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

Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-c

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 is

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 lipopolysaccharide (LPS) from

negative bacteria and to lipoteichoic acid from

Gram-positive bacteria Binding of hemolin to smooth-type forms

of LPS was competed for efficiently by lipoteichoic acid and

by rough mutant (Ra and Rc) forms of LPS, which differ in

polysaccharide length Binding of hemolin to LPS was

partially inhibited by calcium and phosphate Hemolin

bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the 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: hemolin; insect immunity; lipopolysaccharide; lipoteichoic acid; pattern recognition receptor

Upon microbial infection, insects synthesize defensive

plasma proteins, which include antimicrobial peptides and

proteins, lectins, and cell adhesion molecules [1–3] One such

protein is hemolin, a member of the immunoglobulin (Ig)

superfamily Hemolin contains four Ig domains of the I-set

type which are most similar to those in neural cell adhesion

molecules [4–6] Hemolin has been isolated from

hemo-lymph of two immune-challenged lepidopteran insects,

Hyalophora cecropiaand Manduca sexta [7,8] A

hemolin-like cDNA was also cloned from the fall webworm,

Hyphantria cunea[9] Hemolin is synthesized mainly in fat

body in response to microbial challenge [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 at different developmental stages of

M sextadiffers 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 hemolin functions in immune

responses by interacting with insect hemocytes and with

bacteria It binds to hemocytes and bacteria, and 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 has 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 the Ig domains in the structure of H cecropia hemolin suggested a mechanism for homophilic binding of hemolin to molecules

on the surface of hemocytes or micro-organisms [6] However, the biological functions of hemolin in 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 set of proteins known as pathogen-recognition receptors These proteins bind to conserved features of microbial surfaces such as LPS, lipoteichoic acid (LTA) and peptidoglycan from bacterial cell walls, and b-1,3-glucan from fungal cell walls [19,20] Such recognition may initiate a variety of immune responses in insects, including prophenoloxidase activation, phagocytosis, nodule formation, and encapsu-lation

In this paper, we focus on the biological functions of hemolin in defense against microbial infection We investi-gated its binding to Gram-negative and Gram-positive bacteria, yeast, and bacterial LPS and LTA Our results indicate that hemolin functions as a pattern-recognition receptor with a broad specificity for diverse pathogens in the defense against micro-organisms

Correspondence to M R Kanost, Department of Biochemistry,

Kansas State University, Manhattan, KS 66506, USA.

Fax: + 785 532 7278, Tel.: + 785 532 6964,

E-mail: kanost@ksu.edu

Abbreviations: KDO, 2-oxo-3-deoxyoctanoate; LPS,

lipopolysac-charide; LTA, lipoteichoic acid.

(Received 22 October 2001, revised 6 February 2002, accepted 8

February 2002)

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 rough

mutants 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 mutant 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 mM Tris/

HCl, 137 mM NaCl and 3 mM KCl, pH 7.0) and used

for the agglutination assay Hemolin at 0.5 mgÆmL)1 or

BSA at 1.0 mgÆmL)1 (as a control) was used in

agglutination of micro-organisms as described previously

[21]

Binding of125I-labeled hemolin to bacteria and yeast

Hemolin was labeled with125I using Iodobead (Piece) as

an iodination reagent One Iodobead was washed with

500 lL iodination buffer (100 mM sodium phosphate

buffer, pH 7.0) The washed bead was added to 1 mCi

Na125I (Dupont NEN) in 434 lL iodination buffer, and

incubated for 5 min at room temperature Then 250 lg

purified hemolin in 66 lL deionized water was mixed

with the solution containing the Iodobead, followed by

incubation for 3 min at room temperature The

Iodo-bead was removed, and 125I-labeled hemolin was

sepa-rated from free 125I by applying the solution to an

equilibrated Sephadex G-25 desalting column (PD10,

Pharmacia) The column was eluted with NaCl/Pi

(25 mM sodium phosphate buffer, 137 mM NaCl and

3 mMKCl, pH 7.0), and 0.6 mL fractions were collected

Samples of 5 lL were removed from each fraction

and counted in a c counter Fractions containing

125I-labeled hemolin were pooled and stored at )20 °C

The specific activity of the labeled hemolin was

3.9· 105c.p.m.Ælg)1

125I-Labeled hemolin at a concentration of 1.0 lM was

incubated with formalin-killed E coli strain XL1-blue,

Micrococcus lysodeikticus, or S cerevisiae (yeast) (each at

3· 105 cells) in 50 lL Tris/NaCl containing 1 mgÆmL)1

BSA, in the absence or presence of 50 lM unlabeled

hemolin The mixture was incubated for 30 min at room

temperature, then centrifuged for 5 min at 10 000 g The

supernatant was removed by aspiration, and the cells were

washed four times with Tris/NaCl, then counted in a

c counter for bound hemolin

diphosphoryl lipid A as described previously [22] Hemolin

at different concentrations prepared in binding buffer (50 mM Tris/HCl, 50 mM NaCl, pH 8.0, 0.1 mgÆmL)1 BSA) containing 0 or 10 mMCaCl2, or in phosphate buffer (50 mM sodium phosphate, 50 mM NaCl, pH 8.0, 0.1 mgÆmL)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] Bound hemolin was measured by first 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 3000-fold with binding buffer), and bound alkaline phosphatase activity was determined by hydrolysis of p-nitrophenyl phosphate, all as described previously [22] The A405value of 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 wells of a 96-well plate were coated with LPS from

E coli026:B6 (2 lg per well) Hemolin at a concentration of

30 lgÆmL)1 was preincubated with 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 mgÆmL)1), glucose, galactose, mannose, GlcNAc, GalNAc, or xylose (each at 0.4 mM) in 50 lL binding buffer for 3 h at room temper-ature The mixture was then added to S-LPS (E coli 026:B6)-coated wells, and the binding was allowed to occur

at room temperature for 6 h before washing and detection

of bound hemolin as described above

R E S U L T S

Agglutination of bacteria and yeast by hemolin

To investigate whether hemolin can bind to bacteria or yeast and cause aggregation of these micro-organisms, we performed an agglutination assay When E coli, S aureus,

or S cerevisiae cells were incubated with hemolin at a concentration of 0.5 mgÆmL)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, BSA, no obvious aggregates were observed (Fig 1) Agglutination of bacteria by hemolin was not affected by addition of EDTA (data not shown), indicating that hemolin does not require bivalent cations for its agglutination 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 sextahemolin was reported to bind to E coli [23] and to enhance the association of E coli with hemocytes [14]

To assess the binding of hemolin to different types of

micro-organisms, we performed a binding assay using

125I-labeled hemolin Radioactively labeled hemolin bound

to a Gram-negative bacterium, E coli, a Gram-positive

bacterium, M lysodeikticus, and to a yeast (S cerevisiae)

(Fig 2) Specific binding (demonstrated by competition

with unlabeled hemolin) accounted for 70% of the hemolin

binding to M lysodeikticus, 46% of the binding to E coli,

and 25% of the binding to S cerevisiae Thus, binding of

hemolin to Gram-positive and Gram-negative bacteria as

well as yeast appears to involve specific 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 binding of

M sextahemolin to immobilized LPS Hemolin at different

concentrations was added to wells of a microtiter plate

coated with S-LPS from E coli (strain 026:B6) After an

incubation period and washing, the bound hemolin was

detected using antiserum 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 (R2 ¼ 0.92), with a high-affinity site (Kd1 ¼ 0.041 ± 0.065 lgÆmL)1) and a low-affinity site (Kd2 ¼ 53.2 ± 20.1 lgÆmL)1) As these binding studies were performed under nonequilibrium conditions, the calculated binding constants should be considered rough estimates Binding of hemolin to LPS in buffer containing 10 mM CaCl2 was approximately half of that observed in the absence of calcium (Fig 4) When the binding assay was performed in 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 is composed of a b-glucosaminyl-(1,6)-a-D-glucosamine

Fig 1 Agglutination of bacteria and yeast by

hemolin BSA (1 mgÆmL)1) or hemolin

(0.5 mgÆmL)1) was incubated with 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 incubation for 45 min at

room temperature, cells were observed by

fluorescence microscopy.

Fig 2 Binding of 125I-labeled hemolin to bacteria and yeast.

125 I-hemolin (1.0 l M ) was incubated with formalin-killed E coli,

M lysodeikticus or S cerevisiae (each at 3 · 10 5 cells) in Tris/NaCl in

the presence or absence of 50 l M unlabeled hemolin The cells were

washed four times and counted in a c counter to detect bound hemolin.

Total binding represents the amount of hemolin bound in the absence

of unlabeled hemolin Specific binding was calculated by subtracting

the amount of hemolin bound in the presence of a 50-fold excess of

unlabeled hemolin (nonspecific binding) from total binding.

Fig 3 Binding of hemolin to immobilized LPS Hemolin at different concentrations prepared in binding buffer was added to LPS-coated microtiter plates and incubated for 6 h at room temperature The binding assay was performed as described in Experimental Procedures Each point represents the mean ± SD from four individual mea-surements The solid line represents a nonlinear regression calculation

of a two-site binding curve (R 2

¼ 0.92), and the dotted line represents the curve calculated for one-site binding (R 2

¼ 0.85).

disaccharide backbone which carries up to seven fatty

acids The core carbohydrate is further divided into an

inner-core and an outer-core subdomain The inner core is

composed of KDO and heptoses, while the outer core

contains hexoses, primarily glucose, galactose, and

GlcNAc The O-specific antigen consists of a distinct

repeating oligosaccharide of up to 40 units [24] O-specific

antigen structures are highly variable compared with other

moieties of LPS LPS from smooth colony forms of

Gram-negative bacteria (S-LPS) contains all of these components,

whereas LPS from rough mutants (R-LPS) lack the

O-antigen and may also lack parts of the outer and inner

core polysaccharide

We performed 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 forms of LPS or lipid A as competitors S-LPS

from E coli strain 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], this result suggests that

hemolin can bind to the O-specific antigen of 0111:B4

Ra-LPS, which 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 to hemolin binding However, Rd2-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 galactose, glucose, or GlcNAc residues in the outer core or to heptose residues in the inner core The finding that glucose and galactose inhibited binding of hemolin to LPS (Fig 7) is consistent with this idea However, KDO, a component of the inner core of LPS, did not inhibit binding of hemolin to LPS (Fig 7) Re-LPS and lipid A alone were approximately equivalent to

Fig 6 Binding of hemolin to LPS in the presence of 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) in 50 lL binding buffer for 3 h at room tem-perature The mixture was then added to wells of LPS-coated microtiter plate and incubated for 6 h at room temperature 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 coli 026:B6) (each at 800 lgÆmL)1) in 50 lL binding buffer for 3 h at room temperature The mixture was then added to wells of LPS-coated microtiter plate and incubated for 6 h at room temperature The binding assay was performed as in Fig 6 The bars represent the mean ± SD from four individual measurements Fig 5 Schematic diagram of bacterial LPS (modified from [24]).

Fig 4 Binding of hemolin to LPS in the presence of calcium or

phos-phate Hemolin at different concentrations was prepared in binding

buffer without calcium (solid line) or with 10 m M calcium (dotted line),

or in phosphate buffer (dashed line) The binding assay was performed

the same as in Fig 3 Each point represents the mean ± SD from four

individual measurements.

Rd2-LPS as competitors for hemolin binding (Fig 6) These

results are consistent with a hypothesis that the binding of

hemolin to Rd2-LPS and Re-LPS is a result of interaction

with their lipid A moiety To further investigate 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 saturated at 80 lgÆmL)1

hemolin When the assay was 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 which components on the surface of

Gram-positive bacteria and yeast hemolin binds, we

performed a competitive binding assay, using microbial

components as competitors 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%, respectively, 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 is consistent with the finding that

more hemolin bound to Gram-positive bacteria than to

Gram-negative bacteria (Fig 2) Hemolin 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 binding of hemolin to LTA also fits a two-site

binding model (R2 ¼ 0.89), with a Kd1 ¼ 0.12 ±

0.11 lgÆmL)1and Kd2 ¼ 110.1 ± 125.3 lgÆmL)1

Yeast cell walls are composed primarily of b-1,3-glucans

and mannans [27] Zymosan, a yeast 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 form of b-1,3-glucan (data not shown), indicating

that hemolin does not bind to b-1,3-glucans Mannan and 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 hemolin may bind to the mannan on the surface of yeast

D I S C U S S I O N

Hemolin synthesis is induced by negative and Gram-positive bacteria, and it is the major protein produced in response to microbial infection in lepidopteran insects such

as H cecropia and M sexta [7,8,28], suggesting that it

Fig 9 Binding of hemolin to LPS in the presence of microbial compo-nents as competitors Hemolin (30 lgÆmL)1) was preincubated with LPS (E coli 026:B6), LTA, peptidoglycan, zymosan, or laminarin (each at 800 lgÆmL)1) in 50 lL binding buffer for 3 h at room temperature The mixture was then added to wells of a LPS-coated microtiter plate and incubated for 6 h at room temperature The binding assay was performed as in Fig 6 The bars represent the mean ± SD form four individual measurements.

Fig 8 Binding of hemolin to lipid A Hemolin at different

concentra-tions prepared in binding buffer or phosphate buffer was added to

diphosphoryl lipid A-coated microtiter plates and incubated for 6 h at

room temperature The binding was performed as described in

Experimental procedures Each point represents the mean ± SD from

four individual measurements.

Fig 10 Binding of hemolin to immobilized LTA Hemolin at different concentrations prepared in binding buffer was added to LTA-coated microtiter plates and incubated for 6 h at room temperature The binding assay was performed as described in Experimental procedures Each point represents the mean ± SD from four individual mea-surements The solid line represents a nonlinear regression calculation

of a two-site binding curve (R2 ¼ 0.89), and the dotted line represents the curve calculated for one-site binding (R 2

¼ 0.78).

been shown to bind to E coli [4,23] and to increase the

association of E coli with hemocytes [14] We have found

that hemolin also binds to Gram-positive bacteria and to a

lesser degree to yeast In these studies we have investigated

the binding of hemolin to LPS and LTA, molecules that are

present on the surface of Gram-negative and 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 truncated forms of LPS makes it possible to identify

the part of the LPS molecule to which a protein binds

H cecropiahemolin binds to wild-type E coli and also to

mutants lacking the core carbohydrate [29] We found that

M sexta hemolin bound to immobilized LPS and to its

isolated lipid A component in a concentration-dependent,

saturable manner Competitive binding experiments

indi-cated that hemolin binds smooth forms of LPS most

efficiently, but rough forms of LPS, lacking the O-antigen

and parts of the inner-core and outer-core polysaccharide,

and lipid A alone could also partially compete for 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 bind 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 for by lipid A but

not KDO Approximately 30 ng hemolin specifically

bound to the surface of 3· 105 E coli cells (Fig 2),

indicating that 106molecules of hemolin bound to each

E colicell Because Gram-negative bacteria contain 106

molecules of LPS per cell [30], this result suggests that, on

average, each LPS molecule was occupied by one molecule

of hemolin

Results of binding curves and competition experiments

suggest that hemolin contains two binding sites for LPS

One site appears to bind to the carbohydrate components

in the O-antigen and outer core, and 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 & Faye [16] found that a large excess of

lipid A decreased hemolin binding to S-LPS by only 42%

The binding of hemolin to lipid A may involve an

interaction with 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 found in the interface of Ig domains 2 and 3 [6]

Perhaps this region of the molecule is part of a binding site

for lipid A

hemolin binding to LPS by about half, very similar to the effect of phosphate Electrostatic interactions of Ca2+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 of Ca2+on hemolin binding to LPS and other hemolin molecules suggest that homophilic binding occurs at a site distinct from 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 binding of hemolin to LPS by 26%, whereas 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 LTA bind to the same sites on hemolin and that hemolin may have a higher affinity for LTA Hemolin was also observed to bind directly to immobilized LTA (Fig 10) LPS and LTA are similar in containing both polysac-charide components and 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 surface of invading micro-organisms

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 bacteria appears to 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 what part of the yeast cell wall is the hemolin-binding site Aggregation of micro-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 and 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] The rapid induction of hemolin to high concentration in hemolymph (1.5 mgÆmL)1 in M sexta larvae) [5,8] and its broad specificity for binding to different types of micro-organisms suggests that it func-tions as a pattern-recognition receptor that participates in detection and elimination of a variety of pathogens in lepidopteran insects

A C K N O W L E D G E M E N T S

We thank Maureen Gorman and Neal Dittmer for helpful comments

on the manuscript This work was supported by National Institutes of

Health Grants AI31084 and GM41247 This is contribution 00-320-J

from the Kansas Agricultural Experiment Station.

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