Immulectin-2, a lipopolysaccharide-specific lectin from an insect, Manduca sexta, is induced in response to gram-negative bacteria

A lipopolysaccharide-specific lectin, immulectin-2, was isolated from plasma of the tobacco hornworm, Manduca sexta. Immulectin-2 has specificity for xylose, glucose, lipopolysaccharide, and mannan. A cDNA clone encoding immulectin-2 was isolated from an Escherichia coli-induced M. sexta larval fat body cDNA library. The cDNA is 1253 base pairs long, with an open reading frame of 981 base pairs, encoding a 327-residue polypeptide. Immulectin-2 is a member of the C-type lectin superfamily. It consists of two carbohydrate recognition domains, which is similar to the organization of M. sexta immulectin-1. Immulectin-2 was present at a constitutively low level in plasma of control larvae and increased 3-4-fold after injection of Gram-negative bacteria or lipopolysaccharide. Immulectin-2 mRNA was detected in fat body of control larvae, and its level increased dramatically after injection of E. coli. The concentration of immulectin-2 in plasma did not change significantly after injection of Gram-positive bacteria or yeast, even though its mRNA level was increased by these treatments. Compared with immulectin-1, immulectin-2 has a more restricted specificity for binding to Gram-negative bacteria. Immulectin-2 at low physiological concentrations agglutinated E. coli in a calcium-dependent manner. It also bound to immobilized lipopolysaccharide from E. coli. Binding of immulectin-2 to lipopolysaccharide stimulated phenol oxidase activation in plasma. The properties of immulectin-2 are consistent with its function as a pattern recognition receptor for detection and defense against Gram-negative bacterial infection in M. sexta.     

A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection.     

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.     

An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.     

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm,Manduca sexta

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.     

亚洲玉米螟C型凝集素CTL6的克隆和功能分析

【目的】鉴定出一种新的亚洲玉米螟Ostrinia furnacalis (Guenée) C型凝集素(C-type lectin),并对其功能进行初步研究。【方法】通过生物信息学分析,从亚洲玉米螟转录组中筛选得到一个可能的C型凝集素基因,命名为CTL6。利用RT-PCR技术分析该基因在亚洲玉米螟不同龄期、不同组织及不同病原物诱导下的表达模式。借助原核及杆状病毒真核表达系统产生重组CTL6蛋白,并利用细菌凝集实验对其功能进行初步研究。【结果】CTL6基因cDNA全长序列为1 034 nt,其中完整开放阅读框为945 nt。推导的CTL6多肽序列包括314个氨基酸残基,N端含有由22个氨基酸残基组成的信号肽。CTL6成熟肽中含有两个串联的糖识别结构域,与烟草天蛾Manduca sexta的IML-2 (Immulectin-2)同源性最高。RT-PCR结果显示,CTL6在亚洲玉米螟5龄幼虫期转录水平最高,卵期其次,不同组织中则是在脂肪体中转录水平最高,呈诱导性表达。纯化的CTL6重组蛋白对大肠杆菌Escherichia coli具有一定的凝集作用。【结论】鉴定到的亚洲玉米螟CTL6是一种典型的C型凝集素,重组CTL6蛋白可能参与了亚洲玉米螟对病原菌的凝集作用。     

Calcium is not required for immulectin-2 binding, but protects the protein from proteinase digestion

Mammalian C-type lectins are calcium-dependent carbohydrate-binding proteins. They serve as cell adhesion molecules in cell-cell interactions, or function as pattern-recognition receptors in innate immunity. Calcium is a direct ligand for carbohydrate binding in mammalian C-type lectins such as mannose-binding proteins and macrophage mannose receptor. In the tobacco hornworm Manduca sexta, a group of lectins named immulectins have been discovered. Each immulectin contains dual carbohydrate-recognition domains. Previously, we showed that immulectin-2 (IML-2) binds to a bacterial lipopolysaccharide, and agglutination of Escherichia coli cells by IML-2 is calcium dependent. In this study, we demonstrated that IML-2 bound to bacterial lipid A, smooth and rough mutants of lipopolysaccharide, lipoteichoic acid and peptidoglycan, as well as to fungal mannan and beta-1, 3-glucan (laminarin and curdlan). Binding of IML-2 to microbial components was calcium independent, and was increased by addition of spermine, a polyamine. In addition, plasma IML-2 bound to mannan-agarose independent of calcium. But trypsin digestion of IML-2 was inhibited in the presence of calcium. Our results suggest that calcium is not required for IML-2 binding but protects IML-2 from trypsin digestion.     

Primary structure and comparative sequence analysis of an insect apolipoprotein. Apolipophorin-III from Manduca sexta

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Manduca sexta, was determined by a combination of cDNA and protein sequencing. The mature hemolymph protein consists of 166 amino acids. The cDNA also encodes for an amino-terminal extension of 23 amino acids which is not represented in the mature hemolymph protein. The existence of a precursor protein was confirmed by in vitro translation of fat body mRNA. Computer-assisted comparative sequence analysis revealed the following points: 1) the protein is composed of tandemly repeating tetradecapeptide units with a high potential for forming amphiphilic helical structures. Compared to mammalian apolipoproteins the repeat units in the insect apolipoprotein show considerable length variability; 2) the sequence has a striking resemblance to several human apolipoproteins including apoE, AIV, AI, and CI. However, the homology seems to be entirely functional since, although the insect and mammalian apoproteins contain very similar types of amino acid residues, the actual degree of sequence identity is quite low. Whether the mammalian and insect apoproteins are derived from a common ancestral amphiphilic helix forming, lipid-binding protein, or arose by convergent evolution can not be determined at present. This represents the first complete amino acid sequence for an insect apolipoprotein.     

Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein

The amino acid sequence of an insect apolipoprotein, apolipophorin-III from Locusta migratoria, has been deduced from the sequence of its cloned cDNA. The mature hemolymph protein consists of 161 amino acids. Optimized alignments of this protein with apolipophorin-III from the tobacco hornworm, Manduca sexta, disclosed an overall sequence identity of only 29%, even though the two proteins are functionally equivalent. The L. migratoria sequence is composed of 12 repeating peptides that are variable in length. Six amphipathic helical segments of varying length were identified in each protein using a newly described algorithm for detecting such secondary structures. The degree of sequence identity between the two insect apoproteins is considerably less than that observed among orthologous mammalian apolipoproteins. However, calculation of the rates of synonymous and nonsynonymous nucleotide substitutions indicates that the insect genes may be evolving at rates similar to the mammalian apolipoprotein genes. Further comparative analyses of insect and mammalian apolipoproteins should provide insights about the limits of sequence diversity tolerated by their predicted amphipathic helical domains.     

A pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, Manduca sexta

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.     

Innate immune responses in peptidoglycan recognition protein L-deficient mice

Peptidoglycan recognition proteins (PGRPs) constitute a family of innate immune recognition molecules. In Drosophila, distinct PGRPs bind to peptidoglycans on gram-positive or gram-negative bacteria and provide essential signals upstream of the Toll and Imd pathways required for immunity against infection. Four PGRPs, PGRP-L, -S, -Ialpha, and -Ibeta, are expressed from three genes in mammals. In this paper, we provide direct evidence that the longest family member, PGRP-L, is a secreted serum protein with the capacity to multimerize. Using gene targeting to create PGRP-L-deficient mice, we demonstrate little contribution by PGRP-L to systemic challenge using gram-negative bacteria (Escherichia coli, slightly less susceptible), Gram-positive bacteria (Staphylococcus aureus), or yeast (Candida albicans). Peritoneal macrophages from PGRP-L-deficient mice produced decreased amounts of the inflammatory cytokines interleukin 6 and tumor necrosis factor alpha when stimulated with E. coli or lipopolysaccharide, but comparable amounts when stimulated with S. aureus, C. albicans, or their cell wall components. Additionally, these cells produced similar amounts of cytokines when challenged with gram-positive or -negative peptidoglycans. In contrast to its critical role in immunity in flies, PGRP-L is largely dispensable for mammalian immunity against bacteria and fungi.     

亚洲玉米螟免疫凝集素基因的克隆、序列分析及组织表达(英文)

C型凝集素作为模式识别分子可以识别部分脂多糖(LPS),进而参与昆虫细胞的防御反应。本文通过RT-PCR和3′/5′RACE技术从亚洲玉米螟Ostriniafurnacalis 5龄幼虫血细胞中克隆得到免疫凝集素基因(OfIML)。OfIMLmRNA全长为1241 bp,其中开放读码框(ORF)为924 bp,编码307个氨基酸(aa),分子量约为34.65 ku。与其它昆虫的C型凝集素比对分析结果显示,OfIML属于鳞翅目免疫凝集素,并且含有一个独特的结构特征,即一前一后2个糖识别域,氨基末端(CRD1,aa#1-135)和羧基末端(CRD2,aa#136-287)。RT-PCR检测OfIML在幼虫组织中的分布结果表明,其在血细胞、表皮、脂肪体、中肠、马氏管和气管中都有表达。OfIML GenBank登录号为ABZ81710。OfIML是一种昆虫免疫凝集素,含有2个糖识别域,根据其分子结构及在组织分布中的结果显示可能在亚洲玉米螟的免疫反应中起重要作用。     

Molecular cloning of a mouse scavenger receptor with C-type lectin (SRCL)(1), a novel member of the scavenger receptor family.

The cDNA clone encoding a mouse scavenger receptor with C-type lectin (SRCL), a novel member of the scavenger receptor family, has been isolated from a mouse embryonic cDNA library. The predicted cDNA sequence contains a 2226 bp open reading frame encoding a coiled-coil, collagen-like, C-type lectin/carbohydrate recognition domain with an overall sequence identity of 92% to human SRCL. In contrast to human, mouse SRCL mRNA was expressed ubiquitously in various adult tissues including the liver and spleen, in which human SRCL mRNA was under detection limits. Mouse SRCL mRNA was expressed in the macrophage cell line J774A.1 cells at a high level and in the embryo as early as E9.     

A novel pattern recognition protein of the Chinese oak silkmoth,Antheraea pernyi,is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]