Journal of Neuroscience Research 33513-518 (1992)
The gp120 Glycoprotein of HIV-1 Binds to Sulfatide and to the Myelin Associated Glycoprotein L.H. van den Berg, S.A. Sadiq, S. Lederman, and N. Latov Departments of Neurology (L.H.B., S.A.S., N.L.) and Medicine (Division of Rheumatology; S.L.), Columbia University, New York
We investigated the binding of the gp120 glycoprotein of the human immunodeficiency virus (HIV-1) to neural glycolipids and glycoproteins by ELISA. The gp120 protein bound to sulfatide (Gals), a sulfated glycolipid autoantigen implicated in sensory neuritis, and to the myelin associated glycoprotein (MAG), an autoantigen in demyelinating neuropathy. Binding of gp120 to MAG was inhibited by the HNK-1 antibody, which recognizes a sulfated glucuronic acid epitope, suggesting that the interaction involves carbohydrate determinants. Sulfatide and MAG are potential receptors for gp120 in peripheral nerve and may have a role in the neuropathy associated with HIV-1 infection. 0 1992 Wiley-Liss, Inc. Key words: ELISA, sulfatide, myelin associated glycoprotein, autoantigen, neuropathy, HIV-1, gp120 glycoprotein INTRODUCTION
and they might serve as receptors for gp120 in peripheral nerve.
MATERIALS AND METHODS Antibodies and Ligands Recombinant gp 120 and mouse monoclonal IgG anti-gp120 antibodies were obtained from American Biotechnologies, Inc. (Cambridge, MA). Mouse monoclonal IgG anti-heparan sulfate proteoglycan (HpS) antibodies and rabbit antigalactocerebroside (GalC) antibodies were obtained from Chemicon International Inc. (Temecula, CA). Mouse monoclonal IgG anti-chondroitin sulfate (ChS) antibodies and peroxidase-conjugated affinity-purified antibodies to mouse IgG, rabbit immunoglobulins, mouse IgM, or human IgM were obtained from Sigma Chemical Co. (St. Louis, MO). The mouse monoclonal IgM HNK- 1 antibody which binds to the myelin associated glycoprotein (MAG) and to the glycolipid 3-sulfated glucuronyl paragloboside (SGPG; Nobile-Orazio et al., 1984; Freddo et al., 1985), the mouse monoclonal IgG anti-MAG antibody GENS-3, and the mouse monoclonal IgG anti-CD4 antibody OKT4 were produced from hybridomas and were purified from ascites as previously described (Nobile-Orazio et al., 1984; Lederman et al., 1992). Human monoclonal IgM antibodies to GMl ganglioside or to Gals were obtained from patients with neurological diseases (Sadiq et al., 1990; Quattrini et al., 1992). Human myelin associated glycoprotein (MAG) and the glycolipid sulfonyl
The human immunodeficiency virus (HIV) has been reported to infect both neural and non-neural cell lines through CD4 independent interactions, but the mechanisms involved are poorly understood (Clapham et al., 1989; Harouse et al., 1989; Li et al., 1990; Tatano et al., 1989; Werner et al., 1990). The possibility that cell surface carbohydrates might serve as receptors for HIV on neural cells was suggested by the observation that the HIV-1 coat protein gp120 binds to sulfated polysaccharides such as dextran sulfate (DxS) and pentosan polysulfate (PPS, Schols et al., 1990; Lederman et al., 1992), and because viral infection is inhibited by antibodies to the carbohydrate determinants of galactocerebroside Received June 19, 1992; revised and accepted July 23, 1992 (GalC) and its sulfated derivative sulfatide (Gals, Ha- Address reprint requests to Norman Latov, M.D., Ph.D., Department Neurology, Columbia University, Black Bldg Rm 3-323, 650 W. rouse et al., 1991). In this study we report that gp120 of 168th Street, New York, NY 10032. binds to Gals with higher avidity than to other glycolipwork was supported in part by NIH grant NS25187 to N.L. and ids and that gp 120 also binds to the sulfated oligosac- This by a grant from the Ahron Diamond Foundation to the Department of charide containing region of the myelin associated gly- Neurology. L.H. van den Berg is a Visiting Fellow from the Univercoprotein (MAG). Both Gals and MAG are putative sity Hospital of Utrecht, the Netherlands, and is the recepient of a antigens for antibody mediated peripheral neuropathy , scholarship from the Dutch Organization for Scientific Research. 0 1992 Wiley-Liss, Inc.
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glucuronyl paragloboside (SGPG) were purified as previously described (Nobile-Orazio et al., 1984; McGinnis et al., 1988). Recombinant CD4 was a gift of Dr. Vicki Sat0 (Biogen Corp, Cambridge, MA). The following were purchased from Sigma Chemical Co. (St. Louis, Moj: sulfatide (Gals), galactocerebroside (GalC), GM 1 ganglioside, chondroitin sulfate (ChS) , heparan sulfate (HpS), pentosan polysulfate (PPS), dextran sulfate MW8000 (DxS), and dextran MW-10,000 (Dx).
ELISA Studies Binding of gp120 to the glycoproteins MAG and CD4, the proteoglycans ChS and HpS, and to the glycolipids Gals, GalC, SGPG, and GM1 was measured by ELISA by modifications of the procedures of Nobile Orazio et al. (1984) for glycoproteins, Freddo et al. (1986) for proteoglycans, and Sadiq et al. (1990) for glycolipids. Briefly, microwells were coated with 5 pg/ ml of the glycoproteins or proteoglycans in PBS (0.14 N NaCl, 0.01 PO,, pH 7.4) overnight, or with the glycolipids in methanol, which was allowed to evaporate. The wells were then saturated with BSA, and increasing concentrations of gp120 in ELISA solution (PBS with 1% BSA and 0.025% Tween-20) were added for 2 hr at room temperature. Wells were washed 6 x with ELISA solution between subsequent steps. The gp120 protein which bound to the ligands in the microwells was measured using mouse monoclonal antibodies to gp120 (1 :2,000), followed by peroxidase-conjugated, affinity-purified goat antibodies to mouse IgG (1:200). All determinations were done in triplicate and control wells were coated with BSA, or gp120 was omitted from ligand coated wells. Readings in the BSA coated wells were subtracted from the corresponding ligand coated wells. Adherence of the ligands to the microwells was confirmed with antibodies to the specific ligands by ELISA. The secondary antibodies used with each ligand were as follows: for GM1, human IgM anti-GM1 antibodies at 1:10,000, followed by peroxidase-conjugated anti-human IgM at 1:200; for Gals, human IgM antiGals antibodies at 1:10,000, followed by peroxidaseconjugated anti-human IgM at 1:200; for SGPG and MAG, the HNK-1 antibody at 1 pg/ml, followed by peroxidase-conjugated anti-mouse IgM at 1:200; for GalC, rabbit anti-GalC antibodies at 1:200, followed by peroxidase-conjugated anti-rabbit antibodies at 1:200; for CD4, the OKT4 antibody at 4 p,g/ml, followed by peroxidase-conjugated anti-mouse IgG; for ChS , mouse anti-ChS antibodies at 1:200, followed by peroxidaseconjugated anti-mouse IgG at 1:200; and for HpS, mouse anti-HpS antibodies at 1:200, followed by peroxidaseconjugated anti-mouse IgG at 1:200. In parallel studies, microwells were coated with gp120 at 5 pg/ml in PBS, and increasing concentrations
of MAG, CD4, ChS, or HpS were added to the microwells and were incubated for 4 hr. Binding of MAG to gp120 was detected with the HNK-1 antibody (1 Kgiml for 1 hr), followed by peroxidase-conjugated antibodies to mouse IgM (1:200) for I hr. Binding of CD4, ChS, and HpS were detected using mouse monoclonal antibodies specific for each ligand (4 pg/ml of OKT4, and 1:200 dilution for anti-ChS or HpS antibodies), followed by peroxidase-conjugated antibodies to mouse IgG (1 : 200). Binding of the glycolipids to immobilized gp 120 in the ELISA system could not be examined because of nonspecific adherence of the glycolipids liposomes to the surface of the microwells.
Binding Inhibition Studies In the binding inhibition studies, increasing concentrations of dextran sulfate (DxS) , pentosan sulfate (PPS), dextran (Dx), HpS, or ChS were added to microwells coated with CD4 or MAG to determine whether these inhibited the binding of soluble gpl20 to CD4 or MAG. The inhibitory agents were added to the microwells immediately preceding the addition of gp120 and were present throughout the incubation period. To determine whether gp120 bound to specific epitopes in MAG, increasing concentrations of the mouse monoclonal antibodies HNK- 1 or GEN-S3, which bind to different epitopes of MAG, were tested for their ability to inhibit the interaction of soluble gp120 with immobilized MAG by ELISA. Western Blot Studies To confirm the specificity of the interaction between gp120 and MAG in the ELISA system, MAG (10 pg/ml) was added to microwells coated with gp120 or with control wells coated with BSA, and after 2 hr the microwells were washed and the adherent proteins were solubilized in PBS with 1% SDS. The solubilized proteins were then separated by SDS-PAGE, transferred to nitrocellulose strips, and immunostained with the mouse monoclonal anti-MAG antibody HNK- 1 or GENS3 (Nobile-Orazio, 1984).
RESULTS In the ELISA system, soluble gp120 bound to Gals immobilized in microwells more avidly than to the other glycolipids tested, including to SGPG, GalC, or GMl (Fig. 1). Binding to Gals was detected at gp120 concentrations of less than 0.5 pg/ml, and 50% of maximal binding was observed at 1 pg/ml of gp120. Binding was specifically inhibited by dextran sulfate (DxS) and pentosan sulfate (PPS) in a concentration-dependent manner. Maximal inhibition was achieved at 1 pM concentrations of the inhibitory agents (Fig. 2), and no
HIV-1 gp120 Binds to Gals and MAG
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Fig. 2. Inhibition of soluble gp120 binding to Gals by DxS or PPS. Increasing concentrations of DxS or PPS were added with gp120 to Gals coated microwells. The gp120 binding was determined by ELISA. Maximal inhibition was seen at 1 p M concentration of the inhibitors. No inhibition was seen with Dx, ChS, or HpS.
inhibition was observed with dextran (Dx), chondroitin sulfate (ChS), or heparan sulfate (HpS) at concentrations of up to 10 pM. DxS and PPS did not inhibit the binding of anti-Gals antibodies to GalS, indicating that they inhibited the interaction of gp120 with GalS. Soluble gp120 also bound to the sulfated glycoprotein MAG immobilized in microwells at concentrations
of less than 1 pg/ml (Fig. 3 ) . Immobilized CD4, which is known to bind to gp120, was used as a positive control for gp120 binding (Eiden and Lifson, 1992). As negative controls, gp120 did not bind to immobilized ChS or HpS, although both ligands were demonstrated to be immobilized in the microwells using ligand-specific antibodies. Binding of gp120 to MAG and CD4 was also inhibited by DxS and PPS, but not by Dx, ChS, or HpS (not shown). DxS and PPS did not inhibit the binding of anti-CD4 or HNK-1 antibodies to their respective ligands. In order to determine whether gp120 binds to the MAG oligosaccharide, the HNK- 1 antibody which recognizes a sulfated glucuronic acid determinant in MAG was tested for its ability to inhibit the interaction. As shown in Fig. 4, binding of gp120 to immobilized MAG was inhibited by the HNK-1 antibody, but not by GENS3, a mouse monoclonal anti-MAG antibody which recognizes an unrelated peptide epitope. The HNK-1 antibody inhibited the interaction of gp120 with MAG in a concentration dependent manner, with maximal inhibition observed at antibody concentrations of 50 pg/ml. As control, HNK-I did not inhibit gp120 binding to CD4. To determine whether soluble MAG also binds to gp120, increasing concentrations of soluble MAG were added to microwells coated with gp120, and the binding was determined by ELISA. As shown in Fig. 5 , both soluble MAG and CD4, which is known to react with gp120, bound to the immobilized gp120. There was no
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Antibody Concentration (pglrnl) Fig. 4. Inhibition of gp120 binding to MAG by HNK-I . Microwells were coated with MAG, and increasing concentrations of the HNK-I anti-MAG antibody were added with gp120. Binding of gp120 to MAG was determined by ELISA. The HNK-I antibody completely inhibited the gp120 binding at a concentration of 50 pg/ml. No inhibition was seen with the mouse monoclonal antibody GEN-S3, which is directed at a different epitope of MAG. 2.000 h
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Fig. 6. Identification of the ligand for gp120 as MAG by Western blot analysis. MAG was added to microwells coated with gp120 or BSA, and after washing the adherent proteins in the wells were solubilized and characterized by Western blot. In A, the eluent from gp120 coated microwells was identified as MAG by its reactivity with monoclonal HNK-1 anti-MAG antibodies and its mobility on SDS-PAGE. In B, no MAG was detected in the BSA coated microwells.
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with the anti-MAG antibodies on nitrocellulose blots and by its characteristic mobility as 2 closely migrating bands of approximately 100 kD (Fig. 6). As a control, MAG could not be detected in the eluent from the BSA coated microwells.
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Concentration (pg/ml) Fig. 5 . Binding of immobilized gp120 to soluble MAG and CD4. Increasing concentrations of MAG and CD4 were tested for binding to microwells coated with gp120.
binding to gp120 by ChS or HpS (not shown). The specificity of the interaction between MAG and gp120 in the ELISA system was confirmed by Western blot analysis. MAG was added to microwells coated with gp120 or BSA, and after washing the proteins which bound to the microwells were eluted and analyzed on Western blots. Following elution from the gpl20 coated microwells, the bound protein was identified as MAG by its reactivity
DISCUSSION In this study, gp120 reacted with the glycoconjugates and MAG, both of which have sulfated carbohydrate determinants. Gals contains a 3-sulfated galactosyl determinant, and MAG contains the HNK-1 epitope which is shared by the 3-sulfated glucuronic acid bearing glycolipid SGPG (Ariga et al., 1987; Ilyas et al., 1990). The binding of these glycoconjugates to gp120 was inhibited by the sulfated polysaccharides DxS and PPS, which inhibit viral binding and infectivity (Schols et al., 1990; McClure et al., 1992; Lederman et al., 1992), but the interactions was not due to nonspecific binding to sulfated carbohydrates, as there was no binding to HpS or ChS. Interactions between gp120 and spe-
HIV-1 gp120 Binds to Gals and MAG
cific carbohydrate determinants on cell surfaces may be important for viral infectivity, as antibodies to carbohydrate determinants of GalC and Gals inhibit HIV-1 infection of CD4- neural cell lines (Harouse et al., 1991), and as lectins that bind to carbohydrate determinants on the surface of CD4 cells inhibit HIV- 1 infection independently of CD4 (Gattegno et al., 1992). MAG is a neural adhesion molecule, and like CD4 is a member of the immunoglobulin gene superfamily (Spagnol et a]., 1989; Williams et al., 1989). It bears the HNK- I carbohydrate epitope, a 3-sulfated glucuronicacid-containing antigenic determinant, which is shared by several other adhesion molecules in the nervous system (Harper et al., 1990). The interaction of gp120 with MAG is inhibited by the HNK-1 antibody but not by the monoclonal antibody GEN-S3, which recognizes an unrelated peptide epitope in MAG, suggesting that the HNK-1 carbohydrate determinant is involved in the interaction with gp120. However, other regions of MAG and conformational determinants may also have a role in the interaction, as gp120 does not bind to MAG denatured by SDS on Western blots (not shown) and gp120 does not bind well to SGPG, which also bears the HNK1 epitope. The observation that HNK-1 binds to MAG in microwells coated with gp120 at low antibody concentrations, whereas it inhibits MAG from binding to gp120 at the higher antibody concentrations of greater than 10 pg/ml, suggests that MAG bears multiple HNK-1 determinants or that MAG forms multimeric complexes in solution. HIV-1 infection may be associated with an acute or chronic demyelinating neuropathy or a painful sensory axonal neuropathy (Miller et al., 1988; Simpson, 1992). Pathological studies show loss of both myelin and axons, and inflammatory cells or complement deposits may be present (Mah et al., 1988; Hays et al., 1988). Since MAG is an autoantigen for monoclonal IgM antibodies in demyelinating neuropathy (Latov et al., 1988), and Gals is a putative autoantigen in sensory ganglioneuritis (Quattrini et al., 1992), they might also serve as receptors for gp 120 or HIV- 1 in peripheral nerve. If MAG and Gals have a role in HIV-1 associated neuropathies, they could be involved by several different mechanisms: 1) HIV-1 could bind to Gals or MAG to produce a low grade infection of neurons or Schwann cells; 2) infected macrophages expressing gp120 might attach to Gals or MAG, and damage the nerve cells; 3) The gp120 protein might bind to Gals or MAG, and directly damage the nerve cells, as has been suggested for cultured rat retinal ganglion neurons (Kaiser et al., 1990; Lipton 1991); or 4) immune reactivity directed at gp120 attached to Gals or MAG may secondarily damage the nerves. The ability of gp120 from different strains of HIV to bind to MAG or Gals might be an important determinant in the devel-
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opment of neuropathy, and similar interactions in the brain might have a role in the central nervous system manifestations of AIDS (Levy et al., 1988). Disruption of the interaction between gp120 and its ligands on neural cells might prevent the neurological disease.
REFERENCES Ariga T, Kohriyama T, Freddo L, Latov N, Saito M, Kon K, Ando S, Suzuki M, Hemling ME, Rinehart KL, Kusonki S Yu RK (1987) Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy. J Biol Chem 252:848-853. Clapham PR, Weber JN, Whitby D, McIntosh K, Dalgleish AG, Maddon PJ, Deen KC, Sweet RW, Weiss RA (1989) Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T-cells and monocytes but not for brain and muscle cells. Nature 337:368-370. Eiden LE, Lifson JD (1992) HIV interactions with CD4: a continuum of conformations and consequences. Immunol Today 13:201206. Freddo L, Ariga T, Saito M, Macala LC, Yu RK, Latov N (1985): The neuropathy of plasma cell dyscrasia: Binding of IgM Mproteins to peripheral nerve glycolipids. Neurology 35: 14201424. Freddo L, Sherman WH, Latov N (1986) Glycosaminoglycan antigens in peripheral nerve; studies with antibodies from a patient with neuropathy and monoclonal gammopathy . J Neuroimmunol 12: 57-64. Gattegno L, Ramdani A, Jouault T, Saffar L, Gluckman JC (1992) Lectin-carbohydrate interactions and infectivity of Human Immunodeficiency Virus Type 1 (HIV-I). AIDS Res Hum Retroviruses 8:27-37. Harouse JM, Kunsch C, Hartle HT, Laughlin MA, Hoxie JA, Wigdahl B, Gonzalez-Scarano F (1989) CD4 independent infection of human neural cells by human immunodeficiency virus type I. J Virol 63:2527-2533. Harouse JM, Bhat S, Spitalnik SL, Laughlin M, Stefan0 K, Silberberg DH, Gonzalez-Scarano F (1991) Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide. Science 253:320-323. Harper JR, Perry SK, Davis RM, Laufer DM (1990) Human neuroectoderm derived cell line secretes fibronectin that shares the HNK-IIIOCS carbohydrate epitope with neural cell adhesion molecules. J Neurochem 54:395-401. Hays PA, Lee SL, Latov N (1988) Immune reactive C3d on the surface of myelin sheaths in neuropathy. J Neuroimmunol 18: 231-244. Kaiser PK, Offermann JT, Lipton SA (1990) Neuronal injury due to HIV-1 envelope protein is blocked by anti-gpl20 antibodies but not by anti-CD4 antibodies. Neurology 40:1757-1761. Ilyas AA, Chou DKH, Jungalwala FB, Costello C, Quarles RH (1990) Variability in the structural requirement for binding of human monoclonal anti-myelin associated glycoprotein immunoglobulin M antibodies and HNK-I to sphingolipid antigens. J Neurochem 55594-601. Latov N, Hays AP, Sherman WH (1988) Peripheral neuropathy and anti-MAG antibodies. CRC Crit Rev Clin Neurobiol 3:301332. Lederman S, Bergmann JE, Cleary AM, Yellin MJ, Fusco PJ, Chess L (1992) Sulfated polyester interactions with the CD4 molecule and with the third variable loop domain (V3) of gp120 are chemically distinct. AIDS and Human Retroviruses, in press.
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Levy RM, Dredesen DE (1988) Central nervous system dysfunction in acquired immunodeficiency syndrome. In Rosenblum ML, Levy RM, Dresden DE (eds): “AIDS and the Nervous System.” New York: Raven Press, pp 29-64. Li XL, Moudgil T, Vinters HV, Ho DD (1990) CD4 independent, productive infection of a neuronal cell line by Human Immunodeficiency Virus type 1. J Virol 64:1383-1387. Lipton SA (1991) Calcium channel antagonists and human irnmunodeficiency virus coat protein mediated neuronal injury. Ann Neurol 30: 1 10-1 14. Mah V, Vatavarian LM, Akers MA, Vinters HV (1988) Abnormalities of peripheral nerve in patients with human immunodeficiency virus infection. Ann Neurol 24:713-717. Miller RG, Kiprov DD, Parry G , Bredessen DE (1988) Peripheral nervous system dysfunction in acquired immunodeficiency syndrome. In: Rosenblum ML, Levy RM, Bredesen DE (eds): “AIDS and the Nervous System.” New York: Raven Press, pp 65-78, McClure MO, Moore JP, Blanc DF, Scotting P, Cook GMW, Keynes RJ, Weber JN, Davies D, Weiss RA (1992) Investigations into the mechanisms by which sulfated polysaccharides inhibit HIV infection in vitro. AIDS Res 8:19-26. McGinnis S , Kohriyama T, Yu RK, Pesce MA, Latov N (1988) Antibodies to sulfated glucuronic acid containing glycosphingolipids in neuropathy associated with anti-MAG antibodies and in normal subjects. Neuroimmunology 17:119-126. Nobile-Orazio E, Hays AP, Perman G , Golier J, Shy M, Latov N (1984) Reactivity of mouse and human monoclonal anti-MAG antibodies; specificity and immunofluorescence studies. Neurology 34:1336-1342.
Quattrini A, Corbo M, Dodd J, Dhaliwal S, Sadiq SA, Lugaresi A, Oliveira A, Uncini A, Abouzhar K, Miller J , Lewis L, Estes D, Cardo L, Hays AP, Latov N (1992) Anti-sulfatide antibodies in neurological disease; binding to rat dorsal root ganglia neurons. J Neurol Sci, in press. Sadiq SA, Thomas FP, Kilidireas K, Protopsaltis S , Hays AP, Lee KW, Romas SN, Kumar N, Van den Berg L, Lange DJ, Younger D, Lovelace RE, Trojaborg W, Sherman WH, Miller JR, Minuk J , Fehr M, Hollander D, Nichols FT, Mitsumoto H, Kelly JJ Jr, Swift TR, Munsat TL, Latov N (1990) The spectrum of neurological disease associated with anti-GM1 antibodies. Neurology 40: 1067-1072. Schols D, Pauwels R, Desmyter J , De Clerco E (1990) Dextran sulfate and other polyanionic anti-HIV compounds specifically interact with the viral gp120 glycoprotein expressed by T-cells persistently infected with HIV-I. Virology 175556-561, Simpson DM (1992) Neuromuscular complications of Human Immunodeficiency Virus infection. Semin Neurol 12:34-42. Spagnol G, Williams M, Srinivasan J , Golier J, Bauer D, Lebo RV, Latov N (1989) Molecular cloning of human myelin associated glycoprotein. J Neurosci Res 24: 137-142. Tatano M, Gonzalez-Scarano F, Levy JA (1989) Human immunodeficiency virus can infect CD4 negative human fibroblastoid cells. Proc Natl Acad Sci USA 86:4287-4290. Werner A, Wnskowsky G , Cichutek K, Norley SG, Kurth R (1990) Productive infection of both CD4+ and CD4- human cell lines with HIV-I, HIV-2, and SIVagm. Aids 4:537-544. Williams AF, Davis SJ, He Q, Barclay AN. (1989) Structural diversity of the immunoglobulin family. Cold Spring Harbor Symp Quant Biol 54:637-647.
The gp120 Glycoprotein of HIV-1 Binds to Sulfatide and to the Myelin Associated Glycoprotein L.H. van den Berg, S.A. Sadiq, S. Lederman, and N. Latov Departments of Neurology (L.H.B., S.A.S., N.L.) and Medicine (Division of Rheumatology; S.L.), Columbia University, New York
We investigated the binding of the gp120 glycoprotein of the human immunodeficiency virus (HIV-1) to neural glycolipids and glycoproteins by ELISA. The gp120 protein bound to sulfatide (Gals), a sulfated glycolipid autoantigen implicated in sensory neuritis, and to the myelin associated glycoprotein (MAG), an autoantigen in demyelinating neuropathy. Binding of gp120 to MAG was inhibited by the HNK-1 antibody, which recognizes a sulfated glucuronic acid epitope, suggesting that the interaction involves carbohydrate determinants. Sulfatide and MAG are potential receptors for gp120 in peripheral nerve and may have a role in the neuropathy associated with HIV-1 infection. 0 1992 Wiley-Liss, Inc. Key words: ELISA, sulfatide, myelin associated glycoprotein, autoantigen, neuropathy, HIV-1, gp120 glycoprotein INTRODUCTION
and they might serve as receptors for gp120 in peripheral nerve.
MATERIALS AND METHODS Antibodies and Ligands Recombinant gp 120 and mouse monoclonal IgG anti-gp120 antibodies were obtained from American Biotechnologies, Inc. (Cambridge, MA). Mouse monoclonal IgG anti-heparan sulfate proteoglycan (HpS) antibodies and rabbit antigalactocerebroside (GalC) antibodies were obtained from Chemicon International Inc. (Temecula, CA). Mouse monoclonal IgG anti-chondroitin sulfate (ChS) antibodies and peroxidase-conjugated affinity-purified antibodies to mouse IgG, rabbit immunoglobulins, mouse IgM, or human IgM were obtained from Sigma Chemical Co. (St. Louis, MO). The mouse monoclonal IgM HNK- 1 antibody which binds to the myelin associated glycoprotein (MAG) and to the glycolipid 3-sulfated glucuronyl paragloboside (SGPG; Nobile-Orazio et al., 1984; Freddo et al., 1985), the mouse monoclonal IgG anti-MAG antibody GENS-3, and the mouse monoclonal IgG anti-CD4 antibody OKT4 were produced from hybridomas and were purified from ascites as previously described (Nobile-Orazio et al., 1984; Lederman et al., 1992). Human monoclonal IgM antibodies to GMl ganglioside or to Gals were obtained from patients with neurological diseases (Sadiq et al., 1990; Quattrini et al., 1992). Human myelin associated glycoprotein (MAG) and the glycolipid sulfonyl
The human immunodeficiency virus (HIV) has been reported to infect both neural and non-neural cell lines through CD4 independent interactions, but the mechanisms involved are poorly understood (Clapham et al., 1989; Harouse et al., 1989; Li et al., 1990; Tatano et al., 1989; Werner et al., 1990). The possibility that cell surface carbohydrates might serve as receptors for HIV on neural cells was suggested by the observation that the HIV-1 coat protein gp120 binds to sulfated polysaccharides such as dextran sulfate (DxS) and pentosan polysulfate (PPS, Schols et al., 1990; Lederman et al., 1992), and because viral infection is inhibited by antibodies to the carbohydrate determinants of galactocerebroside Received June 19, 1992; revised and accepted July 23, 1992 (GalC) and its sulfated derivative sulfatide (Gals, Ha- Address reprint requests to Norman Latov, M.D., Ph.D., Department Neurology, Columbia University, Black Bldg Rm 3-323, 650 W. rouse et al., 1991). In this study we report that gp120 of 168th Street, New York, NY 10032. binds to Gals with higher avidity than to other glycolipwork was supported in part by NIH grant NS25187 to N.L. and ids and that gp 120 also binds to the sulfated oligosac- This by a grant from the Ahron Diamond Foundation to the Department of charide containing region of the myelin associated gly- Neurology. L.H. van den Berg is a Visiting Fellow from the Univercoprotein (MAG). Both Gals and MAG are putative sity Hospital of Utrecht, the Netherlands, and is the recepient of a antigens for antibody mediated peripheral neuropathy , scholarship from the Dutch Organization for Scientific Research. 0 1992 Wiley-Liss, Inc.
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glucuronyl paragloboside (SGPG) were purified as previously described (Nobile-Orazio et al., 1984; McGinnis et al., 1988). Recombinant CD4 was a gift of Dr. Vicki Sat0 (Biogen Corp, Cambridge, MA). The following were purchased from Sigma Chemical Co. (St. Louis, Moj: sulfatide (Gals), galactocerebroside (GalC), GM 1 ganglioside, chondroitin sulfate (ChS) , heparan sulfate (HpS), pentosan polysulfate (PPS), dextran sulfate MW8000 (DxS), and dextran MW-10,000 (Dx).
ELISA Studies Binding of gp120 to the glycoproteins MAG and CD4, the proteoglycans ChS and HpS, and to the glycolipids Gals, GalC, SGPG, and GM1 was measured by ELISA by modifications of the procedures of Nobile Orazio et al. (1984) for glycoproteins, Freddo et al. (1986) for proteoglycans, and Sadiq et al. (1990) for glycolipids. Briefly, microwells were coated with 5 pg/ ml of the glycoproteins or proteoglycans in PBS (0.14 N NaCl, 0.01 PO,, pH 7.4) overnight, or with the glycolipids in methanol, which was allowed to evaporate. The wells were then saturated with BSA, and increasing concentrations of gp120 in ELISA solution (PBS with 1% BSA and 0.025% Tween-20) were added for 2 hr at room temperature. Wells were washed 6 x with ELISA solution between subsequent steps. The gp120 protein which bound to the ligands in the microwells was measured using mouse monoclonal antibodies to gp120 (1 :2,000), followed by peroxidase-conjugated, affinity-purified goat antibodies to mouse IgG (1:200). All determinations were done in triplicate and control wells were coated with BSA, or gp120 was omitted from ligand coated wells. Readings in the BSA coated wells were subtracted from the corresponding ligand coated wells. Adherence of the ligands to the microwells was confirmed with antibodies to the specific ligands by ELISA. The secondary antibodies used with each ligand were as follows: for GM1, human IgM anti-GM1 antibodies at 1:10,000, followed by peroxidase-conjugated anti-human IgM at 1:200; for Gals, human IgM antiGals antibodies at 1:10,000, followed by peroxidaseconjugated anti-human IgM at 1:200; for SGPG and MAG, the HNK-1 antibody at 1 pg/ml, followed by peroxidase-conjugated anti-mouse IgM at 1:200; for GalC, rabbit anti-GalC antibodies at 1:200, followed by peroxidase-conjugated anti-rabbit antibodies at 1:200; for CD4, the OKT4 antibody at 4 p,g/ml, followed by peroxidase-conjugated anti-mouse IgG; for ChS , mouse anti-ChS antibodies at 1:200, followed by peroxidaseconjugated anti-mouse IgG at 1:200; and for HpS, mouse anti-HpS antibodies at 1:200, followed by peroxidaseconjugated anti-mouse IgG at 1:200. In parallel studies, microwells were coated with gp120 at 5 pg/ml in PBS, and increasing concentrations
of MAG, CD4, ChS, or HpS were added to the microwells and were incubated for 4 hr. Binding of MAG to gp120 was detected with the HNK-1 antibody (1 Kgiml for 1 hr), followed by peroxidase-conjugated antibodies to mouse IgM (1:200) for I hr. Binding of CD4, ChS, and HpS were detected using mouse monoclonal antibodies specific for each ligand (4 pg/ml of OKT4, and 1:200 dilution for anti-ChS or HpS antibodies), followed by peroxidase-conjugated antibodies to mouse IgG (1 : 200). Binding of the glycolipids to immobilized gp 120 in the ELISA system could not be examined because of nonspecific adherence of the glycolipids liposomes to the surface of the microwells.
Binding Inhibition Studies In the binding inhibition studies, increasing concentrations of dextran sulfate (DxS) , pentosan sulfate (PPS), dextran (Dx), HpS, or ChS were added to microwells coated with CD4 or MAG to determine whether these inhibited the binding of soluble gpl20 to CD4 or MAG. The inhibitory agents were added to the microwells immediately preceding the addition of gp120 and were present throughout the incubation period. To determine whether gp120 bound to specific epitopes in MAG, increasing concentrations of the mouse monoclonal antibodies HNK- 1 or GEN-S3, which bind to different epitopes of MAG, were tested for their ability to inhibit the interaction of soluble gp120 with immobilized MAG by ELISA. Western Blot Studies To confirm the specificity of the interaction between gp120 and MAG in the ELISA system, MAG (10 pg/ml) was added to microwells coated with gp120 or with control wells coated with BSA, and after 2 hr the microwells were washed and the adherent proteins were solubilized in PBS with 1% SDS. The solubilized proteins were then separated by SDS-PAGE, transferred to nitrocellulose strips, and immunostained with the mouse monoclonal anti-MAG antibody HNK- 1 or GENS3 (Nobile-Orazio, 1984).
RESULTS In the ELISA system, soluble gp120 bound to Gals immobilized in microwells more avidly than to the other glycolipids tested, including to SGPG, GalC, or GMl (Fig. 1). Binding to Gals was detected at gp120 concentrations of less than 0.5 pg/ml, and 50% of maximal binding was observed at 1 pg/ml of gp120. Binding was specifically inhibited by dextran sulfate (DxS) and pentosan sulfate (PPS) in a concentration-dependent manner. Maximal inhibition was achieved at 1 pM concentrations of the inhibitory agents (Fig. 2), and no
HIV-1 gp120 Binds to Gals and MAG
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Fig. 2. Inhibition of soluble gp120 binding to Gals by DxS or PPS. Increasing concentrations of DxS or PPS were added with gp120 to Gals coated microwells. The gp120 binding was determined by ELISA. Maximal inhibition was seen at 1 p M concentration of the inhibitors. No inhibition was seen with Dx, ChS, or HpS.
inhibition was observed with dextran (Dx), chondroitin sulfate (ChS), or heparan sulfate (HpS) at concentrations of up to 10 pM. DxS and PPS did not inhibit the binding of anti-Gals antibodies to GalS, indicating that they inhibited the interaction of gp120 with GalS. Soluble gp120 also bound to the sulfated glycoprotein MAG immobilized in microwells at concentrations
of less than 1 pg/ml (Fig. 3 ) . Immobilized CD4, which is known to bind to gp120, was used as a positive control for gp120 binding (Eiden and Lifson, 1992). As negative controls, gp120 did not bind to immobilized ChS or HpS, although both ligands were demonstrated to be immobilized in the microwells using ligand-specific antibodies. Binding of gp120 to MAG and CD4 was also inhibited by DxS and PPS, but not by Dx, ChS, or HpS (not shown). DxS and PPS did not inhibit the binding of anti-CD4 or HNK-1 antibodies to their respective ligands. In order to determine whether gp120 binds to the MAG oligosaccharide, the HNK- 1 antibody which recognizes a sulfated glucuronic acid determinant in MAG was tested for its ability to inhibit the interaction. As shown in Fig. 4, binding of gp120 to immobilized MAG was inhibited by the HNK-1 antibody, but not by GENS3, a mouse monoclonal anti-MAG antibody which recognizes an unrelated peptide epitope. The HNK-1 antibody inhibited the interaction of gp120 with MAG in a concentration dependent manner, with maximal inhibition observed at antibody concentrations of 50 pg/ml. As control, HNK-I did not inhibit gp120 binding to CD4. To determine whether soluble MAG also binds to gp120, increasing concentrations of soluble MAG were added to microwells coated with gp120, and the binding was determined by ELISA. As shown in Fig. 5 , both soluble MAG and CD4, which is known to react with gp120, bound to the immobilized gp120. There was no
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van den Berg et al. HNK- 1 80
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Antibody Concentration (pglrnl) Fig. 4. Inhibition of gp120 binding to MAG by HNK-I . Microwells were coated with MAG, and increasing concentrations of the HNK-I anti-MAG antibody were added with gp120. Binding of gp120 to MAG was determined by ELISA. The HNK-I antibody completely inhibited the gp120 binding at a concentration of 50 pg/ml. No inhibition was seen with the mouse monoclonal antibody GEN-S3, which is directed at a different epitope of MAG. 2.000 h
1.600
Fig. 6. Identification of the ligand for gp120 as MAG by Western blot analysis. MAG was added to microwells coated with gp120 or BSA, and after washing the adherent proteins in the wells were solubilized and characterized by Western blot. In A, the eluent from gp120 coated microwells was identified as MAG by its reactivity with monoclonal HNK-1 anti-MAG antibodies and its mobility on SDS-PAGE. In B, no MAG was detected in the BSA coated microwells.
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with the anti-MAG antibodies on nitrocellulose blots and by its characteristic mobility as 2 closely migrating bands of approximately 100 kD (Fig. 6). As a control, MAG could not be detected in the eluent from the BSA coated microwells.
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Concentration (pg/ml) Fig. 5 . Binding of immobilized gp120 to soluble MAG and CD4. Increasing concentrations of MAG and CD4 were tested for binding to microwells coated with gp120.
binding to gp120 by ChS or HpS (not shown). The specificity of the interaction between MAG and gp120 in the ELISA system was confirmed by Western blot analysis. MAG was added to microwells coated with gp120 or BSA, and after washing the proteins which bound to the microwells were eluted and analyzed on Western blots. Following elution from the gpl20 coated microwells, the bound protein was identified as MAG by its reactivity
DISCUSSION In this study, gp120 reacted with the glycoconjugates and MAG, both of which have sulfated carbohydrate determinants. Gals contains a 3-sulfated galactosyl determinant, and MAG contains the HNK-1 epitope which is shared by the 3-sulfated glucuronic acid bearing glycolipid SGPG (Ariga et al., 1987; Ilyas et al., 1990). The binding of these glycoconjugates to gp120 was inhibited by the sulfated polysaccharides DxS and PPS, which inhibit viral binding and infectivity (Schols et al., 1990; McClure et al., 1992; Lederman et al., 1992), but the interactions was not due to nonspecific binding to sulfated carbohydrates, as there was no binding to HpS or ChS. Interactions between gp120 and spe-
HIV-1 gp120 Binds to Gals and MAG
cific carbohydrate determinants on cell surfaces may be important for viral infectivity, as antibodies to carbohydrate determinants of GalC and Gals inhibit HIV-1 infection of CD4- neural cell lines (Harouse et al., 1991), and as lectins that bind to carbohydrate determinants on the surface of CD4 cells inhibit HIV- 1 infection independently of CD4 (Gattegno et al., 1992). MAG is a neural adhesion molecule, and like CD4 is a member of the immunoglobulin gene superfamily (Spagnol et a]., 1989; Williams et al., 1989). It bears the HNK- I carbohydrate epitope, a 3-sulfated glucuronicacid-containing antigenic determinant, which is shared by several other adhesion molecules in the nervous system (Harper et al., 1990). The interaction of gp120 with MAG is inhibited by the HNK-1 antibody but not by the monoclonal antibody GEN-S3, which recognizes an unrelated peptide epitope in MAG, suggesting that the HNK-1 carbohydrate determinant is involved in the interaction with gp120. However, other regions of MAG and conformational determinants may also have a role in the interaction, as gp120 does not bind to MAG denatured by SDS on Western blots (not shown) and gp120 does not bind well to SGPG, which also bears the HNK1 epitope. The observation that HNK-1 binds to MAG in microwells coated with gp120 at low antibody concentrations, whereas it inhibits MAG from binding to gp120 at the higher antibody concentrations of greater than 10 pg/ml, suggests that MAG bears multiple HNK-1 determinants or that MAG forms multimeric complexes in solution. HIV-1 infection may be associated with an acute or chronic demyelinating neuropathy or a painful sensory axonal neuropathy (Miller et al., 1988; Simpson, 1992). Pathological studies show loss of both myelin and axons, and inflammatory cells or complement deposits may be present (Mah et al., 1988; Hays et al., 1988). Since MAG is an autoantigen for monoclonal IgM antibodies in demyelinating neuropathy (Latov et al., 1988), and Gals is a putative autoantigen in sensory ganglioneuritis (Quattrini et al., 1992), they might also serve as receptors for gp 120 or HIV- 1 in peripheral nerve. If MAG and Gals have a role in HIV-1 associated neuropathies, they could be involved by several different mechanisms: 1) HIV-1 could bind to Gals or MAG to produce a low grade infection of neurons or Schwann cells; 2) infected macrophages expressing gp120 might attach to Gals or MAG, and damage the nerve cells; 3) The gp120 protein might bind to Gals or MAG, and directly damage the nerve cells, as has been suggested for cultured rat retinal ganglion neurons (Kaiser et al., 1990; Lipton 1991); or 4) immune reactivity directed at gp120 attached to Gals or MAG may secondarily damage the nerves. The ability of gp120 from different strains of HIV to bind to MAG or Gals might be an important determinant in the devel-
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opment of neuropathy, and similar interactions in the brain might have a role in the central nervous system manifestations of AIDS (Levy et al., 1988). Disruption of the interaction between gp120 and its ligands on neural cells might prevent the neurological disease.
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Levy RM, Dredesen DE (1988) Central nervous system dysfunction in acquired immunodeficiency syndrome. In Rosenblum ML, Levy RM, Dresden DE (eds): “AIDS and the Nervous System.” New York: Raven Press, pp 29-64. Li XL, Moudgil T, Vinters HV, Ho DD (1990) CD4 independent, productive infection of a neuronal cell line by Human Immunodeficiency Virus type 1. J Virol 64:1383-1387. Lipton SA (1991) Calcium channel antagonists and human irnmunodeficiency virus coat protein mediated neuronal injury. Ann Neurol 30: 1 10-1 14. Mah V, Vatavarian LM, Akers MA, Vinters HV (1988) Abnormalities of peripheral nerve in patients with human immunodeficiency virus infection. Ann Neurol 24:713-717. Miller RG, Kiprov DD, Parry G , Bredessen DE (1988) Peripheral nervous system dysfunction in acquired immunodeficiency syndrome. In: Rosenblum ML, Levy RM, Bredesen DE (eds): “AIDS and the Nervous System.” New York: Raven Press, pp 65-78, McClure MO, Moore JP, Blanc DF, Scotting P, Cook GMW, Keynes RJ, Weber JN, Davies D, Weiss RA (1992) Investigations into the mechanisms by which sulfated polysaccharides inhibit HIV infection in vitro. AIDS Res 8:19-26. McGinnis S , Kohriyama T, Yu RK, Pesce MA, Latov N (1988) Antibodies to sulfated glucuronic acid containing glycosphingolipids in neuropathy associated with anti-MAG antibodies and in normal subjects. Neuroimmunology 17:119-126. Nobile-Orazio E, Hays AP, Perman G , Golier J, Shy M, Latov N (1984) Reactivity of mouse and human monoclonal anti-MAG antibodies; specificity and immunofluorescence studies. Neurology 34:1336-1342.
Quattrini A, Corbo M, Dodd J, Dhaliwal S, Sadiq SA, Lugaresi A, Oliveira A, Uncini A, Abouzhar K, Miller J , Lewis L, Estes D, Cardo L, Hays AP, Latov N (1992) Anti-sulfatide antibodies in neurological disease; binding to rat dorsal root ganglia neurons. J Neurol Sci, in press. Sadiq SA, Thomas FP, Kilidireas K, Protopsaltis S , Hays AP, Lee KW, Romas SN, Kumar N, Van den Berg L, Lange DJ, Younger D, Lovelace RE, Trojaborg W, Sherman WH, Miller JR, Minuk J , Fehr M, Hollander D, Nichols FT, Mitsumoto H, Kelly JJ Jr, Swift TR, Munsat TL, Latov N (1990) The spectrum of neurological disease associated with anti-GM1 antibodies. Neurology 40: 1067-1072. Schols D, Pauwels R, Desmyter J , De Clerco E (1990) Dextran sulfate and other polyanionic anti-HIV compounds specifically interact with the viral gp120 glycoprotein expressed by T-cells persistently infected with HIV-I. Virology 175556-561, Simpson DM (1992) Neuromuscular complications of Human Immunodeficiency Virus infection. Semin Neurol 12:34-42. Spagnol G, Williams M, Srinivasan J , Golier J, Bauer D, Lebo RV, Latov N (1989) Molecular cloning of human myelin associated glycoprotein. J Neurosci Res 24: 137-142. Tatano M, Gonzalez-Scarano F, Levy JA (1989) Human immunodeficiency virus can infect CD4 negative human fibroblastoid cells. Proc Natl Acad Sci USA 86:4287-4290. Werner A, Wnskowsky G , Cichutek K, Norley SG, Kurth R (1990) Productive infection of both CD4+ and CD4- human cell lines with HIV-I, HIV-2, and SIVagm. Aids 4:537-544. Williams AF, Davis SJ, He Q, Barclay AN. (1989) Structural diversity of the immunoglobulin family. Cold Spring Harbor Symp Quant Biol 54:637-647.
