Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis

Subspecies of T. brucei (T. brucei gambiense and T. brucei rhodesiense) are the causative agents of human African trypanosomiasis (HAT) in sub-Saharan Africa (1). These parasites are transmitted to mammalian hosts through the bites of tsetse flies. They reside and multiply extracellularly in blood and in tissues, continually encountering the immune responses of the host. Remarkably, the parasites can establish a chronic infection for years due to a range of immunomodulatory mechanisms (2).

The surface proteome of trypanosome parasites serves a critical function at the host–parasite interface and is essential for immune evasion (3, 4). In bloodstream forms of the parasites, most of the surface area (>90%) is covered by the GPI-anchored variable surface glycoproteins (VSGs) (5). The VSGs undergo antigenic variation, which involves chromosomal recombination from a reservoir of more than 1,000 genomic VSG genes combined with expression site activation and silencing (6, 7). Together with rapid endocytosis of surface proteins, this enables the parasites to evade attacks by the circulating Abs of the host (8). To complement these defense mechanisms, trypanosomes can reduce the host’s Ab production capacity by exerting detrimental effects on the B-cell compartment (9, 10).

Embedded within the VSG layer is a diverse array of surface proteins that play crucial roles in nutrient acquisition and signal transduction. These surface proteins, likely originating from a common ancestor, exhibit a similar structural composition to the VSGs (11–14). A subset of trypanosome surface proteins known as invariant surface glycoproteins (ISGs) share similar characteristics (15–18). The ISGs are expressed from multigene arrays but defined as invariant, meaning that their expression remains constant regardless of alterations in VSG expression (19). The ISGs are type 1 transmembrane proteins, and their trafficking relies on ubiquitylation of their cytoplasmic tails (20, 21). In addition, the ISGs are mainly expressed in the forms of the parasites that live in the bloodstream, where they encounter the immune system of the host. Although expression levels of ISGs are much lower than the VSGs, the ISGs represent the second most abundant group of trypanosome surface proteins. Furthermore, ISGs are expressed over the entire surface of the parasite and not restricted to the flagellar pocket as observed for receptors involved in nutrient acquisition (22).

The invariant surface glycoproteins ISG65 and ISG75 were identified in the bloodstream form of T. brucei with estimated levels of around 70,000 molecules/cell and 50,000 molecules/cell, respectively (15, 16). Such high levels of surface-exposed proteins suggest that ISG65 and ISG75 serve important functions for the parasites. Indeed, ISG65 was recently discovered to play a role in immune evasion by interfering with the alternative pathway of the complement system. ISG65 binds C3b deposited on the surface of the parasite and promotes its conversion to inactive C3b (iC3b), preventing the complement cascade from progressing into the terminal lytic phase (23–26).

ISG75 is a 58-kDa surface protein consisting of a large ectodomain (residues 29–468), a transmembrane helix (residues 469–489), and a short cytoplasmic tail (residues 490–523) (19). The ISG75 ectodomain has a single predicted N-linked glycosylation site (Asn¹³⁴) and a coiled-coil motif (residues 280–450) (21, 27). Expression of ISG75 appears to be specific for the Trypanozoon subgenus, and genes encoding ISG75 have been identified in subspecies of T. brucei, Trypanosoma evansi, and Trypanosoma equiperdum (27). No homolog of ISG75 has been identified in any other species. Although embedded in the VSG layer, ISG75 is accessible for Ab recognition and is a promising marker for HAT Ab-based diagnostics (28, 29). Due to its invariant nature and high expression levels, ISG75 has also been considered as a target for vaccine development (22). However, although ISG75 is highly immunogenic, the induced Ab responses are nonprotective and have no impact on parasitemia.

In the genome of T. brucei (Lister strain 427), five contiguous ISG75 open reading frame genes are located on one strand (Tb427-7700, Tb427-7800, Tb427-7900, Tb427-8000, and Tb427-8100) and a single ISG75 open reading frame gene on the opposite strand (Tb427-8200) (30). Among these, Tb427-7700 stands out as the most divergent ISG75 sequence of T. brucei, displaying a sequence identity of 64–66% with respect to the other ISG75 sequences (Supplemental Fig. 1). In contrast, the remaining five sequences exhibit a high level of conservation, with over 90% sequence identity to each other.

The physiological function of ISG75 is unknown. However, ISG75 was identified in a genome-scale RNA interference screen as a critical factor for the activity of the anti-trypanosome drug suramin (31), and ISG75 was suggested to be responsible for suramin uptake via receptor-mediated endocytosis. Together with another chemotherapeutic drug, pentamidine, suramin is used to treat the first stage of HAT (32). Suramin is a sulfated napthylamine compound with a strong negative charge, making it unable to cross lipid membranes by diffusion. A recent study reported a direct interaction between a recombinant fragment of ISG75 and suramin with a dissociation constant (K_D) of 5.8 µM (33). However, knockdown of ISG75 expression does not appear to induce suramin resistance, indicating that other surface proteins contribute to suramin uptake, possibly the VSGsur (34).

In this article, we have analyzed the structural properties of ISG75 using both x-ray crystallography and small-angle x-ray scattering (SAXS) and identified a novel interaction of ISG75 with all four isotypes of human IgG. Through this interaction, ISG75 is capable of inhibiting the binding of C1q and CD32/CD16 to IgG and consequently interfering with the classical pathway of the complement system, as well as Ab-mediated cellular phagocytosis (ADCP) and cytotoxicity (ADCC), suggesting that ISG75 serves a role in protecting the parasite against the immune system of the host.

The gene encoding T. brucei ISG75 (Lister strain 427, Tb427-8200) residues 29–468 (ISG75_ecto) (30) was synthesized and cloned into the EcoRI/XbaI sites of the pPICZαA vector (Invitrogen, catalog no. V195-20) for recombinant expression in Pichia pastoris. A construct expressing ISG75_ecto containing the N134A substitution was generated by site-directed mutagenesis using the InFusion HD cloning kit (Clontech, catalog no. 102518). The gene encoding ISG75 residues 29–286 (ISG75_head) was amplified and inserted into the NdeI/XhoI sites of the pET22B(+) vector (Novagen, catalog no. 69744) for recombinant expression in Escherichia coli. Genes encoding ISG75 residues 29–286 and ISG65 residues 23–349 fused to an anti-human CD163 nanobody (denoted nb2) via a (Gly₄Ser)₃-linker was amplified and inserted into the NdeI/EcoRI sites of pET22B(+). The resulting constructs are denoted ISG75_head-nb2 and ISG65-nb2.

Prior to transfection, ISG75_ecto pPICZαA vectors (wild type and N134A) were linearized using the restriction enzyme PmeI (Fermentas, catalog no. ER1341). The linearized vectors were transfected into the competent P. pastoris SuperMan5 strain (BioGrammatics, catalog no. GS10010) using electroporation, and the cells were subsequently plated on yeast extract–peptone–dextrose–sorbitol medium containing 100 µg/ml Zeocin (Invitrogen, catalog no. R25001). Single colonies expressing ISG75_ecto were inoculated in buffered glycerol-complex medium and grown overnight at 30°C/250 rpm in a shaking incubator. The cells were transferred to a buffered methanol-complex medium and grown for 72 h at 30°C/250 rpm in a shaking incubator. Methanol was added to 1.5% every 24 h to induce expression. ISG75_ecto was purified from the growth medium using immobilized anti-ISG75 nanobody (nb20) and eluted with buffer containing 20 mM MES (pH 6.6) and 3.6 M MgCl₂. ISG75_ecto was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL (Cytiva, catalog no. GE17-5175-01) equilibrated in buffer containing 20 mM Tris-HCl (pH 7.6) and 75 mM KCl. Peak fractions were pooled and concentrated to 5 mg ml⁻¹ using Vivaspin centrifugal concentrators (Sartorius, catalog no. VS0102).

ISG75_ecto N134A was crystallized at 8 °C using sitting-drop vapor diffusion, mixing 2 μl of sample with 2 μl of reservoir buffer containing 100 mM HEPES (pH 7.5), 20% PEG3350, and 20 mM sorbitol. Prior to data collection, the crystals were transferred to cryo-protection buffer containing reservoir buffer with 40% PEG3350 and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K and a wavelength of 1.033 Å at the EMBL Hamburg P13 Beamline. The data were processed and scaled with the XDS package (35) and subsequently subjected to ellipsoidal truncation using the STARANISO server (36). Initial phases were calculated by molecular replacement in Phaser using an AlphaFold2-generated model (37) split into two individual search models covering residues 47–286 and 293–423, respectively. The model was refined using iterative cycles of refinement in PHENIX (38) followed by manual model building in COOT (39). The figures were prepared with PyMOL (40). The electrostatic potentials of ISG75 domains were calculated with APBS (41).

SAXS data on ISG75_ecto were collected at the EMBL Hamburg P12 Beamline at a wavelength of 1.239 Å. The data on three different concentrations of ISG75_ecto N134A in buffer containing 20 mM Tris-HCl (pH 7.6) and 75 mM KCl were collected in a batch setup with 20 exposures of 0.045 s at 293 K and a path length of 1.5 mm. Normalization, radial averaging, buffer subtraction, and concentration correction of the data were performed by the automated pipeline at the beamline (42). R_g and I(0) values were calculated using GNOM. Rigid-body refinement was performed in CORAL using standard settings and with ISG75 residues 29–284 and ISG75 residues 292–435 as rigid bodies restrained by the distance of the loop connecting them. The ab initio surface model was calculated by running 20 rounds of reconstruction in DAMMIN followed by averaging using DAMAVER. The plots were prepared with GraphPad Prism version 9.3.0.

ISG75_ecto N134A was analyzed by size-exclusion chromatography in-line with multiangle light scattering using a Wyatt SEC Analytical Column (Wyatt Technology Europe GmbH, catalog no. WTC-030S5) connected with an Optilab T-rEX Refractive Index Detector (model no. WTREX-02) and DAWN 8+ multiangle light scattering detector (Wyatt Technology Europe GmbH, model no. WH2-06). Bovine serum albumin (Sigma, catalog no. A1900) was used to calibrate the system. The experimental data were recorded and processed by ASTRA software (Wyatt Technology Europe GmbH).

Nanobodies against ISG75 and human CD163 were generated by immunizing a llama (Lama glama) four times with a total of 500 µg of ISG75_ecto and human CD163 (capralogics.com). Peripheral blood lymphocytes from a blood sample were isolated, and the total RNA was extracted. cDNA was generated using the SuperScript III first-strand synthesis system (Invitrogen, catalog no. 18080051) with random hexamer primers. The regions corresponding to the heavy-chain variable domains of the Abs were amplified by PCR and inserted into the phagemid vector. The phagemid vectors were transformed into the TG1 E. coli strain (Amid Bioscience, catalog no. TG1-201), and subsequently, cells were infected with M13 helper phages for the generation of the final M13 phage library. For selection of ISG75/CD163 specific nanobodies, biotinylated ISG75_ecto/CD163 was produced according to the instructions of the manufacturer (ChromaLINK, catalog no. B-1001). The biotinylated ISG75_ecto/CD163 was used for two successive rounds of library panning on magnetic beads (Invitrogen, catalog no. 65001) with increasing stringency. Specific monoclonal binders were obtained after ELISA screening on 96-well MaxiSorp plates (Nunc, catalog no. P6991) using HRP/anti-M13 monoclonal conjugate (Cytiva, catalog no. 27–9421-01) together with TMB substrate (Thermo Scientific, catalog no. N301) for detection. For the current study, a nanobody specific for ISG75 (denoted nb20) and a nanobody specific for CD163 (denoted nb2) were used.

Nanobody-coding sequences were cloned into the pET22B(+) vector (Novagen, catalog no. 69744) sites NcoI/XhoI for periplasmic expression and transformed into E. coli Rosetta (DE3) competent cells (Novagen, catalog no. 70954). Expression was induced with 0.5 mM isopropyl β-d-thiogalactoside at OD₆₀₀ = 0.6-0.9 and incubated overnight at 20 °C. Nanobodies were isolated from the periplasmic space by a wash in TES buffer (250 mM Tris-HCl, 5 mM EDTA, 20% [w/v] sucrose [pH 8]) followed by osmotic shock in 5 mM MgSO₄. Bacterial debris was pelleted at 30,000g for 20 min. Next, supernatants were applied to His-tag affinity purification on HisTrap FF Crude columns as described above.

ISG75_head, ISG75_head-nb2, and ISG65-nb2 vectors were transformed into ShuffleT7 E. coli cells (New England Biolabs, catalog no. C3026J). Cell cultures were grown in LB growth medium with 100 µg ml⁻¹ ampicillin and induced overnight at 20 °C with 0.2 mM isopropyl β-d-thiogalactoside. The cells were resuspended in 20 mM HEPES (pH 7.6), 500 mM NaCl, 20 mM simidazole, 10% glycerol (lysis buffer) and lysed by sonication. The lysate was centrifuged at 30,000g for 20 min, and the supernatant was loaded on a 5-ml HisTrap FF Crude column (Cytiva, catalog no. 17528601) equilibrated in lysis buffer. The protein was eluted by running a linear gradient from 20 to 500 mM imidazole, and fractions containing the protein were pooled. The sample was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 GL (Cytiva, catalog no. GE17-5175-01) equilibrated in buffer containing 20 mM Tris-HCl (pH 7.6) and 75 mM KCl.

Human serum was diluted 5-fold in HBS buffer (10 mM HEPES [pH 7.5], 150 mM NaCl, and 2 mM CaCl₂), and 200 µg of ISG75_head was added. After 30 min of incubation, 100 µl of nb20-Sepharose was added to the tube and incubated for another 30 min. The beads were then pelleted by 2 min of centrifugation at 500g and washed three times with 1 ml of HBS. Bound proteins were eluted with 200 µl of 10 mM glycine (pH 2.5) and analyzed by SDS-PAGE.

Samples from the pull-down experiment were loaded onto Vivaspin centrifugal concentrators (Sartorius, catalog no. VS0102) and washed three times with UA buffer (8 M urea in 100 mM tetraethylammonium bromide). Subsequently, 100 µl of 50 mM DTT in UA buffer was added, and each sample was incubated at 37°C for 1 h. After an additional wash with UA buffer to remove excess DTT, 100 µl of 50 mM iodoacetamide in UA buffer was added, and samples were incubated at room temperature for 20 min in the dark. After one more wash with UA buffer and centrifugation to remove all liquids, 10 µl of Lys-C in UA buffer was added (enzyme to protein ratio at 1:100), and the concentration cell was incubated at 37°C for 2 h. Then 100 µl of trypsin in 100 mM tetraethylammonium bromide (enzyme to protein ratio at 1:50) was added, and the samples were incubated overnight at 37°C. The resultant peptides were isolated from the flowthrough of the concentration cell after centrifugation and then vacuum dried. Peptide quantification was done by a fluorometric peptide assay. Peptide samples were subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using an Orbitrap Fusion mass spectrometer (Thermo Fisher) coupled to an easy nLC-1200 (Thermo Fisher). A 105-min gradient with an active 90-min separation window defined by 5–20% buffer B over 60 min followed by 20–35% buffer B over 30 min, in which buffer B containing 80% acetonitrile in 0.1% formic acid was used to separate peptides. Data-dependent acquisition with a cycle time of 3 s was used, with precursor scan range of 375–1500 and a resolution of 120,000. Automatic gain control and maximum injection time were set at auto. Fragment scan was done at a resolution of 15,000 and high collision dissociation energy of 32%. Automatic gain control and maximum injection time were also set at auto. Dynamic exclusion was enabled with an exclusion window of 40 s. Single charged ions, more than six charged ions, and unknown charged ions were excluded from fragmentation. Triplicates for each sample were combined and searched in MaxQuant (version 2.3.1.0) with the iBAQ algorithm enabled. The database searched was the human UniProt database downloaded on July 21, 2023, plus common contaminants. Carbamidomethylation of cysteine was set as a fixed modification, while acetylation of the protein N-terminus and oxidation of methionine were set as variable modifications. Peptide and protein false discovery rates were both set at 0.01. After removing contaminants and reversed protein sequences, the intensity-based absolute quantification (iBAQ) percentage for each protein was calculated by dividing each individual iBAQ value against the sum of all iBAQ values from one sample. The iBAQ percentage represents the relative amount of each protein in one sample.

Surface plasmon resonance (SPR) experiments were carried out on a Biacore 3000 instrument (Cytiva) using 10 mm HEPES (pH 7.5), 150 mm NaCl, 2 mM CaCl₂, and 0.05% Tween 20 as running buffer. Binding analysis was performed at 25°C, and the data were collected at a rate of 1 Hz. Using the BIAevaluation 4.1.1 software (GE Healthcare), recorded signals from the active flow cell were double referenced; the signal from the in-line reference flow cell was subtracted as was the signal from a blank run (0 nM analyte). All ligand immobilizations were prepared by standard 3-(N,N-dimethylamino) propyl-N-ethylcarbodiimide/N-hydroxysuccinimide amine chemistry on CM5 chips according to the manufacturer’s instructions (Cytiva, catalog no. BR100399); i.e., the surfaces were activated by a 7-min injection of a 1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.4 M 3-(N,N-dimethylamino) propyl-N-ethylcarbodiimide. The ligands were diluted to 20 µg/ml in 10 mM sodium acetate (pH 4.5 or 5). Residual reactive groups were blocked by a 7-min injection of 1 M ethanolamine (pH 8.5).

For the analysis of ISG75 interactions with IgG, an anti-ISG75 chip was prepared with ∼600 RU of immobilized nb20 in flow cells FC3 and FC4. ISG75_head was then captured in FC4 only, to a level of 80 RU. Purified analytes were then injected over both surfaces for 180 s followed by a 180-s dissociation phase. At the end of each binding cycle, noncovalently attached molecules were removed from both surfaces by a 90-s regeneration with 10 mM glycine, pH 2.2. A flow rate of 30 μl/min was used in all steps of the experiments.

Fcγ-receptor chips were prepared by direct immobilization of CD16a (Acro Biosystems, catalog no. CD8-H52H4) and CD32a (Acro Biosystems, catalog no. CDA-H5221) in FC2 and FC4, respectively, leaving FC1 and FC3 blank to serve as reference surfaces. Both proteins were immobilized to a density of ∼2,000 RU. To quantify the inhibitory effect of ISG75, 100 nM of purified IgG mixed with a titration series of ISG75_head was injected, at a 10 µl/min flow rate, in active and reference FCs for 120 s followed by a 180-s dissociation phase. Between cycles, the CD16a surface was regenerated by a 3-min injection of 1 µM ISG75_head. The CD32a surface was regenerated by a 30-s injection of 1 M ethanolamine (pH 8.5).

For evaluation of the inhibitory effect of ISG75_head on the interaction between IgG and complement protein C1q, an intricate setup with both ISG75_head and IgG captured on the sensor chip was applied (for illustration, see Supplemental Fig. 4D). In short, the surface was prepared by immobilization of recombinant human CD163 (residues 51–578) to a density of 3,500 RU in FC4, using a blank FC3 as reference. First, an anti-human CD163 Ab of the IgG3 subclass was bound to the surface and next either ISG75_head-nb2 or ISG65-nb2. C1q, at a concentration of 50 nM, was then injected into both flow cells for 120 s followed by 120 s of dissociation. The surfaces were regenerated by sequential injections of 1:50 mM sodium citrate and 10 mM EDTA (pH 4.5) for 120 s, 2:1 M ethanolamine (pH 9.5) for 300 s, and 3:3.6 M MgCl₂ for 120 s.

The effect of ISG75_head on the complement classical pathway was examined by measuring the deposition of C1q and C3 fragments onto IgG-coated microtiter well surfaces. Microtiter wells were coated by overnight incubation with 5 μg/ml of human IgG1 or IgG3 in 100 μl of PBS. Residual protein-binding sites were blocked by incubation of TBS (10 mM Tris-HCl [pH 7.4], 145 mM NaCl) containing 1 mg/ml human serum albumin for 1 h at room temperature, followed by washing three times in TBS/Tween (TBS with 0.05% Tween 20). Thereafter, 100 µl of human serum diluted to a final concentration of 0.4% in 4 mM HEPES (pH 7.5), 145 mM NaCl, 2 mM CaCl₂, and 1 mM MgCl₂, was added to dilutions of ISG75_head, ISG75_head-nb20, or, as a control, nb20 alone. The final concentration of human serum was 0.2%. The concentrations of ISG75_head, ISG75_head-nb20, and nb20 were tested in 4-fold dilutions with final concentrations starting at 5.1 µM and ending at 5 nM. The admixture of serum and reagents was performed at 4°C to avoid possible complement activation. The plates were thereafter transferred to 37°C and incubated for 1.5 h. The wells were then washed three times in TBS/Tween with 5 mM CaCl₂ (TBS/Tween/Ca) and subsequently 100 µl of 1 µg/ml biotinylated anti-hC1q (Agilent Dako, catalog no. A0136) or 0.5 µg/ml of anti-hC3d (Agilent Dako, catalog no. A0063) in TBS/Tween/Ca was added to the wells, followed by incubation overnight at 4°C. The wells were washed three times in TBS/Tween/Ca and then incubated with 0.1 µg/ml streptavidin-Eu (perkinElmer, catalog no. 1244-360) in TBS/Tween, 25 µM EDTA for 1 h at room temperature. Subsequently, the plate was washed three times in TBS/Tween/Ca, followed by incubation with enhancement buffer (Ampliqon) for 2 min. The signal of the europium in the wells (given as counts per second) was subsequently measured by time-resolved fluorometry on a VICTOR 5 plate reader (perkinElmer).

The effect of ISG75 on Ab-mediated phagocytosis was analyzed using a coculturing system with FlpIn293 cell line expressing human CD163 (43) as target cells and Jurkat-Lucia NFAT CD32-expressing cells as effector cells (Invivogen, catalog no. jktl-nfat-cd32). The Jurkat cell line expresses a Lucia luciferase reporter under control of NFAT, which after CD32 engagement drives luciferase expression. In brief, 20,000 FlpIn293 human CD163 cells were plated in a 96-well plate and incubated overnight at 37°C, 5% CO₂. Followingly, the medium was removed, and anti-human CD163 Ab (human IgG3 chimeric mAb) inhibitors and 100,000 Jurkat NFAT CD32-expressing reporter cells (ratio 1:5) in assay buffer (IMDM medium with 4% IgG depleted FBS) were added and incubated for an additional 24 h. The level of luciferase activity was subsequently measured by transferring assay supernatant to a white 96-well plate followed by addition of luciferase detection reagent (Invivogen, catalog no. rep-qlc4lg1) and immediate reading of luminescence on a luminescent microplate reader (perkinElmer).

To investigate the structural and functional properties of ISG75, the entire ectodomain (ISG75_ecto) of T. brucei ISG75 (isoform Tb427-8200, residues 29–468) was expressed in the yeast P. pastoris. To facilitate crystallization, Asn¹³⁴ was mutated to alanine to disrupt the single putative N-linked glycosylation site. Size-exclusion chromatography performed in-line with multiangle light scattering analysis resulted in a monodisperse peak with an estimated molecular mass of around 50 kDa, showing that ISG75_ecto is a monomer in solution (Supplemental Fig. 2A). Crystals of ISG75_ecto were obtained, and a dataset extending to 2.7 Å resolution was collected (Table I). The data were highly anisotropic with diffraction to 2.7 Å along one axis, while diffraction along the two other axes only extended to 3.6 Å resolution and consequently elliptical truncation of the data were applied. The structure was determined by molecular replacement using fragments of an Alphafold2-predicted structure as search models. Two molecules of ISG75_ecto are present in the asymmetric unit, and electron density is observed for almost the entire extent of the molecules, except for small parts of the loop regions (residues 152–158 and 181–182) and the C-terminal region (residues 436–468) (Supplemental Fig. 2B). The final model displays relatively good crystallographic and geometric statistics with R_work/R_free = 0.23/0.27.

The structure of ISG75_ecto is constituted by two domains: a head domain and a coiled-coil domain (Fig. 1A). The structure of the head domain (residues 29–286) is similar to other trypanosome surface receptors with a core formed by three α-helices (H1, H3, and H6) and a distal region formed by an additional α-helix (H5), the N-terminus, and two relatively long loop regions, denoted L1 and L2 (Supplemental Fig. 1). The coiled-coil domain (residues 290–435) is formed by two α-helices (ccH1 and ccH2) packing against each other and forming a 110 Å rod-like structure. The N-linked glycosylation site at Asn¹³⁴ is located in helix 2 of the head domain and is positioned on the opposite face of the coiled-coil domain (Fig. 1A). An additional N-linked glycosylation (Asn¹¹⁵) site found in Tb427-7700 (Supplemental Fig. 1) is located on the same face as Asn¹³⁴ but is closer to the membrane (Fig. 1A).

In the crystal structure, the coiled-coil domain packs against L1 of the head domain. In the crystal structure, several residues (Glu³³⁰, Arg³³³, and Arg³⁹⁰) from the coiled-coil domain form hydrogen bonds with the main chain of L1 in the head domain (Fig. 1B). Tyr¹⁷⁵ and Tyr¹⁷⁶ from L1 engage in extensive van der Waals’ interactions with the coiled-coil domain, and additional hydrogen bonds are formed between Arg⁴⁰⁰ from the coiled-coil domain and Thr¹⁹⁹ from L1.

The two molecules of ISG75_ecto in the asymmetric unit of the crystal are nearly identical, except at the C-terminal end of the ectodomain (Fig. 1C). In molecule A, ccH2 continues all the way to residue 435, whereas in molecule B, the helical structure of ccH2 is disrupted at residue 418, and following a short loop constituted by residues 418–421, an additional helix is formed (ccH3), indicating a structural plasticity in the region connecting the coiled-coil domain with the transmembrane helix.

To assess whether the crystal structure reflects the solution structure of ISG75_ecto, SAXS data were collected at three different concentrations of ISG75_ecto. The scattering curves show nearly identical slopes (Supplemental Fig. 3A), and the Guinier plot does not indicate aggregation even at 9.5 mg/ml (Supplemental Fig. 3B). The derived pair distribution functions indicate a maximal extent (D_max) of ∼120 Å (Supplemental Fig. 3C). This is in good agreement with ISG75_ecto forming a monomer in solution as also indicated by size-exclusion chromatography performed in-line with multiangle light scattering analysis (Supplemental Fig. 2A). However, the scattering curve calculated from the ISG75_ecto in the crystal structure does not fit well to the experimental data (Fig. 2A). In the Alphafold2 prediction, ISG75_ecto adopts an entirely different conformation with H6 from the head domain and ccH1 from the coiled-coil domain fused into a single α-helix, which results in a highly elongated molecule (Fig. 2C). However, as for the crystal structure, the calculated scattering curve for the Alphafold2 prediction of ISG75_ecto does not fit well to the experimental SAXS data (Fig. 2A). Therefore, we separated ISG75_ecto into its two domains and performed a rigid-body refinement, whereby we obtained a model with a reasonable fit to the experimental data. In this model, the coiled-coil domain does not pack against the head domain and the distal tip of the coiled-coil domain is located ∼60 Å away from its position in the crystal structure (Fig. 2B).

Addition of suramin to ISG75_ecto prevented formation of suitable crystals and structure determination of suramin-bound ISG75_ecto was therefore not feasible. However, when mapping the electrostatic potential on the surface of the ISG75 head domain, several positively charged patches were identified (Supplemental Fig. 2C). Considering the length and flexibility of suramin, multiple patches on the ISG75 head domain could be involved in simultaneous interaction with the negatively charged suramin. Prediction of suramin-binding sites was attempted using the molecular docking tool AutoDock Vina (44). In this article, a range of possible binding sites were identified, but none had a score significantly above the background (data not shown). The electrostatic potential mapped on the surface of the coiled-coil domain reveals a polarized charge distribution, with one face having a strong negative charge, while the other contains both negatively and positively charged areas (Supplemental Fig. 2D).

Our structural data revealed the existence of two individual ISG75 domains. In order to analyze the function of the ISG75 domains, we expressed the head domain of ISG75 (ISG75_head, residues 29–286) separately in E. coli. Although ISG75_head expresses well, it has low solubility (∼0.3 mg/ml) and tends to aggregate over time. However, when the anti-ISG75 nanobody (in the following denoted nb20) is added to ISG75_head, its solubility is increased. To identify potential ligands of ISG75, we incubated ISG75_head with normal human serum from individuals that had not been exposed to T. brucei and subsequently added beads conjugated with nb20. In addition to a band containing ISG75_head, a band at around 150 kDa is observed in SDS-PAGE analysis of the proteins eluted from the beads (Fig. 3). Under reducing conditions, the 150-kDa band separates into two smeared bands at 50–60 kDa and 25 kDa, indicating that the ISG75 ligand is a mixture of human IgG subclasses. This was subsequently confirmed by LC-MS/MS. Using iBAQ (45), >96% of the eluted sample could be assigned to IgG heavy chains or λ/κ light chains. Based on the iBAQ data, the relative distribution of IgG subclasses was estimated. In this article, the majority of the ISG75-bound IgG belongs to subclass 1 (69%) followed by subclass 3 (20%) and subclass 2 (10%). Only a small amount of IgG subclass 4 is detected (1%). The relative distribution of IgG subclasses bound by ISG75_head generally reflects the distribution of IgG subclasses in human serum (46, 47), suggesting that in this experimental setting, ISG75_head does not selectively interact with one or more IgG subtypes.

To quantify the interaction between ISG75_head and the individual subclasses of human IgG, we performed SPR experiments with ISG75_head immobilized on the sensor chip (Fig. 4). Each of the four human IgG subclasses was measured in six concentrations, and the K_D for their interaction with ISG75_head was determined by steady-state kinetics (Table II). Human IgG subclass 3 appears to have the strongest affinity for ISG75_head with a K_D estimated to 0.12 µM, followed by human IgG subclasses 2 and 4 with K_D estimated as 4.54 and 4.84 µM, respectively. The human IgG subclass 1 appears to bind ISG75_head with the lowest affinity (K_D estimated as 6.38 µM). In addition to human IgG, we tested binding of mouse IgG subclass 2a (being the most abundant subclass in mice) to ISG75_head, but no binding was observed (Fig. 4E). Using total rabbit IgG as analyte gave a very low signal in the response curve, whereas total cow IgG showed no binding (data not shown). To further characterize the interaction of ISG75 with human IgG, we prepared Fab and Fc fragments by papain digestion of IgG3. In this article, the IgG3-Fc fragment shows binding to ISG75, although at slightly lower affinity compared with intact IgG3 (Fig. 4F). No binding was observed for the IgG3-Fab fragment of IgG3 (data not shown).

The recognition molecule C1q binds to the Fc region of patterns of deposited IgG and then initiates the classical pathway (CP) of the complement system. Based on its interaction with the IgG3-Fc fragment, we hypothesized that ISG75 could function as an inhibitor of the CP by interfering with C1q binding to IgG deposited on the surface of the parasite. In the presence of serum, we tested the influence of soluble ISG75_head on the binding of C1q to IgG bound to microtiter wells. The assay was performed for both IgG1 and IgG3 isotypes, and the effects of ISG75_head were quantified by measuring the deposition of C1q at increasing inhibitor concentrations (Fig. 5). We tested both ISG75_head alone and ISG75_head in complex with nb20 (ISG75_head-nb20). We observe that ISG75_head shows a clear inhibition of C1q deposition on both IgG1- and IgG3-coated surfaces (Fig. 5A, 5B). When in complex with nb20, the activity of ISG75_head is roughly 3-fold higher, likely due to a stabilizing effect of nb20 on the structure of ISG75_head. nb20 alone does not have an effect on C1q deposition. As expected from SPR analysis (Fig. 4), ISG75_head inhibits the binding of C1q to IgG3 more efficiently than to IgG1, likely due to its higher affinity for IgG3. We observed no ISG75_head inhibition in the CP assay when wells were coated with mouse IgG2a (Fig. 5C), confirming the SPR data showing that ISG75 does not bind mouse IgG2a (Fig. 4E). This is despite the fact that human C1q does bind to mouse IgG2a. We also used a similar setup but now with the detection of the deposition of C3 fragment on the surface, and we found an inhibitory activity of ISG75_head on CP, again with a higher inhibitory activity of ISG75 when in complex with nb20 (Supplemental Fig. 4A–C). Although the CP assay does not fully replicate the physiological setting on the parasite’s surface, where the transmembrane ISG75 is located in close proximity to IgGs bound to surface epitopes, it demonstrates that soluble ISG75_head interferes with the C1q–IgG interaction and prevents the downstream steps in the human CP. Using the purified proteins, we further quantified C1q binding to IgG3 using SPR, with and without cocaptured ISG75_head (Fig. 5D, Supplemental Fig. 4D). This showed a clear decrease of C1q binding to IgG3 in the presence of ISG75_head, whereas cocapture of ISG65 as a control did not affect C1q binding to IgG3. Together, these experiments show that ISG75 most likely inhibits CP activation by competing with C1q for binding to the same or an overlapping site on IgG.

C1q and Fcγ receptors (FcγRs) have partly overlapping binding sites on IgG (48, 49), with specific IgG residues, such as Leu²³⁴ and Leu²³⁵, being crucial for activation of all effector functions of IgG (50). Thus, we speculated that ISG75 could also affect the interaction of FcγR (i.e., CD32 or CD16) with IgG and thereby function as a modulator of ADCP and ADCC. To test this hypothesis, we analyzed the effect of ISG75_head in an assay for ADCP based on a Jurkat-Lucia NFAT cell line expressing the FcγRII receptor CD32 (Fig. 6A). In this article, the reporter cell line was cocultured with HEK293 cells expressing a surface Ag (CD163) recognized by a monoclonal IgG3 anti-CD163 Ab. The assay shows a clear inhibitory effect of ISG75 on the activation of the reporter cell line with an IC₅₀ of ∼400 nM, showing that ISG75_head is capable of inhibiting ADCP. It was only possible to perform the assay with ISG75 stabilized by nb20. In SPR experiments using immobilized CD32, ISG75_head inhibits IgG3 binding in a dose-dependent manner (Fig. 6B), supporting that ISG75 competes with CD32 for binding to IgG3. Moreover, ISG75_head also shows inhibition of the FcγRIII receptor CD16 binding to IgG3 (Fig. 6C), indicating that ISG75 is also capable of suppressing ADCC.

Structure determination revealed that ISG75 is composed of two separate domains: an N-terminal head domain and a C-terminal coiled-coil domain. The structure of the head domain resembles other trypanosome surface proteins and likely has evolved from an ancestral VSG. The presence of the coiled-coil domain appears to be a unique feature of ISG75, because this region does not show sequence homology to any other trypanosome proteins.

In the crystal structure, the coiled-coil domain associates with the side of the head domain. The residues engaged in the interactions are completely conserved in the closely related ISG75 sequences (Supplemental Fig. 1), indicating that the interaction, at least within these isoforms, could play a physiological role. The SAXS data on the other hand suggests a more open conformation with the coiled-coil domain separated from the head domain, which could indicate that the interaction between the head domain and the coiled-coil domain is either a transient conformation in solution or due to crystal packing. The Alphafold2 prediction of ISG75 suggests an entirely different conformation with the tip of coiled-coil positioned distal to the head domain. However, when full-length ISG75 is expressed on the surface of the trypanosome and is integrated within the VSG layer, it may adopt a completely different conformation. The electrostatic analysis shows that the coiled-coil domain exhibits a highly polarized charge distribution, with one face having a pronounced negative charge. Hence, it is possible that the coiled-coil domain facilitates interaction with a positively charged patch on the surface of another trypanosome surface protein such as the VSGs. Remarkably, the coiled-coil domain is almost completely conserved in all ISG75 isoforms, indicating a functional role of the domain.

The head domain appears to be the functionally important part of ISG75, and we show that it likely plays a role in immune evasion by binding to human IgG and neutralizing its effector functions. Using a recombinantly expressed ISG75 fragment encompassing the head domain, we demonstrate a specific interaction with all four isoforms of human IgG. The binding of ISG75 to IgG3 appears to be the strongest with a K_D estimated to 0.12 µM. The interaction of ISG75 with remaining IgG isoforms are of similar affinities, with K_D values ranging from 5 to 6 µM. The four human IgG subclasses are highly conserved and differ primarily in their CH2 domains and the hinge regions connecting CH1 and CH2 (47). Notably, IgG3 is distinct from the other subclasses because of its extended hinge region. This hinge in IgG3 is around 50 residues longer than those found in the other IgG subclasses and confers greater flexibility to IgG3, potentially influencing its effector functions (51). The extended hinge of IgG3 may also be the key contributor to the increased affinity of ISG75 for IgG3 compared with the other subclasses.

The CH2 domains and hinge regions are critical structural elements that facilitate the effector functions of IgG, primarily by enabling interactions with C1q and FcγR. Structural analyses have shown that the binding sites for C1q and FcγR on IgG partially overlap (48, 49). Given that ISG75 can inhibit the binding of IgG to both C1q and FcγR (Figs. 5D, 6B, 6C), it is likely that ISG75 binds within the same area. Consequently, ISG75 may exert its effect through steric hindrance, impeding the interaction between IgG and both C1q and FcγR, thereby attenuating the effector functions of IgG.

The affinities of human IgG subclasses for FcγR differ markedly (52). Overall, IgG3 exhibits the highest affinity for FcγR, followed by IgG1. In contrast, IgG2 and IgG4 demonstrate comparatively lower affinities for these receptors. IgG3 has been shown to be particularly important during the acute-phase response following an infection (51). It is conceivable that the activity of ISG75 during its evolutionary adaptation to the human host has primarily been driven toward an activity that targets IgG3 to disrupt its interaction with FcγR.

Generally, the ISG75 affinities for IgG are more or less within the same range as the FcγR affinities for IgG. One might expect an efficient inhibitor to show high-affinity binding, as is the case for both of the well-known bacterial IgG receptors, protein A and protein G. However, it is important to keep in mind that the high copy number of ISG75 on the surface of the trypanosome ensures a high local density of ISG75 in the immediate vicinity of Ag-bound IgGs, whereas here we report the monovalent affinities for the ISG75–IgG interactions. Efficient activation of FcγR signaling requires many simultaneous binding events engaging numerous host cell receptor molecules and pathogen-bound IgGs (53, 54). Consequently, inhibition of FcγR-mediated ADCC/ADCP does not necessarily require ISG75 to completely inhibit all FcγR–IgG interactions.

The major sequence differences of the ISG75 isoforms are located within the head domain (Supplemental Fig. 1). For the current study, we specifically selected the Tb427-8200 sequence and demonstrated a significant binding to human IgG while exhibiting only minimal binding to rabbit IgG and no detectable binding to mouse IgG or cow IgG. We speculate that ISG75 isoforms may have evolved to bind IgG from different host species, and as a result, trypanosomes are capable of counteracting the effector functions of a broad spectrum of IgG by expressing an array of different ISG75 isoforms on their surfaces. Whether, the divergent ISG75 Tb427-7700 isoform is also an IgG neutralizer or has evolved into an entirely different function remains to be uncovered.

Our study demonstrates that ISG75 represents an additional mechanism for inhibition of the effector functions of human IgG. Together with the antigenic switching of VSGs, hampering of host B-cells, and rapid endocytosis, this novel mechanism may have a large impact on parasite clearance. However, 95% ISG75 knockdown in mice does not appear to result in decreased trypanosome infection (33), which questions the role of ISG75 in immune evasion. There could be several explanations for this. First, there are several independent immune evasion mechanisms in play, and partial knockdown of ISG75 may not be sufficient for a decrease in trypanosome titers in the infected ISG75 knockdown mice. Secondly, because the ISG75 isoform analyzed in the current study does not appear to bind mouse IgG, it is possible that ISG75 does not have a neutralizing effect in a murine infection model. Future studies to assess whether other ISG75 isoforms interact specifically with mouse IgGs and whether a complete ISG75 knockdown in mice confers increased trypanosome infection are required.

Pathogens have developed a variety of mechanisms to counteract the effector functions of IgG. Among these, the production of proteins that target the Fc region of IgG is particularly notable. Protein A from Staphylococcus aureus and protein G from group G Streptococcus specifically bind to the IgG-Fc region, yet their binding sites are distinct from those of C1q and FcγR. This binding does not directly inhibit the interaction between C1q and FcγR with IgG, but it does prevent IgG hexamerization. Such hexamerization is vital for initiating C1q-mediated complement activation and enabling FcγR-mediated immune functions (55). In contrast, trypanosomes have evolved a comparable yet distinct approach to suppress the effector functions of IgG attached to their surface. The mechanism of ISG75 seems to directly inhibit the binding of both C1q and FcγR to IgG. While a variety of IgG-binding proteins have been identified in pathogenic organisms, to our knowledge, ISG75 is unprecedented in its ability to directly interfere with the binding of C1q and FcγR to IgG. It may represent a unique mechanism of pathogenic immune evasion strategies.

Trypanosomes exhibit a remarkable ability to evade the host’s immune defenses, enabling them to maintain a chronic infection within the host’s bloodstream. Central to this evasion is the antigenic variation, which occurs through switching the VSGs on their surface. This antigenic shifting results in characteristic parasitemia waves as the immune system continually adjusts to new antigenic profiles. Additionally, trypanosomes possess a sophisticated endocytic system that efficiently removes and degrades Abs bound to their surface, thereby reducing the threat of Ab-mediated clearance. Furthermore, trypanosomes can modulate B-cell differentiation and functionality. This modulation impairs the host’s ability to mount an effective and sustained humoral immune response. Recently, ISG65 was discovered as a novel component of trypanosome immune evasion mechanisms. ISG65 was shown to bind C3b, a key component of the complement system. Upon binding, ISG65 facilitates the conversion of C3b to its inactive form, iC3b, effectively inhibiting the activation of the host’s alternative complement pathway. The discovery of ISG75 as yet another immune evasion component adds another layer to the complex immune evasion strategies employed by trypanosomes, further illustrating their adaptability and resilience in their encounter with the host immune system (Fig. 7).

In summary, our results suggest that ISG75 could be an important component of the trypanosome immune evasion mechanisms by modulating the effector functions of IgG bound to epitopes on the surface of the parasite. Combined with a range of complementary immune evasion strategies, ISG75 likely contributes to the success of the parasites in sustaining a chronic infection within the host bloodstream.

The authors have no financial conflicts of interest.

We are grateful to Gitte Ratz for expert assistance with protein production and the staff at EMBL Hamburg Beamlines P12 and P13 for assistance with data collection.