Activation of [alpha] M [beta] 2-mediated phagocytosis by HF3, a P-III class metalloproteinase isolated from the venom of Bothrops jararaca

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Biochemical and Biophysical Research Communications 322 (2004) 950–956

BBRC

Activation of aMb2-mediated phagocytosis by HF3, a P-III classmetalloproteinase isolated from the venom of Bothrops jararaca

Carlos A. Silvaa,1, Juliana P. Zulianib,1, Marina T. Assakuraa, Reinhard Mentelec,Antonio C.M. Camargoa, Catarina F.P. Teixeirab, Solange M.T. Serranoa,*

a Laboratorio Especial de Toxinologia Aplicada-CAT/CEPID, Instituto Butantan, CEP 05503-900, Sao Paulo, Brazilb Laboratorio de Farmacologia, Instituto Butantan, CEP 05503-900, Sao Paulo, Brazil

c Abteilung fuer Klinische Chemie und Klinische Biochemie, Klinikum Innenstadt, Universitaet Muenchen, D-80336 Munich, Germany

Received 16 July 2004

Abstract

The integrin aMb2 regulates important cell functions in inflammation being the primary phagocytic receptor on macrophages.

HF3, a metalloproteinase isolated from Bothrops jararaca venom, is a potent hemorrhagic toxin. A cDNA encoding HF3 indicated

that it is a multidomain molecule composed of a pro-domain, a catalytic domain with a zinc binding sequence, followed by disin-

tegrin-like and cysteine-rich domains. It is known that metalloproteinases play a relevant role in the pathogenesis of venom-induced

local tissue damage including inflammation. In this study we evaluated the effects of native HF3 and its recombinant disintegrin-like/

cysteine-rich domains (DC-HF3) on aMb2-mediated phagocytosis of opsonized-zymosan particles by macrophages. HF3 and DC-

HF3 significantly increased phagocytosis and this activity was inhibited by anti-aM and anti-b2 antibodies. The data show the ability

of P-III metalloproteinases to activate macrophages for phagocytosis through integrin aMb2 and suggest that the disintegrin-like/

cysteine-rich domains are important for this effect. This is the first report on the activation of phagocytosis via aMb2 integrin by

a metalloproteinase containing disintegrin-like/cysteine-rich domains.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Snake venom metalloproteinase; Disintegrin-like/cysteine-rich domains; Platelet aggregation; Integrin aMb2; Macrophage; Phagocytosis;

Inflammation

Integrins are heterodimeric cell surface receptors

involved in diverse biological processes including adhe-sion of cells to extracellular matrices as well as intercel-

lular interactions that are central to inflammation,

immunity, hemostasis, wound healing, and tumor

metastasis [1]. aMb2 (Mac-1, Mo-1, CR3, CD11b/

CD18) is a member of the b2 integrin subfamily [1,2].

This and other leukocyte integrins are critical for the

adhesion and transmigration of leukocytes through the

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.08.012

* Corresponding author. Fax: +55 11 37216605.

E-mail address: [email protected] (S.M.T.

Serrano).1 These authors contributed equally to this work.

endothelium, and the phagocytosis of foreign materials

[1]. aM b2 recognizes various protein and non-protein li-gands that can be soluble, matrix-bound, or cell-bound.

These ligands include C3bi, a degradation product de-

rived from activation of the complement system [3],

fibrinogen [4], and intercellular cell-adhesion molecule

1 [5]. Phagocytosis, an essential component of innate-

immune response, is performed by macrophages and

polymorphonuclear leukocytes. It consists in the uptake

and destruction of invading microorganisms, insolubleparticles, damaged or dead host cells, cell debris, and

activated clotting factors [6]. The uptake process is

initiated by the interaction of molecules on the surface

of the particles with phagocytic receptors present on

C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956 951

phagocytes. aMb2-mediated phagocytosis results in a

tight-fitting, zippered phagosome, and requires the acti-

vation of PKC and Rho-GTPases, but not of Cdc42 or

Rac, for cytoskeleton reorganization and gene expres-

sion [6]. aMb2 can be activated by inside-out or out-

side-in signaling mechanisms to generate high-affinitybinding sites on the integrin [7,8].

Snake venom metalloproteinases (SVMPs) are found

mainly in viper venoms, and are primarily responsible

for the observed hemorrhage due to the disruption of

blood vessels as well as for inhibition of platelet aggre-

gation [9]. They are all zinc metalloproteinases with a

Zn2+ binding motif of HEXXHXXGXXH and chela-

tion of the Zn2+ ion with EDTA or orthophenanthrolinedeprives them from their proteolytic and hemorrhagic

activities. These proteins are members of the Reprolysin

subfamily of zinc metalloproteinases, which also in-

cludes another group of mammalian homologous pro-

teins, a disintegrin and metalloproteinase (ADAM)

[10]. SVMPs can be divided into four classes depending

on their domain organization. The P-III class of SVMPs

and the ADAMs share homologous metalloproteinase,disintegrin-like and cysteine-rich domains [10]. The dis-

integrin-like/cysteine-rich domains of SVMPs have been

shown to play a role in the inhibition of collagen-stimu-

lated platelet-aggregation promoted by these enzymes

[11,12]. Members of the ADAMs family have been

implicated in the control of membrane fusion, cytokine

and growth factor shedding, and cell migration, as well

as processes such as muscle development, fertilization,and cell fate determination [13].

Local tissue destruction due to snake envenomation

involves activation of the cellular immune response in

which the cascade of events is usually initiated by tissue

macrophages and blood monocytes. Previous studies

have shown that TNF-a antibodies reduced the size of

venom-induced necrotic lesions, suggesting that endoge-

nous mechanisms of the inflammatory response can beactivated by viper venom metalloproteinases [14]. More-

over, a study on the inflammatory action of jararhagin,

a hemorrhagic metalloproteinase from the venom of

Bothrops jararaca, showed that it is a potent pro-inflam-

matory agent dependent on in situ activation of macro-

phages [15].

HF3, a high molecular mass metalloproteinase, is the

most potent hemorrhagic toxin isolated from B. jararaca

venom [16]. It is a single chain protein of 62 kDa that

causes hemorrhage on rabbit skin with a minimun hem-

orrhagic dose (MHD) of 15 ng. In this work, we deter-

mined the complete amino acid sequence of HF3 by

molecular cloning and showed that both native HF3

and a recombinant protein composed of its disintegrin-

like/cysteine-rich domains (DC-HF3) inhibit collagen-

induced platelet-aggregation. In order to investigatethe effect of HF3 and of its disintegrin-like/cysteine-rich

domains on macrophage functions and the possible

interaction of SVMPs with integrins, we analyzed the

phagocytosis of complement-opsonized zymosan by

elicited macrophages through integrin aMb2. Here we

show that HF3 can significantly upregulate the phagocy-

tic activity of peritoneal macrophages and this effect is

inhibited by anti-aMb2 monoclonal antibodies. Takentogether, these data suggest that HF3 activates the

phagocytosis of complement-opsonized particles via

integrin receptor aMb2. Moreover, we demonstrate for

the first time the increase of aMb2-mediated phagocyto-

sis by a metalloproteinase containing disintegrin-like/

cysteine-rich domains.

Materials and methods

Animals. Male Swiss mice (18–20 g) were housed in temperature-

controlled rooms and received water and food ad libitum until used.

The studies described here were approved by the Experimental Ani-

mals Committee of Butantan Institute in accordance with the proce-

dure laid down by the Universities Federation for Animal Welfare.

Molecular cloning of HF3. A B. jararaca cDNA expression library

was constructed in kZAPII from the venom gland poly(A)+ RNA as

described elsewhere [17]. Anti-HF3 antibodies were prepared as de-

scribed by Mandelbaum and Assakura [18]. Absorption of anti-Esch-

erichia coli and anti-phage antibodies was performed as described in

Sambrook et al. [19]. The library was plated on E. coli XL1-Blue

MRF 0 and grown at 37 �C for 8 h. Recombinant protein expression

was induced with 10 mM isopropyl-1-thio-b-DD-galactopyranoside sat-

urated nitrocellulose (Schleicher & Schuell) and the filters were pro-

cessed for immunostaining with anti-HF3 antibodies, as described in

Sambrook et al. [19]. Plasmids were excised from kZAPII vector and

recircularized in the presence of ExAssist (Stratagene) helper phage to

form phagemid pBluescript. Plasmids containing longest inserts were

selected for sequencing of both strands in an ABI Prism 310 Genetic

Analyzer (Applied Biosystems) using an ABI PRISM Dye Terminator

Cycle Sequencing kit (Perkin–Elmer) and oligonucleotide primers to

internal sequence to obtain overlapping sequence information. An

incomplete clone of 1.6 kb contained a cDNA encoding HF3. To ob-

tain the 5 0end of HF3 5 0RACE was performed using a �Marathon

cDNA library� constructed as described by the manufacturer (BD

Bioscience Clontech). An oligonucleotide antisense primer corre-

sponding to the amino acid sequence I-V-N-M-A-S-M-W-I-Q present

in the partial clone (5 0-CGG GAT CCG CGG CCG CCT AGT AGG

CTG TAG C-3 0) was used: PCR was carried out using the following

parameters: 94 �C for 30 s, followed by 5 cycles of 94 �C for 5 s, 72 �Cfor 4 min; 5 cycles of 94 �C for 5 s, 70 �C for 4 min; and 25 cycles of

94 �C for 5 s, 68 �C for 4 min. The PCR product obtained (�1 kb) was

cloned into pGEM-T vector (Promega) and sequenced. The complete

sequence encoding HF3 was deposited to the GenBank database

(Accession No.AF149788)

Expression of the disintegrin-like/cysteine-rich domains of HF3 in E.

coli. The expression plasmid pGEX-4T1-DC, which contains cDNA

encoding amino acid residues 395–606 and includes disintegrin-like

and cysteine-rich domains of HF3, was constructed by amplification of

the cDNA fragment by PCR and subcloning into pGEM-11Zf vector

(Promega). The plasmid was sequenced on both strands to ensure that

the coding sequence was correct. To generate a fusion protein with

glutathione S-transferase (GST), the insert from the pGEM-11Zf

vector was isolated as an EcoRI–NotI fragment and cloned into

pGEX-4T1 (Amersham). The constructed plasmid was transformed

into E. coli strain DH5a and grown in 50 ml Luria–Bertani medium

containing 100 lg/ml ampicillin at 37 �C to a cell density of A600 = 0.6–

0.8. The expression of recombinant protein was induced by adding

952 C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956

0.5 mM isopropyl thio-b-DD-galactopyranoside (IPTG) and incubation

was continued for 3 h at 30 �C. Cells were collected by centrifugation

at 4000g for 5 min and suspended in 1 ml lysis buffer (50 mM Tris–

HCl, pH 7.5, 150 mM NaCl, and 1% Triton X-100). After lysis by

sonication cell lysate was centrifuged and 80 ll of glutathione–

Sepharose suspension was added to supernatant and incubated 16 h at

4 �C. The resin was washed successively with lysis buffer to eliminate

unbound proteins and incubated with thrombin cleavage buffer

(50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) and

4 U thrombin (Sigma) for 2 h at 37 �C. The recombinant protein ob-

tained in the supernatant was analyzed by SDS–PAGE, Western blot

analysis, and N-terminal amino acid sequencing.

Phagocytosis by macrophages. Peritoneal exudate cells were ob-

tained from male Swiss mice, which were killed under halothane

atmosphere four days after intraperitoneal injection of 1 ml thiogly-

collate, by washing with 3 ml phosphate-buffered saline (PBS), pH 7.2.

Cells were plated on 13 mm diameter glass coverslips (Glass Tecnica)

in 24-well plates at a density of 2 · 105 cells per coverslip and allowed

to attach for 30 min at 37 �C under a 5% CO2 atmosphere. Non-ad-

herent cells were removed by washing with PBS. Cell monolayers

(>95% macrophages) were cultured for 1 h with RPMI 1640 supple-

mented with 1% LL-glutamine, 100 U/ml penicillin G, and 100 lg/ml

streptomycin (all from Sigma) at 37 �C and 5% CO2, and then chal-

lenged with complement-opsonized zymosan, prepared as described

below, followed by incubation for 40 min at 37 �C and 5% CO2. Un-

bound particles were removed by washing with PBS. Cells were fixed

with 2.5% glutaraldehyde for 15 min at room temperature and the

coverslips were mounted in microscope slides. The extent of phago-

cytosis was quantified by phase contrast microscopic observation. At

least 200 macrophages were counted in each determination and those

containing three or more internalized particles were considered posi-

tive for phagocytosis. The cell viability in all suspensions exceeded 97%

as detected by trypan blue exclusion test.

For opsonization, 2 ml of zymosan particles (2.9 mg/ml in PBS)

was mixed with 2 ml mice normal serum and incubated for 30 min at

37 �C. The complement-opsonized zymosan particles were then wa-

shed and resuspended in PBS at a concentration of 1 mg/ml.

In a set of experiments, adhered macrophages were incubated

with RPMI containing increasing concentrations of native HF3, or

GST-DC-HF3, or DC-HF3, or native HF3 treated with 5 mM

orthophenanthroline (OPA-HF3) for 1 h before challenging with

complement-opsonized zymosan. As a control, adhered cells were

incubated with RPMI alone or containing GST or 5 mM OPA. To

evaluate the role of a2, aM, b1, and b2 integrin chains on toxin-in-

duced phagocytosis, adhered macrophages were incubated with

10 lg/ml anti-a2 (clone Gi9, Immunotech) or anti-b1 (clone JB1B,

Serotec) or anti-aM (clone 5C6, Serotec) or anti-b2 (clone GAME46,

Pharmingen) monoclonal IgG antibodies anti-integrin subunits [20]

or irrelevant IgG (control), at 37 �C and 5% CO2 for 1 h, before

adding the toxins. For statistical analysis means and SEM of all data

were compared by analysis of variance followed by the Tukey test,

with significance probability levels of p < 0.05.

Platelet aggregation assays. Platelet suspensions were prepared

from fresh citrated human blood obtained from healthy donors,

according to Mustard et al. [21]. The native and recombinant proteins

were incubated with platelet suspensions containing 3 · 108/ml for

5 min at 37 �C before the addition of 0.5 lg/ml collagen (Sigma). The

extent of aggregation was estimated as the percentage increase of light

transmission taking the value obtained for platelets incubated with

control buffer and stimulated with collagen as 100%.

Analytical procedures. Protein concentrations were determined by

the Bio-Rad protein assay kit using bovine serum albumin as a stan-

dard. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE) was carried out according to Laemmli [22]. Western blot

analysis was performed as described by Burnette [23]. HF3 (2 nmol)

was reduced and pyridylethylated and submitted to Lys-C cleavage as

described elsewhere [24]. Peptides were isolated by RP-HPLC on a

Lichrospher 100 RP 18 column and submitted to N-terminal amino

acid sequencing by Edman degradation with a gas-phase sequencer 473

A (Applied Biosystems).

Results and discussion

Molecular cloning of HF3

Several immunoreactive clones were isolated from the

cDNA bank and those with largest inserts were selectedfor sequencing. The deduced amino acid sequence of the

cDNA contained in one incomplete clone (1600 bp)

showed that it encoded HF3 as confirmed by the amino

acid sequence of five peptides obtained by the cleavage

of nativeHF3with endoproteinaseLys-C (Fig. 1A). In or-

der to obtain the entire coding region of HF3 we used

5 0RACE. The fragment of �1.0 kb amplified by PCR

using an specific oligonucleotide primer for HF3 showedan overlap of 134 bp with the sequence of clone 6.1.1 and

completed the full-length cDNA sequence of HF3 (Fig.

1A). The precursor of HF3 consists of 2306 bases includ-

ing a 77 base 5 0-end non-coding region, an open reading

frame of 1818 bases, and a 3 0-end non-coding region of

411 bases with termination codon, AATAAA sequence,

and polyadenylation sites. The cDNA sequence of HF3

predicts a pro-protein with striking similarities to precur-sors of the Reprolysin subfamily, belonging to class P-III

[9]. The putative pro-domain contains the conserved se-

quence P-K-M-C-G-V-T present in the corresponding

positions of other venom metalloproteinase precursors

andwhich resembles a region in the pro-peptide ofmatrix-

ins calledCys-switchwhich is involved in the activation of

these enzymes (Fig. 1B) [25]. HF3 exhibits the character-

istic elongated consensus motif HEXXHXXGXXH, inwhich the three histidine residues are involved in binding

of the catalytic essential zinc ion [25]. Moreover, the same

domain contains a conserved methionine residue, which

in the metzincins is located beneath the active site metal

as part of a superimposable ‘‘Met-turn’’ [26]. In the disin-

tegrin-like domain of HF3 the sequence RGD frequently

found in the disintegrin domains of the P-II class precur-

sors is replaced by SECD (Fig. 1B).

Expression of disintegrin-like/cysteine-rich domains of

HF3

The cDNA sequence coding for the disintegrin-like/

cysteine-rich domains of HF3 (DC-HF3) was subcloned

in the expression vector pGEX-4T1, which allows the

expression of soluble recombinant proteins in fusion withGST. E. coliDH5a cells were transformed with the resul-

tant expression plasmid pGEX-4T1-DC and protein

expression was induced with 0.5 mM IPTG for 3 h. The

recombinant fusion protein GST-DC-HF3 was detected

by SDS–PAGE and byWestern blot using anti-HF3 anti-

Fig. 1. (A) The cDNA and deduced amino acid sequences of HF3. The nucleotide sequence is shown in the upper line and the predicted amino acid

sequence is shown in the lower line of each row. Underlined amino acid residues denote sequences confirmed by protein sequencing of native HF3.

The italic nucleotide sequence shows overlap between the 5 0 end of HF3 cDNA and 6.1.1 clone. Putative N- and O-glycosylation sites are italic and

boldface. (B) Putative domains of the precursor of HF3. In boldface are the sequences PKMCGVT (in pre-pro-domain), HEMGHNLGINH (in

metalloproteinase domain), and SECD (in disintegrin-like domain).

C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956 953

bodies as a �52 kDa protein, which shifted to �28 kDa

after release of GST by thrombin (Figs. 2A and B, lanes

2–3). The N-terminal amino acid sequence of the DC-

HF3 protein was determined to be G-S-P-E-F-L-L-R-T-

D-I-V-S, where the residues L-R-T-D-I . . . correspondto the amino-terminal sequence of the disintegrin-like do-

main of HF3 (Fig. 2C). The yield of the DC-HF3 protein

was estimated as 2.5 mg/L culturemedium.Previous stud-

ies have shown that binding of a2b1 integrin on platelets

by native P-III snake venom metalloproteinases causes

the inhibition of the signaling events normally induced

in collagen-stimulated platelets resulting in inhibition of

Fig. 2. Expression of disintegrin-like and cysteine rich domains (DC-HF3) by E. coli DH5a. (A) SDS–PAGE, (B) Western blot of fraction obtained

by affinity to glutathione–Sepharose 4B using anti-HF3 antibodies. (1) Molecular mass markers; (2) protein fraction bound to glutathione–Sepharose

4B; and (3) protein eluted from resin after thrombin cleavage. (C) N-terminal amino acid sequence of the DC-HF3 protein, after thrombin cleavage

of fusion protein and (D) inhibition of collagen-induced aggregation of platelet suspensions by GST-DC-HF3 and DC-HF3.

954 C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956

platelet-aggregation [11,12,27,28]. The recombinant pro-

tein DC-HF3 was then tested for its ability to inhibit col-

lagen-induced platelet aggregation. As shown in Fig. 2D,

DC-HF3 inhibited aggregationofwashedplatelet suspen-sions in a dose-dependent manner with an estimated IC50

value of 768 nM.

Effect of HF3 on aMb2-mediated phagocytosis of opso-

nized-zymosan

To investigate the effect of HF3 and of its disintegrin-

like/cysteine-rich domains on the function of macro-phages and the possible interaction of SVMPs with

macrophage integrins, we evaluated the phagocytosis

of complement-opsonized zymosan mediated by the

integrin receptor aMb2. Opsonized zymosan (OZ), an

insoluble polysaccharide particle derived from yeast,

was used as a potent physiological stimulant that acti-

vates macrophages through the integrin aMb2 [29]. In

order to access the ability of HF3 to stimulate phagocy-tosis, the uptake of complement-opsonized zymosan

particles was determined in adherent peritoneal macro-

phages treated with non-cytotoxic concentrations of

HF3, OPA-HF3, GST, GST-DC-HF3 or DC-HF3. Pre-

vious studies showed that incubation of macrophageswith these proteins at concentrations from 0.2 to

2.0 lM did not affect cell viability (data not shown).

As shown in Fig. 3A, 45–50% ingestion of comple-

ment-opsonized zymosan (OZ) particles per macro-

phage was observed in control monolayers incubated

with RPMI media alone and RPMI containing GST.

Moreover, the presence of 5 mM OPA in RPMI media

diminished the phagocytosis by 15%, suggesting adependence of divalent cations for this activity. Pre-in-

cubation of adherent cells with HF3 or recombinant

GST-DC-HF3 or DC-HF3 at concentrations of 0.2–

2.0 lM resulted in an average OZ phagocytosis of

75%, which was significantly higher than the controls.

These effects were not related to the concentration of

proteins used. Pre-incubation of cells with HF3 treated

with 5 mM OPA caused a similar effect in the phagocy-tosis of OZ as the non-treated protein, indicating that

Fig. 3. (A) Effect of HF3, OPA-HF3, GST-DC-HF3, and DC-HF3 on

phagocytosis of complement-opsonized zymosan. Adhered macro-

phages were incubated with RPMI containing increasing concentra-

tions of HF3, or GST-DC-HF3, or DC-HF3, or HF3 treated with

5 mM orthophenanthroline (OPA-HF3) for 1 h before challenging

with complement-opsonized zymosan as described under Materials

and methods. As a control, adhered cells were incubated with RPMI

alone or containing GST or 5 mM OPA. (B) Effect of monoclonal

antibodies anti-integrin subunits on HF3-induced increase of phago-

cytosis. Adhered macrophages were incubated with 10 lg/ml anti-aMor anti-b2 integrin monoclonal IgG antibodies or irrelevant IgG

(control) for 1 h, before adding HF3 or GST-DC-HF3. The bars

represent means ± SEM of at least five experiments. *p < 0.05

compared with RPMI; #p < 0.05 compared with irrelevant IgG;

()p < 0.05 compared with OPA; and *,#p < 0.05 compared with GST.

C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956 955

the catalytic activity of HF3 did not contribute to its

ability to stimulate phagocytosis by macrophages. These

results point to a role of the disintegrin-like/cysteine-rich

domains of HF3 in the activation of phagocytosis med-

iated by aMb2 integrin. Similarly, inactivation of the cat-

alytic domain of jararhagin, a P-III metalloproteinase

isolated from B. jararaca venom, with OPA had no ef-fect on the up-regulation of MMP-1 and MT1-MMP

caused by jararhagin upon binding to integrin a2b1 [30].

Effect of monoclonal antibodies against integrin subunits

on HF3-induced increase of phagocytosis

In order to further investigate the effects of HF3 on

the aMb2-mediated phagocytic process, monoclonal

antibodies against selected integrin chains (a2, b1, aM,and b2) were used. Diverse lines of evidence show that

SVMPs of class P-III interact with integrin a2b1[27,28,30,31]. However, pre-incubation of adherent cells

with monoclonal antibodies anti-a2 and anti-b1 integrinsubunits did not modify the HF3-induced increase in the

OZ phagocytosis (data not shown). On the other hand,

pre-incubation of cells with antibodies anti-aM or anti-

b2 integrin or a combination of both antibodies signifi-cantly reduced the phagocytosis of OZ by both HF3

and its recombinant disintegrin-like/cysteine-rich do-

mains DC-HF3. Monoclonal antibodies (mAbs) which

recognize epitopes in aM and b2 have been used to probe

the interaction of ligands with the integrin subunits. In

the case of C3bi binding to aMb2, it was reported that

a mAb, which recognizes an epitope outside the aMI-do-

main, a major ligand recognition site, partially inhibitedC3bi binding by the integrin [32]. Binding to C3bi and

factor X by I-less aMb2 was inhibited by a function-

blocking antibody, which binds to the b-propellerdomain of the aM subunit [33]. Moreover, based on

extensive studies, Li and Zhang [34] proposed a model

where three individual domains—aM-I domain, aMb-propeller, and b2I-domain—contribute to the formation

of C3bi binding site. Our results suggest that both aMand b2 subunits are important for the stimulatory effects

of HF3 and DC-HF3 on phagocytosis of OZ by

macrophages.

Members of the ADAMs family of Reprolysins have

been shown to bind to various integrins [13]. Consider-

ing the structural similarities between the P-III class

SVMPs and the members of the ADAMs family of pro-

teins, this work shows the potential of the disintegrin-like/cysteine-rich domains in the ADAMs as ligands of

integrins in inflammation events such as phagocytosis.

Since phagocytosis is an innate and key event for host

defense, venom P-III metalloproteinases and disinte-

grin-like/cysteine-rich domains may constitute relevant

tools for studies on the physiology of effector cells of im-

mune responses. Further investigations on-going in our

laboratory on the interaction of HF3 with integrinreceptor aMb2 will lead us to additional insights into

the mechanisms of activation of the integrin for

phagocytosis.

Acknowledgments

This work was supported by Fundacao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP), Grants 98/

14307-9, 02/01009-7, and 02/13863-2.

956 C.A. Silva et al. / Biochemical and Biophysical Research Communications 322 (2004) 950–956

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