Multivalent protein-carbohydrate interactions. A new paradigm for supermolecular assembly and signal transduction

Many biological recognition processes involve the binding and clustering of ligand-receptor complexes and concomitant signal transduction events. Such interactions have recently been observed in human T cells in which binding and cross-linking of specific glycoprotein counter-receptors on the surface of the cells by an endogenous bivalent carbohydrate binding protein (galectin-1) leads to apoptosis [Pace, K. E., et al. (1999) J. Immunol. 163, 3801-3811]. Importantly, different counter-receptors associated with specific phosphatase or kinase activities were shown to form separate clusters on the surface of the cells as a result of galectin-1 binding to the carbohydrate moieties of the respective glycoproteins. This suggests that the unique separation and organization of signaling molecules that results from galectin-1 binding is involved in delivering the signal to die. The ability of galectin-1 to induce the separation of specific glycoprotein receptors was modeled on the basis of molecular and structural studies of the binding of multivalent carbohydrates to lectins that result in the formation of specific two- and three-dimensional cross-linked lattices. These latter studies have been recently highlighted by X-ray crystallographic results showing that a single tetravalent lectin forms distinct cross-linked complexes with four different bivalent oligosaccharides [Olsen, L. R., et al. (1997) Biochemistry 36, 15073-15080]. In this report, binding and cross-linking of multivalent carbohydrates with multivalent lectins is shown to be a new paradigm for supermolecular assembly and signal transduction in biological systems.     

Distribution of terminal sugar residues in the testis of the spotted ray Torpedo marmorata

Lectins represent a class of proteins/glycoproteins binding specifically to terminal sugar residues. The present investigation aims to identify lectin-binding sites in testis of Torpedo marmorata. Using a panel of lectins coupled with fluoresceine isothiocyanate, we demonstrated that germ and somatic cells present in Torpedo testis contain glycoconjugates, whose distribution at the level of the surface, the cytoplasm and the nucleus changes during germ cell differentiation. Moreover our observations demonstrate that the germ cells undergoing apoptosis (Prisco et al., 2003a: Mol Reprod Dev 64:341-348) overexpress a residual sugar recognised by WFA lectin that can be considered a specific marker for apoptotic germ cells. Finally, our results indicate that there is a progressive increase in glycosilation during spermatogenesis, especially at the level of the acrosome in the spermatocyte-spermatid step, and that Leydig cells are differently stained in relation to the spermatogenetic cycle.     

Macrophage C-type lectin on bone marrow-derived immature dendritic cells is involved in the internalization of glycosylated antigens

Bone marrow-derived dendritic cells (DCs) were examined for the expression of the murine macrophage C-type lectin specific for galactose and N-acetylgalactosamine (mMGL). Flow cytometric analysis after double staining for MHC class II and mMGL with specific monoclonal antibodies indicated that mMGL was expressed on immature DCs with low to moderate levels of MHC class II and down-regulated during maturation. Immature DCs bound and internalized alpha-N-acetylgalactosaminides conjugated to soluble polyacrylamide (alpha-GalNAc polymers), whereas mature DCs and bone marrow cells did not. The two-color flow cytometric profiles indicated that the degree of alpha-GalNAc polymer bindings exactly coincided with the intensity of the binding of a mMGL-specific monoclonal antibody LOM-14. The internalized alpha-GalNAc polymers seemed to be transported to MHC class II compartments. Thus, mMGL is transiently expressed on bone marrow-derived DCs during their development and maturation and suggested to be involved in the uptake of glycosylated antigens for presentation.     

Differential binding of lectins IL-2 and CSL to candida albicans and cancer cells

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2- dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).      

Specific glycan elements determine differential binding of individual egg glycoproteins of the human parasite Schistosoma mansoni by host C-type lectin receptors

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite’s eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/α1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/α1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/α1 is bound via the fucosylated Galβ1-4(Fucα1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells via all three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors. Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcβ1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcβ1-4(Fucα1-3)GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcβ1-4(Fucα1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcβ1-4(Fucα1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response.     

Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic acidalpha2-3/6galactose-, fucosealpha1-2galactose- and galactosealpha1-3galactose- were not detected. Terminal alpha-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies.     

Involvement of the endogenous lectin CSL in adhesion of Chinese hamster ovary cells.

Immunochemical localization of an endogenous mannose-binding protein, the cerebellar soluble lectin (CSL; Zanetta et al., J. Neurochem. 49, 1250-1257 (1987)), in Chinese hamster ovary cells indicated its high concentration in areas of contact between cells. This suggested its role in cell adhesion. The pattern of staining differed significantly in the cells cultured in suspension from that grown as monolayer. In cells maintained for a short time as suspension, the extracellular CSL immunoreactivity was found mainly in close apposition to the plasma membrane including contact areas. In cells cultured as monolayer, extracellularly, the lectin was found both at the cell surface and in a 75-nm thick layer between two cells, apparently adhering to the cell surface through bridges. Endogenous glycoprotein ligands of CSL were present in the cultures of CHO cells, both as membrane-bound glycoproteins and as glycoprotein ligands soluble in the presence of mannose in the absence of detergent. The lectin CSL induced adhesion between these cells as evident by low concentration of anti-CSL Fab fragments inhibiting such adhesion. These data suggested that adhesion between CHO cells occurs, in part, through a glycobiological recognition system involving CSL. This mechanism should be taken into account for the interpretation of experiments of transfection in CHO cells of the genes of glycoproteins involved in cell adhesion.     

Identification of functional receptors for vasoactive intestinal peptide and neurotensin in the human submandibular gland duct cell line, HSG-PA.

The HSG-PA human submandibular gland adenocarcinoma cell line has attracted attention recently as a potentially useful cell culture model for studies of salivary duct cell function and regulation. These cells possess a variety of morphological and biochemical markers found in salivary duct cells. Recently, muscarinic cholinergic receptors coupled to inositol intracellular Ca2+ mobilization (He et al., Eur. J. Physiol., 413 (1989) 505-510) and K+ fluxes (Ship et al., Am. J. Physiol., 259 (1990) C340-C348) have been identified in HSG-PA cells. In this study, we report the presence in these cells of functional receptors for two neuropeptides, vasoactive intestinal peptide (VIP) and neurotensin. Receptors for both peptides were labeled in intact cell radioligand binding studies and exhibited pharmacological profiles similar to receptors found in other tissues. There was close agreement between binding Ki values and the ED50 values for stimulation of second messenger production and modulation of K+ efflux, with all values between 1 and 5 nM. Whereas neurotensin stimulated K+ efflux dramatically, VIP alone had no effect but enhanced the response to neurotensin. These studies thus represent the initial documentation of functional receptors for VIP and neurotensin in a cell line of salivary duct cell origin.     

FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic fibroblasts, Yeh et al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryonic fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or FADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.     

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.     

Role of chimeric murine leukemia virus env beta-turn polyproline spacers in receptor cooperation

We have previously reported a set of Moloney murine leukemia virus derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insertion of the Pit-2 binding domain (BD) at the N terminus of the ecotropic retroviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2059-2064, 1996). The resulting chimeric envelopes share two BDs: an additional N-terminal BD (Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 BD). By inserting a variety of different amino acid spacers between the two binding domains, we showed that retroviruses can potentially use the targeted cell surface receptor Pit-2, the ecotropic retroviral receptor mCAT-1, or both receptors cooperatively for entry into target cell (S. Valsesia-Wittmann et al., EMBO J 6:1214-1223, 1997). An extreme example of receptor cooperativity was encountered when envelopes with specific proline-rich interdomain spacers (PRO spacers) were tested: both receptors had to be coexpressed at the surface of the targeted cells to cooperatively allow infection. Here, we characterized the role of PRO spacer in the cooperation of receptors. We have shown that the particular organization of the PRO spacer-a beta-turn polyproline-was responsible for the cooperative effect. In the native configuration of the viruses, the structure masked the regions located downstream of the PRO spacer, thus the mCAT-1 BD. After interaction with the targeted Pit-2 receptor, the BD of the backbone envelope became accessible, and we demonstrated that interaction between the mCAT-1 BD and the mCAT-1 receptor is absolutely necessary. This interaction leads to natural fusion triggering and entry of viruses into targeted cells.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

Expression of endogenous receptors for neoglycoproteins, especially lectins, that allow fiber typing on formaldehyde-fixed, paraffin-embedded muscle biopsy specimens. A glycohistochemical, immunohistochemical, and glycobiochemical study

A panel of biotinylated (neo)glycoproteins was used for specific detection of endogenous sugar receptors, especially lectins, in formaldehyde-fixed, paraffin-embedded muscle biopsy specimens from human deltoid, quadriceps, and biceps muscles, tibial and quadriceps muscles of rat, and bovine masseter muscle. The glycohistochemical probes used consisted of conjugates of a labeled, histochemically inert carrier protein and various covalently linked, histochemically crucial sugar moieties. Specific binding of alpha-L-fucoside, beta-D-galactoside, beta-D-xyloside, and alpha-D-mannoside to muscle sections was detected, showing no species-specific differences. The presence of receptors for the N-acetylated sugars in natural glycoconjugates, and for sugars with a phosphate group, i.e., mannose-6-phosphate and galactose-6-phosphate, was demonstrated glycohistochemically. However, these binding specificities revealed species-specific differences, e.g., the absence of N-acetyl-D-galactosamine-specific receptors or galactose-6-phosphate-specific receptors in rat muscle. Other charged sugars included glucuronic acid and sialic acid, which bound only to ox and rat muscle or failed to reveal their respective receptors in all types of muscle investigated. This different extent of staining with anionic probes served as a further control to ascertain carbohydrate binding specificity. Positive glycohistochemical reaction developed within sarcomeres only at the level of A-bands. Granular staining was observed in the sarcoplasm among the myofibrils and also in the subsarcolemmal regions. Differences in expression of glycohistochemically detectable sugar receptors were noted between type 1, type 2A, and type 2B fibers. The molecular properties of one type of glycohistochemically detectable sugar receptor were inferred both immunohistochemically and biochemically. An antiserum against an endogenous beta-galactoside-specific lectin from muscle tissue localized this lectin within sections consistently similar to (neo)glycoproteins, detecting beta-galactoside-specific receptor(s). This similarity of binding patterns strongly supports the assumption that (neo)glycoproteins with beta-galactoside termini indeed bind to the respective endogenous lectin. The lectin-specific antiserum enabled us to ascertain that glycohistochemical fiber typing corresponds to enzyme histochemical typing. Moreover, biochemical purification using affinity chromatography and subsequent affinity elution revealed only the immunohistochemically detectable beta-galactoside-specific lectin. Consequently, use of a panel of neoglycoproteins, when frozen sections for histochemical analysis are not available, co     

Interaction between ATBP and DmUbc9 in the expression of the Sarcophaga lectin gene

We previously demonstrated that (A+T)-stretch binding protein (ATBP) and Dorsal-related immunity factor (Dif) are required for the expression of the Sarcophaga lectin gene in SL-2 cells (Aozasa et al., Eur. J. Biochem. 268, 2506-2511, 2001). The present study demonstrates that DmUbc9 interacts with ATBP, and cotransfection of the DmUbc9 vector with ATBP and Dif vectors greatly enhances the expression of the luciferase reporter of the Sarcophaga lectin gene in SL-2 cells. These results suggest that sumoylation of ATBP is involved in the expression of the Sarcophaga lectin gene in this system.     

Structural/functional relationships between internal and external MSH receptors: modulation of expression in Cloudman melanoma cells by UVB radiation

Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142:129-136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bolognia et al: J. Invest. Derm., 92:651-656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.     

Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling

Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan-binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand.     

FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts

BC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF-b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al., J. Cell Biol., 103:1799-1805, 1986; Lathrop et al., J. Cell Biol., 100:1540-1547, 1985, and J. Cell Biol., 101:2194-2198, 1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.     

Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73-77, 2001; Colebunders and Borchert, J. Infect. 40:16-20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148-S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643-1645, 2005; Schornberg et al., J. Virol. 80:4174-4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163-175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae.