A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.     

家蚕不同茧色品种中cbp基因的结构和表达分析

类胡萝卜素结合蛋白(CBP)是唯一已被确认与家蚕黄色茧形成儡切相关的主要蛋白质。文章选择12个有色茧和白茧蚕品种, 调查了cbp基因结构、转录产物mRNA类型和丝腺类胡萝卜素紫外可见光吸收特征与其茧色的关系。结果表明: 黄色茧蚕品种含有2种或3种cbp基因结构, 同时转录具有CBP功能性的完整 mRNA和缺少第2外显子的mRNA; 绿茧品种间cbp基因结构存在差异, 转录缺少第2外显子的mRNA; 白茧蚕品种cbp基因只有1种结构, 转录缺少第2外显子的mRNA。文章在黄茧品种中新发现的cbp基因第1内含子序列可能具有茧色品种特异性, 家蚕黄茧品种丝腺的紫外可见吸收光谱特征显著区别于绿茧和白茧品种, cbp基因的结构和表达特征与家蚕茧色密切相关。     

Isolation,characterization, and cDNA sequence of a carotenoid binding protein from the silk gland of Bombyx mori larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.     

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.     

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.     

家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

Functional characterization of the MENTAL domain

Human metastatic lymph node (MLN) 64 is composed of two conserved regions. The amino terminus contains a conserved membrane-spanning MENTAL (MLN64 NH(2)-terminal) domain shared with an unique protein called MENTHO (MLN64 NH(2)-terminal domain homologue) and targets the protein to late endosome. The carboxyl-terminal domain is composed of a cholesterol binding steroidogenic acute regulatory-related lipid transfer domain exposed to the cytoplasm. MENTHO overexpression leads to the accumulation of enlarged endosomes. In this study, we show that MLN64 overexpression also induces the formation of enlarged endosomes, an effect that is probably mediated by the MENTAL domain. Using an in vivo photocholesterol binding assay, we find that the MENTAL domain of MLN64 is a cholesterol binding domain. Moreover, glutathione S-transferase pull-down or co-immunoprecipitation experiments demonstrate that this domain mediates homo- and hetero-interaction of MLN64 and MENTHO. In living cells, the expression of paired yellow fluorescent and cyan fluorescent fusion proteins show MENTHO homo-interaction and its interaction with MLN64. These data indicate that within late-endosomal membranes, MLN64 and MENTHO define discrete cholesterol-containing subdomains. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s).     

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).     

家蚕黄血抑制基因的SSR定位

家蚕黄茧性状主要由3个基因控制, 分别是黄血基因(Yellow blood, Y), 黄血抑制基因(Yellow inhibitor, I)和黄茧基因(Out-layer yellow cocoon, C)。I基因阻止类胡萝卜素从中肠上皮细胞到血淋巴的转运, 是天然黄茧形成过程中的重要控制基因。利用家蚕雌性不发生交换的特点, 采用黄血黄茧品系KY和白血白茧品系巴格达特(Ba)组配正反交群体(Ba×KY)×KY和KY×(Ba×KY), 分别记作BC1F和BC1M, 根据已经构建的家蚕SSR分子标记连锁图谱对I基因进行了定位及连锁分析。筛选出3个与I基因连锁的SSR标记。BC1F群中的所有白血个体均表现出与(Ba×KY) F1相同的杂合型带型; 而所有黄血个体带型与亲本KY一致, 为纯合型。利用另一个群体BC1M构建了关于I基因的遗传连锁图, 连锁图的遗传距离为38.4 cM, 与I基因最近的引物为S0904, 图距为7.4 cM。     

N-218 MLN64, a protein with StAR-like steroidogenic activity, is folded and cleaved similarly to StAR

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer to inner mitochondrial membrane in adrenal and gonadal cells, fostering steroid biosynthesis. MLN64 is a 445-amino acid protein of unknown function. When 218 amino-terminal residues of MLN-64 are deleted, the resulting N-218 MLN64 has 37% amino acid identity with StAR and 50% of StAR's steroidogenic activity in transfected cells. Antiserum to StAR cross-reacts with N-218 MLN64, indicating the presence of similar epitopes in both proteins. Western blotting shows that MLN64 is proteolytically cleaved in the placenta to a size indistinguishable from N-218 MLN64. Bacterially expressed N-218 MLN64 exerts StAR-like activity to promote the transfer of cholesterol from the outer to inner mitochondrial membrane in vitro. CD spectroscopy indicates that N-218 MLN64 is largely alpha-helical and minimally affected by changes in ionic strength or the hydrophobic character of the solvent, although glycerol increases the beta-sheet content. However, decreasing pH diminishes structure, causing aggregation. Limited proteolysis at pH 8.0 shows that the C-terminal domain of N-218 MLN64 is accessible to proteolysis whereas the 244-414 domain is resistant, suggesting it is more compactly folded. The presence of a protease-resistant domain and a protease-sensitive carboxy-terminal domain in N-218 MLN64 is similar to the organization of StAR. However, as MLN64 never enters the mitochondria, the protease-resistant domain of MLN64 cannot be a mitochondrial pause-transfer sequence, as has been proposed for StAR. Thus the protease-resistant domain of N-218 MLN64, and by inference the corresponding domain of StAR, may have direct roles in their action to foster the flux of cholesterol from the outer to the inner mitochondrial membrane.     

Structure and expression of the silk adhesive protein Ser2 in Bombyx mori

Sericins are soluble silk components encoded in Bombyx mori by three genes, of which Ser1 and Ser3 have been characterized. The Ser1 and Ser3 proteins were shown to appear later in the last larval instar as the major sericins of cocoon silk. These proteins are, however, virtually absent in the highly adhesive silk spun prior to cocoon spinning, when the larvae construct a loose scaffold for cocoon attachment. We show here that the silk-gland lumen of the feeding last instar larvae contains two abundant adhesive proteins of 230 kDa and 120 kDa that were identified as products of the Ser2 gene. We also describe the sequence, exon–intron structure, alternative splicing and deduced translation products of this gene in the Daizo p50 strain of B. mori. Two mRNAs of 5.7 and 3.1 kb are generated by alternative splicing of the largest exon. The predicted mature proteins contain 1740 and 882 amino acid residues. The repetitive amino acid sequence encoded by exons 9a and 9b is apparently responsible for the adhesiveness of Ser2 products. It has a similar periodic arrangement of motifs containing lysine and proline as a highly adhesive protein of the mussel Mytilus edulis.     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.     

MENTHO,a MLN64 homologue devoid of the START domain

MLN64 is a late endosomal membrane protein containing a carboxyl-terminal cholesterol binding START domain and is presumably involved in intracellular cholesterol transport. In the present study, we have cloned a human cDNA encoding a novel protein that we called MENTHO as an acronym for MLN64 N-terminal domain homologue because this protein is closely related to the amino-terminal half of MLN64. MLN64 and MENTHO share 70% identity and 83% similarity in an original protein domain encompassing 171 amino acids that we designated as the MENTAL (MLN64 N-terminal) domain. By translation initiation scanning MENTHO is synthesized as two isoforms of 234 (alpha) and 227 (beta) amino acids that can be phosphorylated. As MLN64, MENTHO is ubiquitously expressed and is located in the membrane of late endosomes, its amino and carboxyl-terminal extremities projecting toward the cytoplasm. We show that MENTHO overexpression does not rescue the Niemann-Pick type C lipid storage phenotype. However, MENTHO overexpression alters severely the endocytic compartment by leading at steady state to the accumulation of enlarged endosomes. These results indicate that in addition to its previously established function in addressing and anchoring proteins to the membrane of late endosomes, the MENTAL domain possesses an intrinsic biological function in endocytic transport.     

利用CRISPR/Cas9双元转基因体系探究家蚕配子生成素结合蛋白基因Bmggnbp2的功能

[目的]配子生成素结合蛋白2(gametogenetin binding protein 2,ggnbp2)在哺乳动物配子发生等生殖相关过程中发挥重要作用.本研究以家蚕Bombyx mori为模型,探究驯化基因ggnbp2的生物学功能,为鳞翅目昆虫中该基因的深入研究提供线索.[方法]在NCBI GenBank数据库通过...     

天宫二号航天蚕后代小茧突变体sc的发现与基因定位

【目的】从2016年天宫二号空间实验室经历33 d后返回的1头成活“秋丰×白玉”杂交后代雌蚕Bombyx mori与地面“白玉”雄蚕交配的后代个体中,发现有结小茧突变体,进而分离并建立了飞天蚕(space silkworm)正常茧品系TG和小茧突变体品系sc。本研究通过对sc进行遗传分析和基因定位,旨在揭示产生小茧突变体的基因。【方法】对TG和sc进行表型分析;以sc, TG和正常大茧品系0223V1为试验材料,组配(sc♀×0223V1♂)F₁及回交群体BC₁F——(sc♀×0223V1♂)F₁♀×sc♂和BC₁M——sc♀×(sc♀×0223V1♂)F₁♂。以sc, 0223V1和F₁基因组DNA为模板,每个连锁群随机选10个SSR引物进行PCR扩增,筛选多态性SSR标记。利用雌性家蚕减数分裂染色体不交换的特点,用BC₁F确定sc基因所属连锁群;再根据家蚕SSR分子标记连锁图谱,用BC1M进行基因定位。【结果】表型分析表明sc幼虫体型小于TG幼虫的,蚕茧重约为TG的1/2。遗传分析表明突变受一对隐性基因sc控制;基因定位结果表明该基因位于家蚕基因组第3连锁群S2930-363和S2930-289 SSR标记之间,物理距离为684 kb,包含33个候选基因。【结论】飞天蚕小茧突变受位于家蚕基因组第3连锁群的一对隐性基因sc控制。     

Effects of cadmium chloride and nonylphenol on the expression of StAR-related lipid transfer domain containing protein (START1) gene in aquatic midge, Chironomus riparius

We identified and characterized a partial cDNA of StAR-related lipid transfer domain containing protein gene from Chironomus riparius (CrSTART1) having homology with human MLN64 and Drosophila melanogaster START1 (DmSTART1) and evaluated the effects of cadmium chloride (Cd) and nonylphenol (NP) on its expression. Pfam analysis identified the presence of two StAR-related lipid transfer (START) domains in CrSTART1 having several conserved amino acid residues, characteristic of the MLN64 and DmSTART1. The mRNA expression of CrSTART1 was observed in all developmental stages. The modulation in the mRNA expression of CrSTART1 was investigated after exposure to different concentrations Cd (0, 2, 10, and 20 mg/L) and NP (0, 10, 50, and 100 μg/L) for different time intervals in fourth instar larvae of C. riparius. Significant downregulation of CrSTART1 mRNA was observed after exposure to 2, 10 and 20 mg/L of Cd for 24, 48 and 72 h. Significant upregulation of CrSTART1 was observed after exposure to 10 and 50 μg/L of NP for 24, and 48 h period. At 100 μg/L of NP significant upregulation of CrSTART1 was observed after 12 h and downregulated after 24, 48 and 72 h.