Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Cloning of cDNA for cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), and characterization of recombinant CBBP

Previously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.     

Purification of the 200 kDa hemocyte membrane protein of Sarcophaga peregrina and its specific interaction with fat body

A 200 kDa protein specifically expressed on the surface of pupal hemocytes of Sarcophaga peregrina was purified from the hemocyte membrane. This protein has been suggested to participate in dissociation of the fat body in the pupal stage of this insect. This protein was found to inhibit the dissociation of the fat body in vitro. Furthermore, it was shown to bind to the fat body and the binding could be saturated. These results suggested that pupal hemocytes expressing the 200 kDa protein interact directly with specific binding sites on the basement membrane of the fat body when they disintegrate this tissue.     

Wound Responses in Insects

For over a century, the study of specific antipathogenic strategiesin insects has been confounded by non-specific responses tointegumental invasion. Experimental injury to diapausing Hyalophoracecropia silkmoth pupae elucidated some of the events inherentin this response—increased oxygen consumption and DNAand RNA synthesis leading to de novo synthesis of proteins,some of which are constituents of the adult protein cohort aswell as some injury-specific ones. The mechanism which enforcesdiapause is apparently released by integumental injury as wellas by normal developmental stimuli. Recent work has concentratedon purification of antipathogenic and injury-specific proteins,the possible involvement of lectins in the immune response,and localization of synthesis of these proteins in hemocytesand fat body cells. At least ten different hemolymph proteinswhich are synthesized by fat body cells in response to inoculationof lepidopteran species with bacteria currently are being isolated.The hemolymph of H. cecropia contains lectins which are synthesizedby hemocytes. Analysis of in vitro incorporation of ['H]leucineby hemocytes into proteins reveals that these lectins apparentlyare not constituents of the secreted injury response proteincomplex in fifth instar caterpillars or diapausing pupae, norare hemolymph lectin titers significantly different in healthyversus diseased or injured animals. However, intracellular lectinconcentrations may increase upon injury. Increased lectin titerand induction of bactericidal activity coincide in another holometabolousspecies, the fleshfly Sarcophaga peregrina. Pursuit of thesestudies may elaborate our knowledge of insect cellular immunity.     

Participation of a 200-kDa hemocyte membrane protein in the dissociation of the fat body at the metamorphosis of Sarcophaga

Three monoclonal antibodies were raised against a 200-kDa protein specifically expressed on the surface of hemocytes when Sarcophaga larvae pupated. One of these antibodies significantly inhibited dissociation of the fat body in the presence of pupal hemocytes, indicating that the 200-kDa protein is essential for dissociation of the fat body by the hemocytes. When the hemocytes were treated with a mixture of the monoclonal antibodies, they were found to secrete a significant amount of chymotrypsin-like proteinase. Moreover, the number of hemocytes expressing the 200-kDa protein increased significantly after puparium formation. These results suggested that some signal is transferred to the hemocytes via the 200-kDa protein when they interact with the basement membrane of the fat body and that this induces their secretion of a chymotrypsin-like proteinase essential for decomposition of the fat body.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

Two subunits of the insect 26/29-kDa proteinase are probably derived from a common precursor protein

We previously identified the 26/29-kDa proteinase in the hemocytes of Sarcophaga peregrina (flesh fly) that appears to participate in elimination of foreign proteins in this insect [Eur. J. Biochem. 209, 939-944 (1992)]. Here, we report the cDNA cloning of this proteinase. The cDNA encodes a protein which includes both the 26- and 29-kDa subunit, strongly suggesting that the both subunits are derived from a single precursor protein. The 26- and 29-kDa subunit located at the amino-terminal and carboxyl-terminal of the precursor protein. The 29-kDa subunit itself appeared to be a proteinase, for this subunit had 52% sequence identity with Sarcophaga cathepsin L, while 26-kDa subunit had no significant similarity. We also showed that 26/29-kDa proteinase was insensitive to specific inhibitors of cathepsin L. These results indicate that this proteinase is a novel member of the papain family. We isolated similar cDNAs from Drosophila melanogaster and Periplaneta americana (cockroach), suggesting that this proteinase is conserved in a wide variety of insects and participates in their defense mechanisms.     

Interaction between ATBP and DmUbc9 in the expression of the Sarcophaga lectin gene

We previously demonstrated that (A+T)-stretch binding protein (ATBP) and Dorsal-related immunity factor (Dif) are required for the expression of the Sarcophaga lectin gene in SL-2 cells (Aozasa et al., Eur. J. Biochem. 268, 2506-2511, 2001). The present study demonstrates that DmUbc9 interacts with ATBP, and cotransfection of the DmUbc9 vector with ATBP and Dif vectors greatly enhances the expression of the luciferase reporter of the Sarcophaga lectin gene in SL-2 cells. These results suggest that sumoylation of ATBP is involved in the expression of the Sarcophaga lectin gene in this system.     

Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al. , Dev. Biol. 144 , 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.     

Purification and characterization of a lectin-like molecule specific for galactose/N-acetyl-galactosamine from tumoricidal macrophages

A lectin-like molecule (macrophage lectin) was purified from murine peritoneal exudate macrophages which had been induced with an antitumor streptococcal preparation, OK-432. The purified macrophage lectin from both 3H-labeled and unlabeled macrophages after rechromatography on a beta-D-galactose-Bio-Gel P-100 column gave a broad single band corresponding to 45-60 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The broadness of this band was due to high N-glycosylation of the lectin, because the lectin gave a compact band corresponding to 35 kDa on SDS-PAGE after deglycosylation. The lectin required Ca2+ for binding and showed an optimum pH of around 6. The sugar specificity of the lectin was examined by means of an inhibition assay using simple sugars and neoglycoproteins. The lectin was found to be specific for D-galactose/N-acetyl-D-galactosamine, and not inhibited with D-mannose or N-acetyl-D-glucosamine at all. The lectin was detected on the surface of OK-432-elicited and thioglycolate-elicited macrophages, but it was not detected on resident macrophages. Moreover, the binding of tumor cells to macrophages was inhibited by the addition of the purified lectin to the binding mixture. These results suggest that this lectin is expressed on the surface of activated macrophages, and that it participates in the interaction between tumoricidal macrophages and tumor cells.     

A novel hemocyte-specific membrane protein of Sarcophaga (flesh fly).

Extensive tissue remodeling takes place during metamorphosis of holometabolous insects. It has been shown that hemocytes play crucial roles in the recognition and elimination of apoptotic cells and larval tissue fragments produced during metamorphosis. We report the immunoaffinity purification, cDNA cloning, and characterization of a prepupal hemocyte membrane protein of Sarcophaga (flesh fly) with a molecular mass of 120 kDa. This protein is a novel type I transmembrane protein with 18 repeats of an epidermal growth factor-like domain in the predicted extracellular region. Expression of the protein was restricted exclusively to prepupal hemocytes. This protein is suggested to be a scavenger receptor for tissue remodeling.     

Rapid inactivation of tumor killing factor in vitro

The mechanism of the rapid inactivation of the cytotoxic activity produced by peritoneal macrophages stimulated with Sarcophaga lectin was investigated. Experiments using radioiodinated tumor killing factor (TKF) showed that the main reason for its inactivation was the rapid dissociation of its trimeric structure into 15-kDa subunits. This dissociation was found to proceed spontaneously in the culture medium irrespective of the presence of macrophages.     

Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner

The imaginal discs of Sarcophaga were found not to develop normally in the presence of galactose, a hapten sugar of Sarcophaga lectin, or anti-Sarcophaga lectin antibody. Wing and leg discs cultured with these substances became morphologically abnormal and no imaginal discs reached the stage of terminal differentiation, even in the presence of 20-hydroxyecdysone. The development of the imaginal discs was shown to be autonomously regulated in an autocrine manner by Sarcophaga lectin; namely Sarcophaga lectin was secreted by the imaginal discs in the presence of 20-hydroxyecdysone, and the stimulus of self-induced Sarcophaga lectin seemed to be indispensable for further development of the imaginal discs. Sarcophaga lectin was originally found as a defense protein, but these results show that it plays independent roles in both defense and development.     

Activation of the Sarcophaga lectin gene promoter in transgenic Drosophila

We established transgenic Drosophila strains in which the lacZ gene was expressed under the control of the 5'-upstream regulatory region of the Sarcophaga lectin gene promoter (3.1 kbp). The reporter gene was expressed in the fat bodies of the transgenic larvae when they were immunized by body pricking or treatment with Escherichia coli, which was the same as the Sarcophaga lectin gene expression in Sarcophaga larvae. However, the same reporter gene was found to be expressed constitutively in the digestive tracts of the transgenic larvae even without immunization.     

Targeted disruption of a pupal hemocyte protein of Sarcophaga by RNA interference.

Previously, we purified a transmembrane protein with a molecular mass of 120 kDa (p120) that is exclusively expressed in pupal hemocytes of Sarcophaga. In this study, we demonstrated that double-stranded RNA (dsRNA) injected into the larval body cavity effectively inhibited the expression of p120 in pupal hemocytes. Thus, RNA interference (RNAi) was found to be a useful technique for creating pupal hemocytes with a loss-of-function of a specific protein. The p120-less pupal hemocytes generated by RNAi were found to have lost the ability to take up acetylated low density lipoprotein, indicating that p120 is a scavenger receptor specifically expressed on the surface of pupal hemocytes.