A novel ML protein from Manduca sexta may function as a key accessory protein for lipopolysaccharide signaling

Abstract

Lipopolysaccharide (LPS) present on the outer membrane of Gram-negative bacteria is one of the most important pathogen-associated molecular patterns and a potent elicitor in innate immunity. In human, TLR4 (Toll-like receptor 4) and MD-2 (myeloid differiation-2) form a receptor complex to transduce the LPS signal into cells. However, in invertebrates, receptors that recognize LPS have not been determined. Here we report the purification, characterization and cDNA cloning of an ML (MD-2-related lipid-recognition) protein from the tobacco hornworm Manduca sexta. The full-length cDNA of this M. sexta ML protein, named MsML-1, is 532bp with an open reading frame of 456bp that encodes a polypeptide of 151 amino acids containing an ML domain. MsML-1 is a secreted glycoprotein and its mRNA is expressed in fat body and hemocytes. The expression level of MsML-1 mRNA in fat body and hemocytes as well as MsML-1 protein in hemolymph are not induced by immune challenge. Recombinant MsML-1 protein specifically binds to LPS from several Gram-negative bacteria and LPS Re mutant, as well as to lipid A, but not to KDO (2-keto-3-deoxyoctonate). Our results suggest that MsML-1 may function as a key accessory protein for LPS signaling in M. sexta against Gram-negative bacterial infection.

Fig. 1

Analysis of M. sexta ML-1 (MsML-1) protein purified from hemolymph. (A) SDS-PAGE analysis of MsML-1 purified from hemolymph. MsML-1 and a 46-kDa protein were purified to homogeneity from hemolymph after several chromatographies. Co-purified MsML-1 and the 46-kDa protein (lane 1, 3µg) and purified MsML-1 (lane 2, 2µg) and the 46-kDa protein (lane 3, 2µg) were analyzed by 15% SDS-PAGE, and the gel was stained by Coomassie Brilliant Blue. (B) Amino-terminal sequences of MsML-1 and the 46-kDa protein. The amino-terminal sequences of MsML-1 and the 46-kDa protein (band a) were determined by Edman degradation, and the sequence of leureptin (residues 19–31) was obtained from the Genbank database (accession number: AAO21503). (C) Deglycosylation of MsML-1. MsML-1 (2.0µg) purified from hemolymph was heated to 100 °C for 3 min in 20 mM sodium phosphate buffer, pH 7.2. The denatured protein was then incubated with or without 1U of N-glycosidase F (PNGase F) (Sigma) in 50µL of 50 mM phosphate buffer, pH 7.2, 0.1% SDS, 0.5% (v/v) Nonidet P-40 and 0.5% (v/v) 2-mercaptoethanol for 24 h at 37 °C. Both N-glycosidase F treated and untreated MsML-1 samples (1.0µg each) were analyzed by 15% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. (D) Immunoblotting analysis of MsML-1 under reducing and non-reducing conditions. MsML-1 (0.5µg each) purified from hemolymph was dissolved in the sample loading buffer in the presence (reducing) or absence (non-reducing) of β -mercaptoethanol and heated to 95 °C for 5 min. Protein samples were separated by 15% SDS-PAGE, and MsML-1 was identified by immunoblotting using rabbit polyclonal antiserum against recombinant MsML-1. The arrowhead indicates the 46-kDa protein (band a) while the arrows indicate native MsML-1.

Fig. 2

Alignment of MsML-1 with homologous insect proteins, mite allergen Der f2, and human and mouse MD-2. The deduced amino acid sequence of MsML-1 is aligned with homologous proteins from other insect species, D. melanogaster (CG11314 and CG11315), B. mori (accession number: AB030701), A. gambiae (accession number: EAA00385), A. aegypti (accession number: XP 001648436), A. mellifera (accession number: XP 392711), as well as with mite allergen Der f2 (accession number: CAI05848), human MD-2 (hMD-2) (accession number: BAA78717) and mouse MD-2 (mMD-2) (accession number: BAA93619) proteins. Cysteine residues that are conserved in all the proteins are labeled with asterisks above the alignment; two cysteine residues that are conserved only in insect proteins are labeled with filled diamonds; a cysteine residue that is conserved in insect proteins and mite allergen Der f2 but absent in MD-2 is labeled with a filled triangle; and two cysteine residues that are conserved only in MD-2 proteins are labeled with open triangles. Identities between MsML-1 and other proteins are also indicated in the parentheses.

Fig. 3

Tissue distribution of MsML-1. Total RNAs (1µg each) from midgut (Gut), epidermis (Ep), Malpighian tubule (Mt), hemocytes (Hc) and fat body (Ft) of M. sexta naïve larvae were reverse-transcribed into cDNA using Generacer™ Oligo-dT primer. The cDNA was used as a template for PCR reactions with MsML-1 specific primers (32 and 35 cycles) or RPS3 specific primers (30 cycles). PCR products were analyzed on an agarose gel and stained with ethidium bromide. The asterisks indicate a non-specific amplification product in the Malpighian tubule sample.

Fig. 4

Expression of MsML-1 mRNA in fat body and hemocytes and MsML-1 protein in hemolymph after immune challenge. Day 2 fifth instar M. sexta larvae were injected with heat-killed E. coli (10⁸ cells/larva), M. luteus (100µg/larva), yeast (S. cerevisiae) (10⁷ cells/larva) or saline (as a control). Hemocytes and fat body were collected at 24 h post-injection. Total RNAs were prepared from these hemocytes and fat body, and then transcribed into cDNAs using GeneRacer™ Oligo-dT primer. Expression of MsML-1 mRNA in microbial challenged hemocytes or fat body was determined by real-time PCR in three replicas (A and B). The bars represent the mean of three individual measurements±S.E.M. For MsML-1 protein expression in hemolymph after immune challenge (C and D), day 2 fifth instar M. sexta larvae were injected with saline (as a control), heat-killed E. coli, M. luteus or yeast (S. cerevisiae) as described above, or with LPS (E. coli 026:B6), LTA or laminarin (20µg each per larva) (four larvae for each group). Hemolymph was collected from each larva at 0 and 24 h after injection and equal volumes of the plasma samples were mixed, diluted (1:1 in water), and then added to the 2 × SDS loading buffer. For electrophoresis, diluted plasma samples (each corresponding to 2 µL of the original mixed plasma) were loaded to 15% SDS-PAGE. MsML-1 in each plasma sample was detected by immunoblotting using rabbit polyclonal antibody to recombinant MsML-1. The arrows indicate MsML-1 protein in hemolymph.

Fig. 5

Recombinant MsML-1 binds to immobilized LPS and lipid A. A 96-well microtiter plate was coated with LPS (E. coli strains 0111:B4 and 026:B6, P. aeruginosa, S. minnesota, Re mutant of S. minnesota Re595) (50 µL/well, 2 µg/well), mono- or di-phosphoryl lipid A (1 µg/well), or KDO (2 µg/well) (panels A and B), and renatured recombinant MsML-1 (rMsML-1) or CP36 (rCP36), diluted to 20 µm in the binding buffer (50 mM Tris–HCl, 50 mM NaCl, pH 8.0) containing 0.1 mg/mL BSA, was added to the LPS (or lipid A)-coated plate (50 µL/well) and incubated at room temperature for 3 h. A 96-well microtiter plate was also directly coated with renatured rMsML-1 or rCP36 (50 µL/well, 20 and 100 µm) (panel C). Then, the total binding (panel A) of recombinant proteins and the specific binding of MsML-1 (panel B) to immobilized LPS or lipid A were determined by ELISA using the rabbit polyclonal antiserum against rMsML-1 or rCP36. The specific binding of recombinant MsML-1 to LPS and lipid A was calculated by subtracting the total binding of rCP36 from the total binding of rMsML-1. Reaction of antibodies to recombinant proteins directly coated on the plate (panel C)was also measured by ELISA. Each point represents the mean of three individual measurements±S.E.M. Significance of difference was calculated using a Student’s T-test.

Fig. 6

Recombinant MsML-1 specifically binds to immobilized lipid A. A 96-well microtiter plate was coated with diphosphoryl lipid A (E. coli F598 Rd mutant) (50 µL/well, 1 µg/well) and blocked with BSA. Renatured rMsML-1 or rCP36, diluted in the binding buffer at different concentrations (0–200 µm), was added at 50 µ/well and incubated at room temperature for 3 h. Binding of the recombinant proteins to the immobilized lipid A was determined by ELISA as described above in Fig. 5. Each point represents the mean of three individual measurements ± S.E.M.

Fig. 7

Structures of human MD-2, mite allergen Der f2 and MsML-1. Cartoon representations of human MD-2 (PDB: 2e56) (A) and mite allergen Der f2 (PDB: 1wrf) (B) structures and a model structure of MsML-1 (C) based on the structure of mite allergen Der f2. The electrostatic potential surfaces of human MD-2 (D), mite allergen Der f2 (E) and MsML-1 (F) were also calculated using the PyMOL program and positive and negative potentials are shown in blue and red, respectively. The potential surface of MD-2 (D) is viewed from a 90° rotation with respect to (A), whereas (E) and (F) are the same views of (B) and (C), respectively.