蜜蜂表皮蛋白apidermin (apd)基因家族3个新成员的特性鉴定及昆虫APD家族序列特征的分析

Apidermin (APD)蛋白家族是一个新的昆虫结构性表皮蛋白家族。本研究结合生物信息学和RT-PCR扩增, 对意大利蜜蜂Apis mellifera ligustica（简称“意蜂”）的apd-1-like, apd-3-like和中华蜜蜂Apis cerena cerena（简称“中蜂”）的apd-2 等3个新的apd基因的结构特征和表达进行了分析, 并分析了昆虫APD蛋白家族的序列特征。结果显示, 在西方蜜蜂Apis mellifera（简称“西蜂”）中, apd基因家族的6个成员串联排列在基因组序列第4号连锁群上, 它们在A. m. ligustica雄蜂头部中的转录水平差异明显, 且其启动子序列所含顺式元件也不同。中蜂apd-2和意蜂apd-1-like都含有3个外显子和2个内含子, 而意蜂apd-3-like则由4个外显子和3个内含子组成。蛋白序列分析结果显示, 目前已知的10条APD蛋白序列N末端均具有相似的信号肽序列, 其成熟蛋白分子量为6.0~37.0 kD, pI为6.2~10.8。其中西蜂的APD1-3、APD-like和东方蜜蜂Apis cerena的APD-2等5条较短的多肽中疏水氨基酸残基达52%~67%, 且Ala含量最为丰富（占25%~34%）; 而丽蝇蛹集金小蜂Nasonia vitripennis的APD 1-3和西蜂APD-1-like, APD-3-like等另外5条APD多肽富含Gly（21%~30%）, 其序列中疏水氨基酸残基含量为35%~41%。多肽序列多重比对和系统进化分析结果显示, APD家族可划分为2个亚家族。亚家族Ⅰ含有西蜂APD 1-3和东方蜜蜂APD-2等4条较短的多肽序列, 其N末端为一个长33 aa的保守基序; 亚家族Ⅱ由另外6条相对较长的多肽序列组成, 其N末端保守基序长50 aa, C末端保守基序长16 aa。本文所描述的APD蛋白家族序列特征有助于以后从其他昆虫中鉴定新的apd基因。     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.     

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta,Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Surfactant cholesterol metabolism of the isolated perfused rat lung

The cuticle (0.15 to 0.5 microns thick) of the microscopic free-living nematode Panagrellus silusiae was isolated intact by incubating worms with 1% sodium dodecyl sulfate at 37 degrees C overnight. After shearing and further treatment with detergent, electron microscopy revealed that the cuticular pieces were free of contaminating material and retained their characteristic in situ ultrastructure. From amino acid determinations, the cuticle is collagen-like with high levels of glycine (approximately equal to 31 residue %), proline (approximately equal to 20 residue %) and alanine (approximately equal to 21 residue %) although the hydroxyproline (2.6 residue %) content is low. Half-cystine (approximately equal to 1 residue %) is present in purified cuticles. Treatment with 8 M guanidine hydrochloride-2% beta-mercaptoethanol can solubilize more than 85% of the cuticular preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized cuticles from juvenile, adult and old dead worms revealed, at least, 18 discrete components. Estimated molecular weights ranged from about 26 000 (peak 1) to 250 000 (peak 18).     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

Cuticular proteins: The neglected component

This paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

Is beta-pleated sheet the molecular conformation which dictates formation of helicoidal cuticle?

Over 100 sequences for cuticular proteins are now available, but there have been no formal analyses of how these sequences might contribute to the helicoidal architecture of cuticle or to the interaction of these proteins with chitin. A secondary structure prediction scheme (Hamodrakas, S.J., 1988. A protein secondary structure prediction scheme for the IBM PC and compatibles. CABIOS 4, 473-477) that combines six different algorithms predicting alpha-helix, beta-strands and beta-turn/loops/coil has been used to predict the secondary structure of chorion proteins and experimental confirmation has established its utility (Hamodrakas, S.J., 1992. Molecular architecture of helicoidal proteinaceous eggshells. In: Case, S.T. (Ed.), Results and Problems in Cell Differentiation, Vol. 19, Berlin-Heidelberg, Springer Verlag, pp. 116-186 and references therein). We have used this same scheme with eight cuticular protein sequences associated with hard cuticles and nineteen from soft cuticles. Secondary structure predictions were restricted to a conserved 68 amino acid region that begins with a preponderance of hydrophilic residues and ends with a 33 amino acid consensus region, first identified by Rebers and Riddiford (Rebers, J.F., Riddiford, L.M., 1988. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. J. Mol. Biol. 203, 411-423). Both classes of sequences showed a preponderance of beta-pleated sheet, with four distinct strands in the proteins from 'hard' cuticles and three from 'soft'. In both cases, tyrosine and phenylalanine were found on one face within a sheet, an optimal location for interaction with chitin. We propose that this beta-sheet dictates formation of helicoidal cuticle.     

Cuticular Proteins in Insects and Crustaceans

Comparisons between crustacean and insect cuticles are hamperedby the paucity of cuticular protein sequences for the former.Sufficient complete sequences are available for insect cuticularproteins to allow recognition of conserved motifs and relationshipsamong proteins that reflect the type of cuticle from which theyhave been extracted. All five sequences from an arachnid andtwo of 14 from crustaceans have a motif found in the largestgroup of insect cuticular proteins. Numerous insights have beengained from studying insect cuticular proteins and their genes.These insights have been summarized in hopes of encouraginginterest in building on the foundations laid by Dorothy Skinnerwith the exoskeleton of Gecarcinus.     

Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies. Immunoblotting analysis and ultrastructural immunolocalization in a beetle, Tenebrio molitor

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.     

Investigations on epiphytic living Pseudomonas species from Malus domestica with an antagonistic effect to Venturia inaequalis on isolated plant cuticle membranes

In order to understand better the survival and mutual interaction of epiphytic bacteria and fungi on apple plants, bacteria collected from these plants were cultivated on intact adaxial, stoma free cuticle membranes originally obtained from apple. The bacteria were labelled with luciferase genes from Vibrio harveyi in order to follow up their development and activity on the isolated cuticles. Our finding was that the epiphytic bacteria can have access to nutrients below the cuticle without causing damage to these cuticular membranes. Bacterial proteins may enable this nutrient mobilization and we found, indeed, that more than 46 proteins that must have been delivered by the bacteria in response to interaction with the cuticles as they could be found below the cuticle membrane. Eight major representatives of this group of external proteins have been sequenced with electron spray quadrupole time of flight mass spectrometry and subsequently identified by data base homology search as a flagellin, a porin type protein and proteins that are involved in amino acid recruitment and metabolism.     

The cuticle of the nematode Caenorhabditis elegans: A complex collagen structure

The cuticle of the nematode Caenorhabditis elegans forms the barrier between the animal and its environment. In addition to being a protective layer, it is an exoskeleton which is important in maintaining and defining the normal shape of the nematode. The cuticle is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively crosslinked. Although it also contains other protein and non-protein compounds that undoubtedly play a significant part in its function, the specific role of collagen in cuticle structure and morphology is considered here. The C. elegans genome contains between 50 and 150 collagen genes, most of which are believed to encode cuticular collagens. Mutations that result in cuticular defects and grossly altered body form have been identified in more than 40 genes. Six of these genes are now known to encode cuticular collagens, a finding that confirms the importance of this group of structural proteins to the formation of the cuticle and the role of the cuticle as an exoskeleton in shaping the worm. It is likely that many more of the genes identified by mutations giving altered body form, will be collagen genes. Mutations in the cuticular collagen genes provide a powerful tool for investigating the mechanisms by which this group of proteins interact to form the nematode cuticle.