Báo cáo khoa học: Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid An immunoglobulin superfamily member from insects as a pattern-recognition receptor doc

Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid An immunoglobulin superfamily member from insects as a pattern-recognition receptor Xiao-Qiang Yu and Michael R. Kanost Department of Biochemistry, Kansas State University, Manhattan, KS, USA Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis i s induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopo lysaccharide (LPS) from Gram-negative b acteria and to lipoteichoic acid from G ram- positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and R c) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS w as partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely b locked by free phosphate. O ur results suggest that hemolin has t wo binding sites for LPS, one that interacts with t he phosphate groups of lipid A and one that interacts w ith t he O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro- organisms. Keywords: h emolin; i nsect immunity; lipopolysaccharide; lipoteichoic a cid; pattern recognition r eceptor. Upon microbial infection, insects synthesize d efensive plasma proteins, which include antimicrobial peptides and proteins, lectins, and cell a dhesion molecules [1–3]. One such protein is hemolin, a member of the i mmunoglobulin (Ig) superfamily. H emolin contains four Ig do mains of the I-set type which are most similar t o t hose in neural cell adhesion molecules [4–6]. Hemolin has been isolated from hemo- lymph of two immune-challenged lepidopteran insects, Hyalophora cecropia and Manduca sexta [7,8]. A hemolin- like cDNA was also cloned from the fall webworm, Hyphantria c unea [9]. Hemolin is synthesized mainly in fat body in response to microbial chal lenge [4,8], but it is also synthesized in the absence of infection in embryos [10] and in fat body and midgut during metamorphosis [3,11,12]. Hemolin expressed a t d ifferent developmental s tages o f M. sexta differs in carbohydrate content. Hemolin isolated from adult moths and from bacteria-induced larvae con- tains noncovalently bound carbohydrates, whereas hemolin from wandering stage (prepupal) larvae lacks carbohydrates [11]. Available data suggest that hem olin functions in immune responses by interacting with insect hemocytes and with bacteria. It binds to hemocytes and bacteria, a nd its binding to hemocytes inhibits hemocyte aggregation [5,13–15]. Hemolin from H. cecropia interacts with bacterial lipopoly- saccharide (LPS) and its lipid A component [15,16] and binds to hemocytes in a calcium-dependent manner [17]. A membrane-bound form of hemolin has also been reported [18]. It h as been suggested that hemolin may modulate hemocytic activities in development and during immune responses [12], and may function as an opsonin or as a pathogen-recognition molecule in the defense against infec- tion [14–16]. The horseshoe-shape arrangement of t he Ig domains in the structure of H. cecropia hemolin suggested a mechanism for homophilic binding of hemolin to mole cules on the surface of hemocytes or micro-organisms [6]. However, the b iological functions of hemolin i n insects are still not well understood. Recognition of nonself plays an essential role in initiating immune responses. The vertebrate innate immune system and invertebrate immune responses rely on a s et of proteins known as pathogen-recognition receptors. These proteins bind to conserved features o f m icrobial surfaces such as LPS, lipoteichoic acid (LTA) a nd peptidoglycan from bacterial cell walls, and b-1,3-glucan from f ungal cell walls [19,20]. Such recognition may initiate a variety of immune responses in insects, including prophenoloxidase activation, p hagocytosis, nodule formation, and encapsu- lation. In this paper, we focus on t he biological functio ns of hemolin in de fense against microbial infection. We investi- gated its binding to Gram-negative and Gram-positive bacteria, yeast, and bac terial LPS and LTA. Our results indicate that hemolin functions as a pattern-recognition receptor with a broad specificity for diverse pathogens in t he defense against micro-organisms. Correspondence to M. R. Kanost, Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA. Fax: + 7 8 5 532 7278, Tel.: + 785 532 6964, E-mail: kanost@ksu.edu Abbreviations: KDO, 2-oxo-3-deoxyoctanoate; LPS, lipopolysac- charide; LTA, lipoteichoic acid. (Received 2 2 October 200 1, revised 6 February 2002, a ccepted 8 February 2 002) Eur. J. Biochem. 269, 1827–1834 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02830.x EXPERIMENTAL PROCEDURES Hemolin and microbial components Hemolin from hemolymph of naive wandering stage larvae (hemolin form W1 which lacks bound carbohy- drate) was purified as described previously [11], and used for all experiments. Smooth LPS (S-LPS) from Escherichia coli strains 026:B6 and 0111:B4, LPS r ough mutan ts Ra (E. coli EH100), Rc (E. coli J5) and Rd2 (E. coli F583), diphosphoryl lipid A (E. coli F583), LTA from Staphylococcus aureus, 2-oxo-3-deoxyoctanoate (KDO), laminarin, curdlan, zymosan, chitosan, mannan, glucose, galactose, mannose, xylose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc) were purchased from Sigma. The Re m utant of LPS (E. coli D31m4) was from List Biological Laboratory Inc. (Campbell, CA, USA). Peptidoglycan (from S. aureus) was purchased from Fluka. Agglutination of bacteria and yeast by hemolin Fluorescein isothiocyanate-labeled S. aureus, E. coli,or Saccharomyces cerevisiae (Molecular Probes) were sus- pended in Tris-buffered saline ( Tris/NaCl; 25 m M Tris/ HCl, 137 m M NaCl and 3 m M KCl, pH 7.0) and used for the agglutination assay. Hemolin at 0.5 m gÆmL )1 or BSA at 1 . 0 mgÆmL )1 (asacontrol)wasusedin agglutination of micro-organisms as described previously [21]. Binding of 125 I-labeled hemolin to bacteria and yeast Hemolin was labeled with 125 I using Iodobead (Piece) as an iodinatio n reagent. One Iodobead was washed with 500 lL iodin ation buffer (100 m M sodium phosphate buffer, pH 7.0). The washed bead was added to 1 mCi Na 125 I (Dupont NEN) in 434 lL iodination buffer, and incubated for 5 min at room temperature. Then 250 lg purified hemolin in 66 lL deionize d water was mixed with the s olution containing the Iodobead, f ollowed by incubation for 3 min at room temperature. The Iodo- bead was r emoved, a nd 125 I-labeled he molin was sepa- rated from free 125 I by applying the solution to an equilibrated Sephadex G -25 d esalting c olumn (PD10, Pharmacia). The column was eluted with NaCl/P i (25 m M sodium phosphate buffer, 137 m M NaCl and 3m M KCl, pH 7.0), and 0.6 mL fractions were collected. Samples of 5 lL were removed from each fraction and c ounted in a c counter. F ractions containing 125 I-labeled hemolin were pooled and stored at )20 °C. The specific activity of the labeled hemolin was 3.9 · 10 5 c.p.m.Ælg )1 . 125 I-Labeled hemolin at a concentration of 1.0 l M was incubated with formalin-killed E. coli strain XL1-blue, Micrococcus lysodeikticus,orS. cerevisiae (yeast) (each at 3 · 10 5 cells) in 50 lL Tris/NaCl containing 1 m gÆmL )1 BSA, in the absence or presenc e of 50 l M unlabeled hemolin. The mixture was incubated for 30 min at room temperature, then centrifuged for 5 min at 10 000 g.The supernata nt was removed by aspira tion, and the cells were washed four times with Tris/NaCl, then counted in a c coun ter for bound hemolin. Binding of hemolin to immobilized LPS, LTA or lipid A Wells of a flat-bottom 96-well assay plate (Costar, Fisher) were coate d with 2 lgLPS(E. coli 026:B6), LTA or diphosphoryl lipid A as described p reviously [22]. H emolin at different concentrations prepared in binding buffer (50 m M Tris/HCl, 50 m M NaCl, pH 8.0, 0.1 mg ÆmL )1 BSA) co ntaining 0 or 10 m M CaCl 2 , o r in phosphate buffer (50 m M sodium phosphate, 50 m M NaCl, pH 8.0, 0.1 m gÆmL )1 BSA) was added at 50 lL per well, and binding was allowed to occur for 6 h at room temperature, before washing as described by Yu & Kanost [22]. B ound hemolin was m easured by fi rst incubating wells with rabbit anti-hemolin serum (diluted 1000-fold with binding buffer), then with alkaline phosphatase-conjugated goat anti-(rabbit IgG) Ig (Bio-Rad) (diluted 3 000-fold with binding buffer), and bound alkaline phosphatase activity was determined by hydrolysis of p-nitrophenyl phosphate, all as described previously [22]. The A 405 value o f each well was determined using a microtiter plate reader (Bio-Tek Instrument, Inc.). Binding of hemolin to immobilized LPS in the presence of competitors The w ells of a 96-well plate w ere coated with LPS from E. coli 026:B6 (2 lg per well). Hemolin at a concentration of 30 lgÆmL )1 was p reincubated w ith S-LPS (E. coli strains 026:B6 and 0111:B4), LPS from rough mutants (Ra, Rc, Rd2 and Re), diphosphoryl lipid A, LTA, peptidoglycan, zymosan, laminarin, KDO (each at 0 .8 mg ÆmL )1 ), glucose, galactose, mannose, GlcNAc, GalNAc, or xylose (each at 0.4 m M )in50lL binding buffer for 3 h at room temper- ature. The mixture was then added to S-LPS (E. coli 026:B6)-coated w ells, and the binding was allowed t o occur at room temperature f or 6 h before washing and detection of bound hemolin as described above. RESULTS Agglutination of bacteria and yeast by hemolin To inve stigate whether hemolin can bind t o bacteria or yeast and cause aggregation of t hese micro-organisms, we performed an agglutination assay. When E. coli, S. aureus, or S. cerevisiae cells we re incubated with hemolin at a concentration of 0.5 mg ÆmL )1 , aggregates of bacteria and yeast were observed (Fig. 1) . The size of the aggregates correlated with hemolin concentration, with higher hemolin concentration resulting in larger aggregates (data not shown). When these micro-organisms were incubated with a control protein, B SA, no obvious aggregates were observed ( Fig. 1). A gglutination of bacteria by hemolin was not affected by addition of EDTA (data not shown), indicating that hemolin does not require bivalent cations for its a gglutination activity. The observed agglutination of bacteria and yeast by hemolin may be due to binding and crossing-linking of these micro-organisms by hemolin. Binding of hemolin to bacteria and yeast cells M. sexta hemolin was reported to b ind to E. coli [23] and to enhance the association o f E. coli with hemocytes [ 14]. To assess the binding of hemolin to different types of 1828 X Q. Yu and M. R. Kanost (Eur. J. Biochem. 269) Ó FEBS 2002 micro-organisms, we performed a binding assay using 125 I-labeled hemolin. Radioactively labeled h emolin bound to a Gram-negative b acterium, E. coli, a Gram-positive bacterium, M. lysodeikticus,andtoayeast(S. cerevisiae) (Fig. 2 ). Specific binding (demonstrated by competition with unlabeled hemolin) accounted f or 70% of the h emolin binding to M. lysodeikticus, 46% of the binding to E. coli, and 25% of the binding to S. cerevisiae . Thus, b inding of hemolin to Gram-positive and Gram-negative bacteria as well as yeast appears to involve specifi c binding sites, with a lower degree of specific binding to yeast cells than to bacteria. Binding of hemolin to immobilized LPS Daffre & Faye [16] reported that H. cecropia hemolin interacts with bacterial LPS. We performed an enzyme- linked immunosorbent assay to measure bind ing of M. sexta hemolin to immobilized LPS. Hemolin at different concentrations was a dded to w ells of a m icrotiter plate coated with S-LP S from E. coli (strain 026:B6). After an incubation period and washing, the bound hemolin was detected using a ntiserum to hemolin. Binding of hemolin to LPS was concentration-dependent and saturable, reaching a maximum at 80 lgÆmL )1 hemolin (Fig. 3). Nonlinear regression analysis of the binding data showed that binding of hemolin to LPS fits a two-site binding model (R 2 ¼ 0.92), with a high-affinity site (K d1 ¼ 0.041 ± 0.065 lgÆmL )1 ) and a low-affinity site (K d2 ¼ 53.2 ± 20.1 lgÆmL )1 ). As these binding studies were performed under nonequilibrium conditions, the calculate d binding constants s hould be c onsidered rough estimates. Binding of hemolin to LPS in buffer containing 10 m M CaCl 2 was approximately half of that observed in the absence of calcium ( Fig. 4). When the binding assay was performed i n phosphate buffer instead of Tris buffer, we also observed an  50% decrease in binding of hemolin to LPS (Fig. 4). Hemolin binds to the O-specific antigen, outer-core, and lipid-A moieties of LPS Bacterial LPS consists of three moieties: lipid A, the core carbohydrate, and the O-specific antigen (Fig. 5) [24]. Lip- id A i s composed of a b-glucosaminyl-(1,6)-a- D -glucosamine Fig. 1. Agglutination of bacteria and yeast by hemolin. BSA ( 1 mgÆmL )1 ) or h emolin (0.5 mg ÆmL )1 ) was incubated w ith fluorescein isothiocyanate-labeled E. coli (1.0 · 10 9 cellsÆmL )1 ), S. aureus (1.0 · 10 9 cellsÆmL )1 )or S. cerevisiae (yeast) (1.0 · 10 8 cellsÆmL )1 )in Tris/NaCl. After i ncubation for 45 min at room temperature, cells we re observed by fluorescence microscopy. Fig. 2. Binding of 125 I-labeled hemolin to bacteria and y east. 125 I-hemolin (1.0 l M ) w as incubated with formalin-killed E. coli, M. lysodeikticus or S. cerevisiae (each at 3 · 10 5 cells) in T ris/NaCl in the presence or absence of 50 l M unlabeled hemolin. The c ells were washed four times and counted in a c c ounter to detec t bound hemolin. Total binding re presents the amount of hemolin bo und in the absence of unlabeled hemolin. Specific binding was calculated by subtracting the amount of h emolin bo und in t he pre sence of a 5 0-fo ld excess of unlabeled hemolin (nonspecific binding) from t otal binding. Fig. 3. Binding of h emolin to immobilized LPS. He mo lin at different concentrations prep ared in binding buffer was a dded to LPS-coated microtiter plates and incubated for 6 h at room temperature. The binding assa y was performed as describ ed in Experime ntal Proce dure s. Each point represents the mean ± SD from four i ndividual mea- surements. The solid line represents a nonlinear regression calculation of a two-site b inding curve (R 2 ¼ 0.92 ), and the dotted line represents the curve calculated for one-site binding ( R 2 ¼ 0.85). Ó FEBS 2002 M. sexta hemolin as a pattern-recognition protein (Eur. J. Biochem. 269) 1829 disaccharide backbone which carries up to seven fatty acids. The c ore carbohydrate i s further divided into an inner-core and an outer-core s ubdomain. The inner core i s composed of KDO a nd heptoses, while the outer core contains hexoses, primarily glucose, galactose, and GlcNAc. The O-specific antigen consists of a distinct repeating o ligosaccharide of up to 40 units [24]. O-specific antigen structures are highly variab le compared with other moieties of LPS. LPS from s mooth c olony forms of Gram- negative bacteria (S-LPS) c ontains all of t hese components, whereas LPS from rough m utants (R-LPS) lack the O-antigen and may a lso lack parts of the outer and inner core polysac charide. We per formed competitive binding assays to test binding of different moieties of LPS to M. sexta hemolin. Binding of hemolin to immobilized LPS from a smooth strain of E. coli (026:B6) was measured in the presence of a 20-fold excess of different f orms of LPS o r lipid A as competitors. S-LPS from E. coli st rain 0111:B4 competed more efficiently (82%) for hemolin binding than did S-LPS from strain 026:B6 (64%) (Fig. 6). Because these two types of LPS differ in O-specific antigen structure [25,26], t his result suggests t hat hemolin can bind to the O-specific antigen of 0111:B4. Ra-LPS, w hich lacks an O -specific antigen, and Rc-LPS, which also lacks part of the outer core, competed for hemolin binding (59%) about as well as did S-LPS from strain 026:B6 (Fig. 6), indicating that the O-specific antigen of 026:B6 may not contribute significantly t o hemolin binding. However, Rd 2 -LPS, which lacks the entire outer core and two heptose residues from the inner core, was significantly less efficient as a competitor (30%), suggesting that hemolin may bind to g alactose, glucose, or GlcNAc residues in the outer cor e or to heptose residues i n the inner core. The finding that glucose and galactose inhibited binding of hemolin to LPS (Fig. 7) is consistent with this idea. H owever, KDO, a component of the inner core of LPS, did not inhibit binding of hemolin to LPS (Fig. 7). Re-LPS and lipid A a lone were approximately e quivalent to Fig. 6. Binding o f hemolin t o LPS in the presence o f different forms of LPS as competitors. Hemolin (30 lgÆmL )1 ) was preincubated with S-LPS (from E. coli strains 026:B6 and 0111:B4), Ra-LPS, Rc-LPS, Rd2-LPS, Re-LPS, or diphosphoryl lipid A (each at a final concen- tration of 0.8 mgÆmL )1 )in50lL binding buffer for 3 h at room tem- perature. Th e mixture was then ad de d to wells of LPS-coated microtiter plate and incubated for 6 h at room temperatu re. The binding assay was performed as described in Experimental procedures. The bars represent the mean ± SD from four individual measurements. Fig. 7. Binding of hemolin to LPS in the presence of saccharides as competitors. Hemolin (30 lgÆmL )1 ) was preincubated with glucose, galactose, mannose, GlcNAc, GalNAc, xylose (each at final 400 m M ), KDO, or LPS (E. c oli 026:B6) (each at 800 lgÆmL )1 )in50lLbinding buffer for 3 h at room temperature. The mi xture was th en ad ded to wells of L PS-coated microtite r plate and incubated for 6 h at room temperature. The binding assay was performed as i n Fig. 6 . The bars represent the mean ± S D from four individual m easuremen ts. Fig. 5. Schematic d iagram of ba cterial LPS (mod ified from [24 ]). Fig. 4. Binding of h emolin to LPS in the presenc e o f calcium or phos- phate. Hemolin at differen t c on centrations was prepared in binding buffer without c alcium (solid line) o r with 10 m M calcium (dotted line), or in phosphate buffer (dashed line). The binding assay was performed the same as i n Fig. 3 . Each point represents th e mean ± SD from fo ur individual measurements. 1830 X Q. Yu and M. R. Kanost (Eur. J. Biochem. 269) Ó FEBS 2002 Rd 2 -LPS as competitors for hemolin binding (Fig. 6). These results are consistent with a hypothesis t hat the binding of hemolin to Rd 2 -LPS and Re-LPS is a result of interaction with their lipid A moiety. T o further i nvestigate the interaction of M. sexta hemolin with lipid A, we assayed direct binding of hemolin to immobilized lipid A (Fig. 8). Hemolin binding to lipid A in Tris buffer was concentra- tion-dependent, but was not satu rated at 80 lgÆmL )1 hemolin. When t he assay w as carried out in phosphate buffer, binding of hemolin to lipid A was nearly eliminated (Fig. 8). Hemolin binds to LTA on Gram-positive bacteria To investigate to w hich components o n the sur face of Gram-positive bacteria and yeast h emolin binds, w e performed a competitive binding assay, using microbial components a s c ompetitors for binding of hemolin to S-LPS (Fig. 9 ). LTA and peptidoglycan, both cell-wall compo- nents of Gram-positive bacteria, decreased binding of hemolin to LPS by 86% and 26%, r espectively, suggesting that they bind to hemolin at the same site as LPS. The observation that LTA was a more efficient competitor than was LPS itself suggests that hemolin has a higher affinity for LTA than for LPS. This i s consistent with the finding that more hemolin bound to Gram-positive bacteria than to Gram-negative bacteria (Fig. 2). H emolin bound directly to immobilized LTA (Fig. 10). The binding was concentra- tion-dependent and was not saturated at 50 lgÆmL )1 hemolin. Nonlinear regression analysis of the binding data showed that bin ding o f h emolin to LTA also fits a two-site binding model ( R 2 ¼ 0.89), with a K d1 ¼ 0.12 ± 0.11 lgÆmL )1 and K d2 ¼ 110.1 ± 125.3 lgÆmL )1 . Yeast cell walls are composed primarily of b-1,3-glucans and mannans [27]. Zymosan, a y east cell-wall preparation that contains glucan, mannan and chitin, decreased binding of hemolin to LPS by 61% (Fig. 9 ). But laminarin, a soluble form of b-1,3-glucan, did not inhibit hemolin binding to LPS (Fig. 9), and hemolin did not bind to curdlan, an insoluble f orm o f b-1,3-glucan ( data not shown), indicating that hemolin does not bind to b-1,3-glucans. Mannan a nd chitosan (deacetylated chitin) also did not inhibit hemolin binding to LPS (data not shown). However, mannose inhibited binding of hemolin to LPS by 28% (Fig. 7), which suggests that h emolin may b ind to the mannan on the surface of yeast. DISCUSSION Hemolin synthesis is i nduced by Gram-negative a nd Gram- positive bacteria, and it is the major protein produced in response to m icrobial infection i n l epidopteran insects such as H. cecropia and M. sexta [7,8,28], s uggesting that i t Fig. 9. Binding o f hemolin to LPS in the presence of microbial c ompo- nents as competitors. Hemolin (30 lgÆmL )1 ) was preincubated with LPS (E. coli 026:B6), LTA, peptidoglycan, zymosan, or lamin arin (each at 800 lgÆmL )1 )in50lL binding buffer for 3 h at room temperature. The mixture was then added to we lls of a LPS -co ated microtiter plate and in cubated for 6 h at ro om temperature. T he binding assay was performed as in Fig. 6. The bars r epresent the mean ± SD f orm four individual measurements. Fig. 8. Binding of hemolin to lipid A. Hemolin at different concentra- tions prepared in binding buffer or phosphate buffer w as added to diphosphoryl lipid A-coated microtiter plates and incubated for 6 h at room te mperature. The binding was performed as described in Experimental procedures. E ach point represents t he mean ± SD from four individual measurements. Fig. 10. Binding of hemol in to immobilized LTA. Hemolin at different concentrations prepared in binding buffer was added t o L TA-coat ed microtiter plates and incubated for 6 h at room temperature. The binding a ssay was p erformed as described in Experimental procedures. Each point represents the mean ± SD from four i ndividual mea- surements. The solid line represents a nonlinear regression calculation of a two-site b inding curve (R 2 ¼ 0.89 ), and the dotted line represents the curve calculated for one-site binding ( R 2 ¼ 0.78). Ó FEBS 2002 M. sexta hemolin as a pattern-recognition protein (Eur. J. Biochem. 269) 1831 functions in the i mmune response o f these insects. However, hemolin does not display direct antibacterial activity. Instead, its role may be related to its ability t o bind to the surface of hemocytes [5,13–15] a nd to bacteria. Hemolin has beenshowntobindtoE. coli [4,23] and t o increase t he association of E. coli with hemocytes [ 14]. W e h ave found that hemolin als o binds to Gram-positive bacteria and to a lesser degree to yea st. In these studies we ha ve invest igated the binding of hemolin to LPS and LTA, molecules that are present on the surface of Gram-negative a nd Gram-positive bacteria, respectively. LPS on the surface of Gram-negative bacteria is a potential target for binding of pattern-recognition recep- tors. The availability of E. coli mutants expressing differ- ently t runcated forms of LPS makes it possible to identify the part of the LPS molecule to which a protein binds. H. cecropia hemolin binds to wild-type E. coli and also to mutants lacking the c ore carbohydrate [29]. We f ound that M. sexta he molin bound to immobilized LPS and to its isolated lipid A component in a concentration-dependent, saturable manner. Competitive b inding experiments indi- cated that hemolin binds smooth forms of LPS most efficiently, but rough forms of LPS, lacking the O-antigen and p arts of the i nner-core and outer-core p olysaccharide, and lipid A alone could also p artially compete f or hemolin binding to smooth LPS. Rough mutants Rd and Re, containing only the lipid A moiety and part of the inner core, competed no better than lipid A alone, suggesting that hemolin does not bin d to KDO in the inner core. This is consistent with the observation that KDO did not compete for hemolin binding to LPS and with the results of Daffre & Faye [16], who showed by photoaffinity labeling that hemolin from H. cecropia bound to S-LPS and that the binding could be competed f or by lipid A but not KDO. Approximately 30 n g hemolin specifically bound to the surface of 3 · 10 5 E. coli cells (Fig. 2 ), indicating that  10 6 molecules of hemolin bound to each E. coli cell. Because Gram-negative b acteria c ontain  10 6 molecules of LPS per cell [30], this result suggests that, on average, each LPS mole cule w as occupied by one molecule of hemolin. Results of binding curves and competition experiments suggest that hemolin contains two binding sites for LPS. One site a ppears to bind to the carbohydrate components in the O-antigen and outer core, a nd the other site binds to lipid A. Even though isolated lipid A binds to hemolin, it could only partially compete for LPS binding to S-LPS. Similarly, Daffre & F aye [16] f ound that a large excess of lipid A decreased hemolin binding to S-LPS b y only 42%. The b inding of hemolin to lipid A may involve an interaction w ith the phosphate groups on lipid A. Free phosphate decreased hemolin binding to S-LPS by approximately half and nearly eliminated hemolin binding to lipid A. An interpretation of these results is that phosphate disrupts binding of lipid A by competing for a site that interacts with phosphate groups, and that a separate binding site that interacts with carbohydrate components of LPS is not affected by phosphate. In the crystal structure of H. cecropia hemolin, a phosphate ion was f ound in the interface of Ig d omains 2 and 3 [6]. Perhaps this r egion o f t he molecule is part of a binding site for lipid A. Because the homophilic binding of hemolin to o ther hemolin molecules was shown to require Ca 2+ [17], we tested the effect of Ca 2+ on LPS binding. Rather than enhancing h emolin binding, 10 m M Ca 2+ decreased hemolin binding to LPS by a bout half, very similar to the effect of phosphate. Electrostatic interactions of Ca 2+ with phosphate groups of lipid A may mask these groups and interfere with the lipid A-binding site but not the carbohy- drate-binding site. The opposing effects o f C a 2+ on hemolin binding to LPS and other hemolin molecules suggest that homophilic binding occurs at a site distinct f rom LPS binding. More hemolin bound to the Gram-positive bacterium M. lysodeikticus , which does not contain LPS, than to E. coli (Fig. 2). When we tested whether cell surface components of Gram-positive bacteria can compete with LPS for hemolin binding, we found that peptidoglycan, the major cell-wall component of Gram-positive bacteria, inhibited b inding of hemolin t o L PS by 26%, w hereas LTA, another surface component of Gram-positive bacteria, inhibited hemolin binding to LPS by 86%. LTA was more effective than LPS itself as an inhibitor of hemolin binding to LPS, suggesting that LPS and LTAbindtothesamesitesonhemolinandthathemolin may have a higher affinity for LTA. Hemolin was also observed to bind d irectly to i mmobilized LTA (Fig. 10). LPS and LTA are s imilar in containing both polysac- charide components a nd lipid components associated with phosphate groups [31], and these may occupy the same binding sites in hemolin. Another insect plasma protein that has been shown to interact with LTA is apolipophorin-III of Galleria mellonella, which presum- ably binds to the hydrophobic components of LTA [32]. To function as a pattern-recognition receptor, a protein must bind to the s urface of i nvading micro-organis ms. We showed that hemolin binds to the surface of Gram- negative and Gram-positive bacteria and yeast, and caused aggregation of these micro-organisms. Binding of hemolin to the surface of b acteria appears t o be due to specific interactions with LPS on Gram-negative bacteria and to LTA and perhaps also peptidoglycan from Gram-positive bacteria. Binding of hemolin to yeast was less efficient, and it is not clear from our experiments w hat part o f the yeast cell wall is t he hemolin-binding site. Aggregation of m icro-organisms by hemolin and the ability of hemolin to bind to hemocytes may promote phagocytosis and the formation of hemo- cyte nodules to clear micro-organisms from the insect hemolymph. Recognition of micro-organisms by pattern-recognition receptors is a crucial function of the innate immune system of vertebrates a nd invertebrates [19,20]. Pattern- recognition receptors identified in M. sexta and other insect species include C-type lectins [9,21,22,33–35], b-1,3- glucan-binding proteins [36,37], and peptidoglycan-binding proteins [38–41]. 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It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin