ADONIS 030094759100090

Scatid. J. Immunol. 33, 81 88, 1991

Conglutinin Binds The HIV-1 Envelope Glycoprotein gpl60 and Inhibits its Interaction with Cell Membrane CD4 O, ANDERSEN. A.-M. S0RENSEN, S,-E. SVEHAG & E. FENOUILLET Departmetit of Medical Microbiology, Institute of Medical Biology, Odense University, Denmark; and the Laboraioire de Biologic ct Genetique des Deficits Immunitaires. Hopital de \a PitieSalpetricre, Paris, France

Andersen, O., S0rensen, A.-M.. Svehag, S.-E. & Fenouillet, E. Congluiinin Binds the HiV-l Envelope Glyuoprotein gpl60 and Inhibits its Interaction with Cell Membrane CD4. Scand. J. Immunol. 33, HI 88, 1991 The highly glycosylated envelope glycoprotein (gpl60) of human immunodeficiency virus (HIV) interacts wtth the CD4 molecule present on the membrane of CD4 ' cells and is involved in the pathobiology of HIV inlection. Lectins bind glycoproteins through non-covalent interactions with specific hexose residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact wilh recombinant gpl60 (rgpl60) produced in vaccinia virus-infected BHK2I cells. Specific binding of conglutinin to rgpl60 was demonstrated by ELISA. The interaction of bovine conglutintn with rgplW) was calcium-dependent, which is characteristic of the binding of a C-type leetin tc its iigand, and the binding was inhibited in a dose-dependent manner with A'-acetyl-D-glucosamine. Deglycosylalion of rgpl60 abrogated the congluiinin binding. In addition, congtutinin exerted a dose-dependent inhibition of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that congluiinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein. O. Andersen. Department of Medical Microbiology, Institute of Medical Biology, Odense University, J. B. Win.-ilow,sTet 19. DK-5000 Odense C, Denmark

A component capable of agglutinating erythrocytes reacted with antibody and complement was demonstrated in bovine serum in 1906 [4]. This component, the first described vertebrate lectin, was named conglutinin by Bordel & Streng [5]. The discovery of a wide variety of mammalian lectins, both membrane-bo und and soluble, during the last decades called for a redefinition of the lectins [6]. Goldstein in 1980 [18) defined lectins as carbohydrate-binding proteins of non-immune origin that agglutinate cells or precipitate polysaccharides and glycoproteins. The current interest in mammalian lectins has been stimulated by the belief that iectin-carbohydrate interactions can trigger different cellular functions by recognition of the numerous oligomeric carbohydrates present on soluble as well as membrane-bound proteins [15, 41]. Furthermore, they may act as opsonins and contribute to the host defence against certain pathogens [7, 16]. Conglutinin,

demonstrated in eows [4] and humans [2, 44]. is a member of the mammalian C-type lectins, defined by Drickamer [8] and distinct from the mannose-binding protein (MBP), Bovine conglutinin binds to high-mannose type oligosaeeharides. widely present in the cell walls of many pathogens [40]. but also to complex- and hybridtype chains with terminal A'-acetylglucosamine and mannosc residues [35]. Conglutinin binds in a calcium-dependent manner to the high-mannose oligosaccharides on the i' chain of the degradation product iC3b of the major complement component C3 [19]. Whether this binding has any physiological function has yet to be established, but we have shown that conglutinin exhibits a phagocyte- and complement-dependent bactericidal activity [16]. The binding of conglutinin to its ligand is inhibitabie with ,V-acetytglucosamine and to a lesser degree with mannose and fucose [31,46].

82

O, Andersen et al.

The human immunodeficiency virus (HIV) envelope precursor glycoprotein, gpl60, is cleaved to yield the outer membrane gp 120, which binds to its cellular receptor {CD4) present on CD4' cells, and the transmembrane gp4l molecule, whieh anchors gpl2() to the viral lipid membrane [10]. gpi60 is a highly glycosylated molecule with more than 30 ;V-linked gtycans [I, 36. 37]. Several reports indicate that the interaction of gp!20 with the CD4 molecule, present on T-helper lymphocytes, macrophages and monocytes, accounts for HIV's tropism and cytopathic effect [22, 23. 43]. The importance of the glycan moieties, especially the high-mannose oligosaccharides of the gpI20, in HIV's interaction with its target cell is indicated in studies which show that the mannose-specifie lectins eoneanavalin A and MBP inhibit HIV infection and syncytia formation in vitro [II, 42]. However, we have recently shown that the carbohydrate moieties of major gpl20 do not play a critieal role in its interaction with CD4 [13, 14]. The aim of the present study was to investigate whether bovine congluiinin could bind to ,V-linked glycans of gpl60 and possibly interfere with the binding of gpl60to CD4+ cells,

MATERIALS AND METHODS 1I) Reagent.-!. Saceharomyces cerevisiae mannan was prepared to Ihe procedure of Nakajima & Ballou [39] and coupled to CNBr- aclivaled CL4B Sepharose (Pharmacia, Uppsala, Swcdeti). Zytnosan was prepared as described by Lachmanii [21] from commercial baker's yeast. A'-acetyl-D-glucosamine (GlcN Ac) and Dgalactose (GAl) were obtained from Sigma (St Louis, Mo.. USA), Para-nitropheny! phosphate (PNPP) and Endo F ,V-glycanase were from Boeliringer (Mannheim Biochemicals, FRG) and streptavidin-phycoeryllirin from Bectiin Dickinson (Mountain View, Calif., USA). DEA hufTer comprised 9.1"'.-. diethanolamine (vol vol) (DEAl, 0.5 niM NaN,, 0.5 mM MgCN. pH 9.K. Coating butler comprised 50 niM Na:C03, pH 9.6. Veronal butler saline (VBS) comprised 4 niM sodium barbitai. 145 mM NaCi, 0.5"';, NaN,, 0.05'"., Twccn 20. pH 7.4. while Me' * VBS was VBS containing 2 mM CaCl: and I mM MgCI;. In the immunncytometric analyses Me-'VBS without Tween 20 was employed. EDTAVBS was VBScontainint! 10 mM EDTA. Recombinant gpl60 (rgpl60) produced in recombinant vaccinia virus-infected BHK2I cell.s was a gift from Pasteur Vaccins (Val do Reuil., France). (2) Antibodies. Alkaline piiosphatase-cnnjugated goat anli-rabbit IgG (AP-anti-rabbit IgG) and alkaline phosphalase-eonjugatcd rabbit anti-mouse IgG (APanti-mouse IgG) were obtained from Sigma (USA). Biotinylated sheep anli-mnuse IgG was from Amer-

sham International (Amersham, UK). Anti-gpl60 murine monoclonal antibody (MoAbl 10 4) was purchased from Genetic Systems (Seattle, Wash., USA), and anliCD4 MoAb (anti-leu-3a) from Becton Dickinson. Anlibovine conglutinin antibody was produced m rabbits by immunization wilh a 4.1-kDa band obtained from gotdslaincd western blots of bovine congluiinin, reduced and fractionated on SDS-PAGE. (.^) Purification and characierization of conglutinin. Ox serum healed at 56 C for 45 min, wa.s incubated with zymosan, and bound proteins were eluted with EDTAVBS. Calcium chloride was added at a tinal concentration of 2 mM of free unchelated calcium, before application to a mannan CL4B Sepharose column. The column was washed wilh Me-"^ VBS. 0.5 M NaCl. and eluled with EDTA-VBS. The eluate was treated as above and applied to a second column of mannanCL4B Sepharose. The bound proteins were eluted as described and the eluate was lested for conglutinin activiiy by ELISA [2|. The fractions with the highest congluiinin activity were analysed by SDS PAGE on 4-20".I. acrylamidc-bisacrylamide gel [26]. Proteins were stained wilh silver nitrate [38]. (4) ELISA analysis of the binding of conglutinin to rgpl60. Assay I: The optimal concentration of rgpl60 for coaling microtitre plates (PolysorbR. Nunc, Copenhagen. Denmark), was determined by incubating microwells overnighl al 4 C with increasing amounts of rgpI60 (10 3000 ng) diluted in 100 /il of coating buffer in duplicate. The wells were washed in Me-'VBS, conglutinin (300 ng, 100 /d Me'-* VBS) was added, and the cells were incubated overnighl at 4 C. After washing the wells were filled with rabbit anti-bovine congluiinin antibody (100 ng/lOO /JI) in Me'* VBS and incubated for I h al 20 C. The wells were wa.shed again and incubated wilh AP-iinti-rabbit [gG (50 ng/lOO ;jl) in Me-' VBS for I h ai 20 C. After washing and further incubation wilh PNPP (100 ;ig/IOO tA) in DEA buffer for 30 min at 37 C, optical densiiy (405 nm) was read. In controls, weils were coated with BSA (a non-glyeosylated protein), human IgG (Miles. USA; known to contain complc.K-type oligosaccharides). and human IgM (Cappci, USA, containing high mannose-type oligosaccharides). In additional controls conglutinin was omitted or congluiinin was incubated in EDTAVBS or VBS only. Assay 2: In order to study binding of conglutinin to wells coated with a fixed amount of rgpl60 (250 ng), increasing concentrations of bo vine conglutinin (3 300 ng) in 100 /d of Me-' VBS were added and incubated overnight at 4 C. The staining procedure was as described above. Controls: (1) incubation with conglutinin in increasing concentrations (3 300 ng. 100 fd) in M e ' ' VBS containing I niM. 10 niM, and 50 mM GlcNAc. 10 mM GAl, or in 10 mM EDTA-VBS: (2) coating with rgp 160 (250 ng) treated with Endo E ;Vglycanase (0.25 U) for 20 h at 37 C or rpgl60 treated with hcat-inactivalcd Endo F ,V-glycanase. In order to inactivate Endo F ;V-glyeanase completely, it was necessary to heat for 10 min at 100 C. (5) ELISA analysis of the binding of Mo Ah 110-4 to rgpl60 and rgpl6() preincubated with conglutinin. In order to demonstrate that ihe clTcct observed in the immunocyiometric studies of bovine conglutinin was

Conglutinin Binds to gpl60 of HIV-1 not the result ofbiocking by conglutinin of the epitopes on rgpl60 recognized by MoAb 110-4, wells were coated with rgpl60 (10 .100 ng.'lOO n\). and incubated with excess of bovine conglutinin (H' /Jg/100 /i!) in Me-"^ VBS at 4 C overnight. After washing, MoAb ! 10 4(1:1000) in M e ' ' VBS was added and incubaied for 1 h at 20 C. The wells were washed and incubated for i h at 20 C with AP-anti-mouse IgG (50 ng/KX) /il) in Me- *" VBS. Staining was performed as described above. In controls, rgpl60 was preincubated with Me'^VBS only, (6) Culture ofCD4 ' lymphoid cells. Cells of clone 13 (CEM 13) derived from the CEM line (American Type Cullure Collection, Rockville, Md, USA) were cullured at 37 C in RPMI 1640 medium (f'low Laboratories, Irvine. UK), supplemented with iO"(j fetal calf scrum, !"/> glutamine, and I".;, streptomycin-penicillin, in a humified atmosphere with 5".'n CO;. (7) Immunocytometric analysis ttfthe binding to CD4 * cells ofrgpI60andofrgp}f}Opremcuhated\vith congtutinin. One microgram of rgp 160 was incubalcd with or without 50 |(g of conglutinin in 100 /il Me-' VBS overnight at 4 C. In parallel. I /ig of rgplfiO was incubated with 50 ;ig of congluiinin in Me-'VBS supplemented with 20 niM GlcNac or 5(1 niM GlcNAc in order to inhibit the binding of rgpl60 to conglutinin. After this incubation, increasing amounts of rgp 160 from the four different samples were incubated wilh 3x 10^ CEM 13 cells for 2 h at 20 C in 100 ^^l final volume of Me"' VBS containing O.l"(. BSA. Controls were performed with GlcNAc in Me"' VBS. Afler washing and further incubalion at 4 C. for I h with lOO n\ of MoAb 110^ (1:100 dilution) in bulTer, the cells were washed and incubated at 4 C for 1 h with biotinylated anti-mouse IgG (1:25 dilution). After washing, the ceils were further incubated at 4 C for 30 min with 100/d of streptavidin phycoerythrin (1:1000 dilution) in buffer. The ceils were washed and resuspended tn 500 /jl oT Me' ^ VBS containing 1 "••» paraformaldehyde. Cell membrane fluorescence intensity was measured by the FACS analyser (Beclon Dickinson). The following controls were perlbrmed in parallel: (I) incubation of 3x10- CEM 13 cells with 20 /ig of conglutinin m 100 /il Me-' VBS and 0.1 'v,, BSA for 2 h al 20 C in order to study a possible modification o\ the auto fluorescence of the cells: (2) background fiuorescence obtained when thecells were onK submitted to the staining procedure was compared with that obtained when cells were preinciibated with congiulinin (2(1 fig) and then submitfed to the staining procedure: (3) after incubation ol" 3 X 10^ CEM 13 cells wilh or wilhout 20 Ug conglutinin in 100 ^1 Me"' VBS and 0.1"., BSA for 2 h at 4 C, the cells were washed and the binding ofantiCD4 MoAb anli-leu-3a( 100 ng/100/(I) was determined. This MoAb reacts with an epitope on CD4 near the region recognized by rgpI60 (33].

RESULTS Conglutinin preparation Bovine conglutinin was purified as described in Malerials and Methods. The purified preparation

83

showed a major band af 43 kDa and a minor band at 86 kDa under reduced conditions. The 86 kDa band represented incompletely reduced dimers of conglutinin. Both bands reacted wilh rabbit antibovine conglutinin antibody on western blots (Fig- 1)-

Binding of conglutinin to rgplOO Binding of eonglutinin to rgpl60 was investigated by ELISA (see Materials and Methods). When wells were incubated with increasing concentrations (10 3000 ng, 100 fi\) of rgp 160. significant binding of conglutinin (300 ng/100 /il) was observed at rgp 160 concentrations above 100 ng/ 100 fi\ and a plateau was reached at rgpl60 concentrations exceeding 1000 ng/100//I (Fig. 2). The binding was calcium-dependent, as it was abolished in the presence of 10 mM EDTA and when congiutinin was diluted in calcium-free bufter (VBS), The affinity of conglutinin for rgpl60 was sufficient to resist the washing procedure. No staining was observed when conglufinin was omitted, or in wells coated with BSA or human IgG (data not shown). Conglutinin showed a calcium-dependent binding to igMeoated wells. In thelight of these results, we chose a rgp 160 concentration of 250 ng/100 fd for further experiments. We next examined the binding of increasing amounts of conglutinin {3 300 ng/100 n\) to rgp 160 (250 ng/100 /d) coated wells. Conglutinin showed a dose-dependent binding to rgpl60 in this sy.stem (Fig. 3). The binding of conglufinin was abrogated by 10 mM EDTA and reduced in a dose-dependenl fashion when congluiinin was incubated with rgp 160 in the presence of increasing concentrations (I, 10, and 50 mM) of GlcNAc. a monosaccharide with high affinity for conglutinin. The binding was not inhibited in presence o( 10 niM Gal (a monosaccharide with low alTmity for congluiinin) under the same experimental conditions. Deglycosylation of rgpl6D by pretreatment with Endo F ,Vglycanase totally abolished the binding ofconglutinin. Binding of conglutinin to rgp 160 was not affected when rgpl60 was preincubated with heat-inactivated Endo F N-glycanase (data not shown). No reaction was seen when the anticonglulinin aniibody was replaced by preimmune rabbit sera or when the secondary antibody was omitted (data not shown). The observations were reproducible in that similar results were obtained

84

O. Andersen et ul.

-OS

I- 0 I

I

S 10 Fraction number FIG. I. Purilicalion of bovine conglutinin. A preparation obtained from he:it-inactivatcd ox serum was fractionated on a man[ian-Seph:irose column as described in Materials and Methods. The concentration of proiein (OD^NH) eluled with 10 mM EDTA ( A ) was measured. Conglutinin wa.s measured by ELISA {Eit)f) forconglulinin epitopes (Ag-F.LISA) (•) and for conglutinin bioactivity (Bio-ELISA) (O). The fraelions that conUiined mn.ximtim conglulinin iictivity were pooled, reduced, and analysed by SDS PAGE and stained with silver nilrale (insert, left lane). A reduced sample was also analysed on western blots and stained with biotinylated rabbit anti-bovine congltiiinin (insert righl lane).

3 0 -

7 0 -

10-

100

300

1000

BOOO

rgp 160/WP

FIG. 2. Binding of bovine conglutinin (300 ng) to microweiis eoaled with increasing doses (10 ng 3000 ng) of rgpI60 measured by ELISA. Binding in the presence of 2 mM CaCI: (•) in 10 mM FDTA ( A ) and without addition of conglutinin (O) is shown. Vertical bars indicate! 1 SD.

Conglulinin Binds lo gpl60 ofHIV-l

85

H05 2Si141208-

50 rgpl50/we(l |ng|

[onglutinin/usll (ngl

FIG. 3. Binding of bovine conglutinin (3 ng 300 ng) to wells ineubated with recombinant gpl60 (250 ng) measured by ELISA. Binding in the presence of 2 mM CaCl:(»), lOmMEDTA(D). I mM(B), lOmM(O), and 50 niM ClcNAc (,AJ, or 10 mM galactose-D* (AJ is shown.

in independent assays performed on different days. Analysis of the binding of rf;p/60 und rgpI60 preincubaled with conglufinin ro CD4 ' cells Preineubation of rgpl60 (1 /(g/lOO /il) with conglutinin (50 /(g/100 /j|) diminished ihe binding of rgpl60 to CEM 13 ecll.s by one log of magnitude. However, we still observed a weak membrane fluorescence because of binding of the rgpl60-conglutinin complexes to the CD4* cells {Fig. 4). Inhibition of the binding of rgpl60 to the

300

FIG. 5. BLISA analysis of binding of MoAb 110-4 lo wells incubated either with increasing amounts of rgpl60 (0-300 ng) {•) or with rgpl60 (0 300 ng) preincubaled with excess of bovine conglutinin (10 /(g/ 100 ^i)(O}. Vertical bars indicate +1SD.

eells by eonglulinin was significantly reduced on addition of 20 mM GlcNAc and abolished by 50 mM GlcNAc. With 50 mM GlcNAc, the membrane fluorescence was the same as that obtained when rgp 160 was not preineubated wilh conglutinin. When CEM 13 eells were preineubaled with or without conglutinin (as described in Materials and Methods), no difference in the binding of the anti-CD4 MoAb anli-leu-3a to the CD4' eells was observed (data not shown). Furthermore, preincubalion of the CEM !3 eells with conglutinin did not affect cither the autofluorescenee or the background fluorescenee because of the labelling proeedure (data not shown). Finally, preincubation of rgpl60-coated mierowells with an exeess of conglutinin (10 /f bovine conglutinin. Scand. J. ImmwwI. 26. 355. 1987. 3 Bahraoui. E.. Clerget-Raskiin. B.. Chapuis, F., Olivier, R., Parravicini. C , Yagello. M.. Montagnier. L. & CJluckman. J.C. A molecular mechanism of inhibition of HIV-1 binding to C D4-(- cells by monoclonal antibody to gpl 10 .MOS 2, 165. 1988. 4 Bordet, M.J, & Gay. F,P, Sur les relations des scnsibilisatricesavec falexine. .4tiii. In.st. /•«,v(f»r30, 467. 1906. 5 Bordet. H.J.&Streng, P. Lesphenomcnesd'absorption de la eonglutinine du serum de boeuf. Zentr. Bakterioi. Parasimik. .Ahi. I (hif^. 49. 260. 1909, 6 Boyd. W.C. & Shapleigh, E. Specific precipitating activity of plant agglutinins (lectins). Scienrc 119, 419. 1954. 7 Coombs. R.R.A.. Coombs. A.M. & Ingram. D.G. The St'roldf^y «/ Conglutinntinn and i/.s Relation to Pi.fetise. Blackwell Scientilic Publications. Oxford. 1961. 8 Drickamer. K. Two distinct classes of carbohydrate-recognition domains in animal leetins. J. Bud. Chem. 263,9557. 1989. 9 Drickamer. K. Structure and biosynthesis of membratie receptors which mediate cndocytosis of glyeoproteins. KUlfiey Ini. 32 (SuppI, 23). 67. 1987, ' 10 Dimarzo Veronese. F.. DeVico. A.L.. Copeland. T.D.. Orozlan, S.. Gallo, R.C & Sarngadharan. M.G. Characterization of gp4I as the transmembrane protein coded by the HTLV-UI LAV envelope gene. SiiencelVi, 1402. 1985. 11 Ezekowitz. R A.B.. Day. L.E. & Herman. G.A. A human mannose-binding protein is an acute-phase reactani that shares sequence homology with other vertebrate leetins. J. K\p. Med. 167. 1034. 1988. 12 Ezekowitz. R.A,B., Kuhlman, M . Groopman, J.E. & Byrn. R.A. A human serum mannose-hinding protein inhibits in vitro infection by the human immunodeficiency virus. J. E.\p. Mvd. 169. 185, 1989. 13 Fenouillei. E..CIerget-Raslain, B., Gluckman. J . C Guctard. D.. Montagnier. L. & Bahraoui. E. Role of N-linked glyeans in the interaction between the envelope glyeoprotein of human immunodeficiency virus and its CD4 cellular reeeptor. J. E\p. Metl. 169,807. 1989. 14 Fenouillet, E..Gluekman. J.C. & Bahraoui. R. Role of N-linked glucans iy\' envelope glycoproteins in infectivily of human immunodeliciency virus type I. J. Tim/. 64. 6. 2841. 1990. 15 Feizi. T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproleins and glycolipids are onco-developmenlal antigens. Nature 314, 53. 1985. 16 Friis-Christiansen. P.. Thiel. S.. Svehag. S.-E.. Dessiiu. R.. Svendsen. P.. Andersen, O.. Laursen. S.B. & Jensenius. J,C, In vivo and in vitro antibacterial activity of conglutinin. a mammalian plasma lectin. SiWul. J. Immuuol. 31. 453. 1990. 17 Gitelman. A.K.. Bere/in, V.A. &. Kharitonenkov. I.G. The role of carbohydrate in detertnining the immunocbemical propertiesof the hemagglutinin of intluenzii A virus. Ardt. ViroL 67. 253. 1981.

87

18 Goldslcin. I.J.. Hughes. R.C. Monsigny, M., Osarta. T. &L Sharon, N. What should be called a lectin? (correspondence). ,\iiiLirc 285. 66, 1980. 19 Hirani, S., Lambris, J.D. & Muller-nberhard. J. Localization of the conglutiniii binding site on the third component of human complemenl. J. liiimuncl. 1.14. li()5. 14X5. 20 Hogle. J.M.. Chow. M., Filman, D.J. Three-dimensional structure of poliovirus at 2.9 A resolution. Snemr 229, 1358. 1985. 21Ikeda. K.. Takayoshi. S.. Kawasaki. N.. Kawasaki. T. & Yamashina, J. Serum lectin with known structure activates complement through the classical pathway. / Biol. Chem. 262, 7451. 19K7. 22 Klat/mann. D.. Barre-Sinoussi. V.. Nugeyre. M.T.. Dauglict.C.. Vilmer. E..Griscelli.C.. Brun-Vezinei. I.. Rou/iou>i. C . Gluckman. J.C .. Chermann, J.C. & Montagnier. L. Selective tropism of lympbiidenopathv associated virus (LAV) for he I per-induced Tlymphocytes Siiciue 225, 59. 1984. 2} Klat/man. D.. Champagne, E.. Chamaret, S., Gruest. J.. Guetard. D.. Hercend. T.. Gluckman. J C. & Moniagnier. L. T-i>mphocvte T4 molecule behaves as tbe receptor for human retrovirus LAV. .Vtj/Hr< 312. 767. 1984 24 Kuhlman, M., Joiner, K. & E/cktJwit/, R.A.H. Tbe human mannose-binding protein functions as an opsonin. J- e.\(>. Mvd. 169. 1733. 1989. 25 Lachmann. P.J. Conglutinin and imniunoconglutinin. Adr. Immmul. 6,479, 1967. 26 Laetnmli. U.K Cleavage of structural proteins during the assembly of the head of baeteriophage T4. ,V