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人源类溶菌酶蛋白6的功能研究及生理特性分析 *
The Functional Studies of Human Lysozyme-like Protein 6 and Characterization of Its Physiological Properties
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}对人源类溶菌酶蛋白6(human lysozyme-like protein 6,LYZL6)在受精过程中的作用进行研究,并对重组LYZL6蛋白(recombinant LYZL6,rLYZL6)的生理特性进行分析,从而揭示其生理功能。细胞免疫荧光法确定LYZL6定位于成熟精子头部的顶体后区域,反转录PCR(RT-PCR)分析表明精子表面的LYZL6蛋白来源于睾丸和附睾的分泌,Western blot法分析表明精子获能前后表面LYZL6的量无明显改变。半透明带结合实验和精子穿透实验分析表明兔抗LYZL6血清未明显抑制人精子结合透明带,但可明显抑制精卵融合。利用毕赤酵母表达系统成功表达了rLYZL6,使用甲壳素亲和层析和凝胶过滤层析可从发酵上清中纯化到具有生物活性的rLYZL6。酶联免疫吸附法(ELISA)分析显示rLYZL6不具有透明质酸结合能力、透明质酸水解能力和自由基清除活性,但具有较强的肽聚糖结合能力和异肽酶活性。LYZL6由睾丸和附睾分泌后定位于成熟精子头部的顶体后区域,可以参与精卵融合,并具有肽聚糖结合能力和异肽酶活性,提示LYZL6可能通过多种机制参与精子功能。
The aim is to identify the possible performance of human lysozyme-like protein 6 (LYZL6) in fertilization and to characterize its physiological properties. Immunofluorescent staining with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa, which was secreted by testis and epididymis as demonstrated by the reverse transcription polymerase chain reaction (RT-PCR). No significant decrease of LYZL6 after capacitation was observed by Western blot analysis. Immunoneutralization of LYZL6 showed no effect on the binding of spermatozoa to the hemizona, but significantly decreased the numbers of human spermatozoa fused to zona-free hamster eggs in a dose-dependent manner. The Pichia expression system was utilized to produce recombinant LYZL6 (rLYZL6). After induction with methanol, rLYZL6 was purified from the fermentation supernatant by chitin affinity in combination with gel-filtration chromatography. In vitro assays indicated that rLYZL62 possessed no hyaluronan-binding ability, hyaluronidase activity and free radical scavenging activity, but peptidoglycan-binding ability and isopeptidase activity. In conclusion, LYZL6, a human sperm-related protein is reported, not only plays a role in sperm-egg fusion but also has peptidoglycan-binding ability and isopeptidase activity, suggesting it might contribute to diverse sperm functions.
人源类溶菌酶蛋白6 / 顶体 / 受精 / 毕赤酵母 / 异肽酶活性 {{custom_keyword}} /
Human / lysozyme-like / protein / 4 / Acrosome / Fertilization / Pichia / pastoris / Isopeptidase / activity {{custom_keyword}} /
表1 研究所用引物序列Table 1 Primers used in this study |
| Primer name | Primer sequence (5'-3') |
|---|---|
| RT-LYZL6-F | ATGACAAAGGCGCTACTCATC |
| RT-LYZL6-R | GAAGGTTGGGATTCAGCAGATC |
| GAPDH-F | CGTGGAAGGACTCATGACC |
| GAPDH-R | GAGGCAGGGATGATGTTCTG |
图6 发酵液上清SDS-PAGE电泳Fig.6 SDS-PAGE of the fermentation supernatant |
| [1] |
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Lysozyme-like proteins (LYZLs) belong to the class of c-type lysozymes and are not well characterized in many species including the rat. In this study, using in silico and molecular biology techniques, we report the identification, cloning and characterization of rat Lyzl4 gene and also determine the expression pattern of Lyzl1, Lyzl3 and Lyzl6. The rat Lyzl genes were found to be distributed on three chromosomes and all of them retained the characteristic eight cysteine signature of c-type lysozyme. Homology modeling of rat LYZL4 indicated that its structure is similar to that of the mouse SLLP1. In the male reproductive tract of rat, Lyzl gene expression was confined to the testis. Lyzl1 and Lyzl4 were found to be expressed in tissues beyond the male reproductive tract, whereas Lyzl3 and Lyzl6 were not. Lyzl expression in the developing (10-60 day old) rats was androgen dependent in the testis. Immunodetection using antibodies against rat LYZL4 revealed the presence of LYZL4 protein in the germinal layer of the testes and on the sperm tail. Recombinant LYZL4 did not exhibit antibacterial, muramidase and isopeptidase activities characteristic to c-type lysozyme. To the best of our knowledge, for the first time we report the characterization of Lyzl genes in the rat. Results of our study indicate that rat LYZL proteins may have an important role in male reproductive tract function.
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Abstract The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the 'changed' amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.
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C-type lysozyme genes (Lyzls) belong to the class of lysozymes and are highly expressed in the testis and epididymis. The members Lyzl4 and Spaca3 have been reported to play a role in sperm-egg binding and fertilisation in mice. However, the function of the remaining two mouse c-type lysozyme genes, Lyzl1 and Lyzl6, is still not clear. In the present study, we analysed the tissue expression and androgen-dependent expression of mouse c-type lysozyme genes and the possible contribution of human recombinant LYZL6 (rLYZL6) to immunity. The expression of Lyzls was detected by RT-PCR, Western blots, immunohistochemistry and immunofluorescence. The bacteriolytic activity of rLYZL6 was analysed by a colony-forming assay. In mice, the expression of Lyzlgenes was mainly in the testis and epididymis in a developmentally regulated manner and androgen- or testicular factor-regulated manner. Immunodetection revealed the presence of LYZL6 protein in primary spermatocytes and round spermatids of the testis and on the post-acrosomal area and midpiece of mature epididymal spermatozoa. The rLYZL6 protein exhibited antibacterial activity. From the results, Lyzls may play a role in mitochondrial function of spermatozoa and LYZL6 may contribute to the innate immunity of the male genital tract.
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黄鹏, 李文姝, 谢君 , 等. 人源类溶菌酶蛋白6在毕赤酵母中的重组表达及活性分析. 中国生物工程杂志, 2015,35(8):30-37.
<p>利用毕赤酵母(<em>Pichia pastoris</em>)重组表达人源类溶菌酶蛋白6(human lysozyme-like protein 6,hLyzl6),对其酶学性质进行分析。根据毕赤酵母密码子偏爱性设计并人工合成hLyzl6基因,将其连接至含有乙醇氧化酶启动子(AOX1)的pPIC9K质粒构建重组表达载体pPIC9K-<em>hlyzl6</em>;重组表达载体经线性化后电转化入毕赤酵母GS115感受态细胞,经G418筛选获得高拷贝重组菌株后进行甲醇诱导表达。经甲醇诱导72 h后发酵液上清中酶活性达到最高值,发酵液上清经SDS-PAGE检测在14.8 kDa处有重组hLyzl6蛋白条带,分子量符合预期,通过甲壳素亲和层析可对其进行纯化;采用比浊法测定hLyzl6酶学活性,结果表明hLyzl6对溶壁微球菌(<em>Micrococcus lysodeikticus</em>)有较好的杀灭作用,最适反应温度为40℃,最适pH为5.5,其酶活力为54 700U/mg,Cu<sup>2+</sup>对其活性有明显抑制,EC<sub>50</sub>为30.2799 mg/L。采用基因工程方法首次在毕赤酵母GS115成功表达了重组hLyzl6,证实其在体外具有杀菌活性,初步揭示hLyzl6在男性生殖系统先天性免疫中发挥了一定作用,为进一步研究hLyzl6的功能和应用开发奠定了基础。</p>
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| [9] |
黄鹏, 李文姝, 杨智昉 , 等. 人源类溶菌酶蛋白6 在男性生殖系统中的表达. 中华男科学杂志, 2016,22(7):584-590.
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Lysozyme-like proteins (LYZLs) belong to the c-type lysozyme/-lactalbumin family and are selectively expressed in the mammalian male reproductive tract. Two members, human sperm lysozyme-like protein (SLLP) -1 and mouse LYZL4, have been reported to contribute to fertilization but show no bacteriolytic activity. Here, we focused on the possible contribution of LYZL6 to immunity and fertilization. In humans, LYZL6 was selectively expressed by the testis and epididymis and became concentrated on spermatozoa. Native LYZL6 isolated from sperm extracts exhibited bacteriolytic activity againstMicrococcus lysodeikticus. Recombinant LYZL6 (rLYZL6) reached its peak activity at pH 5.6 and 15 mM of Na+, and could inhibit the growth of Gram-positive, but not Gram-negative bacteria. Nevertheless, the bacteriolytic activity of rLYZL6 proved to be much lower than that of human lysozyme under physiological conditions. Immunodetection with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa. Immunoneutralization of LYZL6 significantly decreased the numbers of human spermatozoa fused with zona-free hamster eggs in a dose-dependent mannerin vitro. Thus, we report here for the first time that LYZL6, an acidic, bacteriolytic and human sperm-related protein, is likely important for fertilization but not for the innate immunity of the male reproductive tract.
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黄鹏, 杨智昉, 徐一新 , 等. 人源类溶菌酶蛋白4的多克隆抗体制备及其表达分析. 中华男科学杂志, 2017,23(1):3-10.
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Capacitation is a complex series of molecular events that occurs in sperm after epididymal maturation and confers on sperm the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of oviductal fluid. In most cases, capacitation media contain energy substrates, such as pyruvate, lactate and glucose, a cholesterol acceptor (usually serum albumin), NaHCO 3 , Ca 2+ , low K + , and physiological Na + concentrations. The mechanism of action by which these compounds promote capacitation is poorly understood at the molecular level; however, some molecular events significant to the initiation of capacitation have been identified. For example, capacitation correlates with cholesterol efflux from the sperm plasma membrane, increased membrane fluidity, modulations in intracellular ion concentrations, hyperpolarization of the sperm plasma membrane and increased protein tyrosine phosphorylation. These molecular events are required for the subsequent induction of hyperactivation and the acrosome reaction. This review discusses the recent progress that has been made in elucidating mechanisms which regulate sperm capacitation.
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| [14] |
Sperm-egg fusion is a cell-cell membrane fusion event essential for the propagation of sexually reproducing organisms. In gamete fusion, as in other fusion events, such as virus-cell and intracellular vesicle fusion, membrane fusion is a two-step process. Attachment of two membranes through cell-surface molecules is followed by the physical merger of the plasma membrane lipids. Recent progress has demonstrated an essential role for an oocyte tetraspanin, CD9, in mouse sperm-egg fusion, and a specific molecular site crucial for CD9 function has been identified. Absence of glycosylphosphatidylinositol-anchored proteins on the oocyte surface also results in loss of oocyte fusion competence in this gamete. These discoveries provide a strong starting point for the identification of additional proteins that have roles in sperm-egg fusion.
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| [15] |
Fertilization includes a series of cellular interactions culminating with the fusion of gamete membranes, creating a zygote. Two ADAM proteins present on sperm, fertilin β and cyritestin, drew much attention. However, gene deletion in mice showed that fusion can happen in their absence. The presence of the integrin α6β1 on egg, a putative fertilin β receptor, is also dispensable. In contrast, sperm lacking Izumo, a molecule with a single Ig domain, are unable to fuse. On the egg side, a role for GPI-anchored molecules has been shown, and in mice lacking both tetraspanins CD9 and CD81 fertilization is completely blocked.
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| [16] |
Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B鈥揝LLP1 as a pair of novel sperm鈥揺gg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
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| [17] |
The last stages of male gamete differentiation occur outside the gonad in a specific environment controlled by the epididymal epithelium. All the fundamental characteristics of a fertile spermatozoon are acquired sequentially during transit through the epididymal tubule. Full understanding of the mechanisms involved in these gamete modifications is a key to understanding and controlling such important stages in male fertility. With the development of new large scale technologies, large amounts of information give hope of identifying the fundamental elements involved in such cellular events and of being able to obtain some markers predictive of male fertility that would be valuable both in human and/or animal reproduction.
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| [18] |
Abstract Testicular spermatozoa acquire fertility only after 1 or 2 weeks of transit through the epididymis. At the end of this several meters long epididymal tubule, the male gamete is able to move, capacitate, migrate through the female tract, bind to the egg membrane and fuse to the oocyte to result in a viable embryo. All these sperm properties are acquired after sequential modifications occurring either at the level of the spermatozoon or in the epididymal surroundings. Over the last few decades, significant increases in the understanding of the composition of the male gamete and its surroundings have resulted from the use of new techniques such as genome sequencing, proteomics combined with high-sensitivity mass spectrometry, and gene-knockout approaches. This review reports and discusses the most relevant new results obtained in different species regarding the various cellular processes occurring at the sperm level, in particular, those related to the development of motility and egg binding during epididymal transit.
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| [19] |
Evaluating the immunocontraceptive potential of sperm-bound proteins is an active area of investigation. In this study, we analyzed the role of prostate- and testes-expressed (PATE) and PATE-F proteins in sperm function. Capacitation was measured as a function of tyrosine phosphorylation of sperm membrane proteins. Ionophore-induced acrosome reaction was assessed by measuring the fluorescence intensity of calcium-bound Fluo 3-AM and sperm-bound PNA-FITC in a flow cytometer. Rat spermatozoa subjected to capacitation and acrosome reaction in vitro displayed changes in the PATE and PATE-F protein localization on their surface, indicating the role of these proteins in sperm function. Capacitation and ionophore-induced acrosome reaction in vitro were inhibited in spermatozoa pre-incubated with antiserum raised in rabbit against PATE or PATE-F. Male rats were immunized with PATE proteins to assess their role in sperm function and fecundity. Antibody titer in the serum, testicular, and epididymal fluid was measured by ELISA. The motility parameters were recorded using CASA. High antibody titer was observed in serum, epididymal, and testicular fluid in rats immunized with PATE or PATE-F protein. Immunization did not cause any structural damage and inflammation in the testis and epididymis. PATE and PATE-F antisera obtained from the immunized rats inhibited acrosome reaction. Motility parameters, capacitation, acrosome reaction, and fecundity were compromised in PATE-F-immunized rats, whereas the same were not affected in rats immunized with PATE. These results suggest that PATE-F might play an important role in sperm function and fecundity and can be explored further to determine its immunocontraceptive potential.
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Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0鈥60 min and measuring the incorporation of the radioactive precursors [methyl- 3 H]thymidine, [5- 3 H]uridine and L-[4,5- 3 H(N)]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean 卤 S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization.
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BIN1b was reported as an epididymis-specific beta-defensin antimicrobial peptide. In this paper, the recombinant BIN1b was expressed and purified by fusing with GB1-His tag. The size-exclusion gel filtration experiment indicated that the fusion protein GB1-BIN1b formed multimers at pH 7.4, and existed as monomer at pH 4.5. The oligomerization of GB1-BIN1b was only related to pH value, neither to NaCl concentration nor protein concentration. Far-UV circular dichroism (CD) spectra also showed the fusion protein had more ordered secondary structures at pH 4.5 than at pH 7.4, as a negative peak appeared around 218聽nm indicative of typical -sheet. The 2D 15 N- 1 H heteronuclear single-quantum coherence (HSQC) spectra suggested that the fusion protein adopted a compact three-dimensional structure at pH 4.5. Colony forming unit (CFU) inhibition assay demonstrated that 25聽渭M fusion protein at pH 7.4 had an antimicrobial activity of 40% against E. coli K 12 D 31 , which might imply the fusion protein functions as multimeric states. In conclusion, the GB1 fusion partner helps BIN1b form a stable homogenous conformation to facilitate subsequent structural determination without a significant effect on the antimicrobial activity.
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| [22] |
The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.
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| [23] |
The aim of this research was to evaluate the role of lysozyme in the phenomenon of seminal hyperviscosity. The enzyme was determined in 142 samples of seminal plasma either leucospermic or not, with or without active macrophages classified according to their consistency (normal or high). The kinetic method with Micrococcus lysodeikticus as substrate was employed. No difference was found in enzymatic concentration expressed in nmol/L of enzymatic protein (mean 00± 2 SEM) on comparing normal and high seminal consistency groups, while differences proved highly significant in batches either leucospermic or not (n = 44, 197.2 00± 51.3 vs. n = 98, 108.3 00± 12.8; p <. 0005). On subdividing the normal and high-consistency groups according to the count of polymorphonuclear leucocytes and the macrophagic response, differences were also significant (p <. 005 in both cases). Lysozyme concentration increases in presence of leucospermic reaction. In vitro lysozyme addition showed no significant effect on samples with high consistency. The results indicate that lysozyme plays no direct role in the phenomenon of seminal hyperviscosity, although its deficiency in cases of chronic infections may prove a factor aggravating the clinical picture.
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Abstract 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-gamma-Glu-pNA, D-gamma-Glu-pNA, and epsilon-(gamma-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.
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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.
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The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
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表1 研究所用引物序列
图1 精子细胞LYZL4免疫荧光染色图2 LYZL6在男性生殖系统中的表达谱分析图3 精子获能前后LYZL6 的Western blot分析图4 抗LYZL6血清对精卵融合的影响 (n=3)图5 抗LYZL6血清对精子透明带结合的影响 (n=8)图6 发酵液上清SDS-PAGE电泳图7 纯化产物SDS-PAGE图8 肽聚糖结合能力测定图9 透明质酸结合能力测定图10 透明质酸水解活性测定图11 自由基清除活性测定图12 异肽酶活性测定/
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