect c‐type lysozymes are antibacterial proteins that are synthesized in different organs with high activity against Gram‐positive bacteria. Because lysozymes possess muramidase activity, they also play an important role in the digestion of bacteria in Diptera. Triatomines express lysozyme‐encoding genes constitutively in the anterior region (cardia and stomach) of the midgut and the fat body after injection of bacteria into the haemocoel. The present study describes the overexpression of the Triatoma brasiliensis lysozyme 1 (lys1) in Escherichia coli. Recombinant T. brasiliensis Lys1 (TbLys1) is purified after solubilization of the inclusion bodies. The protein refolds successfully, showing muramidase activity against Micrococcus lysodeikticus lyophilized cells, after enterokinase cleavage of its thioredoxin fusion protein. In in‐gel zymograms and turbidimetric liquid assays TbLys1 is broadly active under alkaline and acid conditions, indicating a possible digestive function in the two physiologically different midgut regions of the bug: the stomach and small intestine. Muramidase activity is shown in the stomach and small intestine content of unfed bugs and bugs at different days after feeding, respectively. Western blot analysis identifies TbLys1 as lysozyme.     

Co-segregation of alleles at a microsatellite locus within the macrophage expressed lysozyme gene and levels of serum lysozyme activity in two half-sib families of Polish Black and White Lowland cattle

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70–95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.     

Protein purification and nucleotide sequence of a lysozyme from the bacteria-induced larvae of the fall webworm,Hyphantria cunea

A protein with lytic activity against Micrococcus luteus was purified from the hemolymph of the fall webworm, Hyphantria cunea, larvae challenged with live E. coli. A bacteriolytic protein of about 14,000 daltons in mass was purified by cation exchange chromatography and reverse-phased HPLC. The optimum pH and optimum temperature range for activity were around pH 6.2 and 50°C, respectively, in a 100 mM phosphate buffer. The aminoterminal amino acid sequence of this protein was determined and the corresponding cDNA was isolated and analyzed. The deduced protein of 142 amino acid residues was composed of a putative leader sequence of 20 residues and the mature enzyme of 122 residues. The cloned lysozyme gene was strongly induced in response to bacterial injection, implying that the enzyme is a part of the immune response of H. cunea. Comparison with other known lysozyme sequences shows that our lysozyme belongs to the chicken lysozyme. Arch. Insect Biochem. Physiol. 35:335–345, 1997.© 1997 Wiley-Liss, Inc.     

Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity

Calcium-binding equine lysozyme (EL) combines the structural and folding properties of c-type lysozymes and alpha-lactalbumins, connecting these two most studied subfamilies. The structural insight into its native and partially folded states is particularly illuminating in revealing the general principles of protein folding, amyloid formation and its inhibition. Among lysozymes EL forms one of the most stable molten globules and shows the most uncooperative refolding kinetics. Its partially-folded states serve as precursors for calcium-dependent self-assembly into ring-shaped and linear amyloids. The innate amyloid cytotoxicity of the ubiquitous lysozyme highlights the universality of this phenomenon and necessitates stringent measures for its prevention.     

小菜蛾溶菌酶Pxlys的基因克隆及其重组蛋白的抗菌活性分析

[目的]本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路.[方法]利用RACE技术克隆小菜蛾溶菌酶基因.构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys.利用牛津杯法检测重组蛋...     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouse

The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5' flanking region, 1607 bases of coding region, and 309 bases of 3' flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-beta-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45 degrees C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate.     

Cloning, expression, and sequence homologies of cDNA for human carbonic anhydrase II

A cDNA clone for human carbonic anhydrase (CA) II was isolated from a kidney lambda gt10 library. Expression of the cDNA insert in Cos-7 cells produced an immunoprecipitable product and enzymatically active carbonic anhydrase. The cDNA insert is 1551 bp in length and contains an open reading frame which encodes a 260-amino-acid polypeptide. The deduced amino acid sequence is identical to that reported for human CA II. The protein coding region of this cDNA for human CA II shows 81 and 70% nucleotide identity with cDNAs for CA II from mouse and chick, respectively. Even the long 3'-untranslated region of the cDNA for human CA II (703 bp) is 64 and 42% identical to those of CA II from mouse and chick, showing remarkable conservation of the CA II cDNAs in amniotes. The protein coding region of the human CA II cDNA is 64 and 65% identical with those of human CA I and CA III, which are thought to have arisen from a common precursor by gene duplication.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)