Major human schistosome species express different glycans with immunological and diagnostic implications

Domain: biology
Published: 2026-03-23
Editorial outcome: major_revision

Abstract
Schistosomes are parasitic worms responsible for devastating chronic disease including intestinal and urogenital pathologies. In-depth studies of the Schistosoma mansoni glycome have revealed complex, immunogenic and life stage-specific glycans, that are crucial in host-parasite interactions. Despite causing the majority of schistosome infections and specific associated pathology, the glycosylation of S. haematobium remains largely unstudied. Here, we characterize the glycan repertoire of S. haematobium using mass spectrometry-based approaches. We report substantial differences to S. mansoni glycans, most notably in the core structure, fucosylation patterns and glucuronic acid modifications of glycosphingolipid (GSL) glycans. Furthermore, IgG from S. haematobium-infected individuals preferentially bind the acidic GSL glycans compared to IgG from S. mansoni-infected individuals. Our results demonstrate that S. haematobium and S. mansoni are differentially glycosylated and that S. haematobium glycans are involved in parasite-host immunobiology. The specific, immunogenic S. haematobium glycans constitute targets for use in epidemiological studies and potential species-specific diagnostics.

Introduction
Schistosomiasis is a widespread debilitating parasitic disease caused by trematodes of the genus Schistosoma. S. mansoni and S. haematobium are the most prevalent species causing human intestinal and urogenital schistosomiasis, respectively1,2. Disease control programs most commonly rely on mass administration of preventive chemotherapy, supported by safe water and hygiene (WASH) and educational initiatives3,4. There is no vaccine against schistosomiasis although novel (prophylactic) therapies as well as adequate diagnostics are deemed necessary to eliminate schistosomiasis as a global public health problem5. Thus, acquiring a solid fundamental knowledge of schistosome biology is critical to providing the field with the tools required to meet the 2030 elimination target.

Years of research into schistosome glycobiology, with an almost exclusive focus on S. mansoni, have unraveled structural, biological, and immunological features of the parasite’s glycoconjugates. S. mansoni glycans are key players in host-parasite interactions and targets of the host’s innate and adaptive immune system6–8. Extensive structural characterization has shed light on the large diversity of N-linked, O-linked and glycosphingolipid (GSL) glycans expressed by S. mansoni, that exhibit unique motifs such as dimeric \alpha(1-2)-linked fucose (Fuc) residues and distinct sex- and life stage-specific features9–11. A strong and specific antibody response to a subset of S. mansoni glycans is raised by infected mammalian hosts both in natural and experimental infections12–15. These studies highlighted the potential of schistosome glycan antigens as targets for diagnostic serology and as vaccine candidates16–19. Such structural or functional glycomics studies have not been performed for S. haematobium despite the specific and highly significant public health impact associated with urogenital schistosomiasis. S. haematobium infections can lead to disease such as male and female genital schistosomiasis20,21 and bladder cancer22. Furthermore, S. haematobium is the human schistosome species most frequently involved in the worrisome zoonotic hybridizations that are increasingly observed with cattle schistosome species23. Compared to S. mansoni, the S. haematobium life cycle is more challenging to maintain in the laboratory resulting in lesser availability of species-specific material for research, including glycomics. Thus, general knowledge of S. haematobium glycobiology is limited to a few glycomic traits shared between Schistosoma species24, including the excretion of the Circulating Cathodic Antigen (CCA) and Circulating Anodic Antigen (CAA); two diagnostic carbohydrate antigens25,26. Although many (putative) glycosylation-related genes of S. mansoni27 have orthologues in S. haematobium28, differences between the two species are expected in view of their specific biology.

Here, we studied the lipid-linked GSL glycans and the protein-linked N- and O-glycans expressed by S. haematobium cercariae, adult worms and eggs in detail. We combined mass spectrometry (MS)-based approaches with enzymatic and chemical glycan sequencing to structurally define glycans in these three key life-stages of the parasite. We found that the S. haematobium glycome comprises many glycan elements shared with S. mansoni but expressed with major quantitative differences. Importantly, the identified S. haematobium GSL glycans also harbor species-specific structural features including a trihexosyl GSL core, cercarial branched structures with \beta-linked galactose (Gal), fucosylation patterns and glucuronic acid (GlcA) modifications in the eggs. A microarray with a specific focus on the differentially expressed native S. haematobium and S. mansoni glycans was constructed and screened with sera from schistosome-infected individuals, which revealed differential binding of IgG from S. mansoni and S. haematobium infected individuals to acidic GSL glycans in particular.

This study provides a detailed analysis of the hitherto unknown glycome and glycan antigen repertoire of S. haematobium. Our findings indicate that many S. haematobium glycan antigens, including some that are unique to the species, are immunogenic. This suggests that S. haematobium-specific glycans play a role in S. haematobium-host interactions that could be exploited as species-specific serology targets for epidemiological studies.

Results
I] Structural analysis of S. haematobium GSL, N- and O-linked glycans reveals major structural differences compared to S. mansoni

Lipid- and protein-linked glycans from S. haematobium cercariae, adult worms and eggs were released enzymatically using endoglycoceramidase II (rEGCase II) for the GSL glycans and N-glycosidases (PNGases) for the N-glycans, or chemically, via reductive \beta-elimination for the O-glycans. Released glycans were analyzed by Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)-MS, revealing complex spectra with many molecular ions (Figure S1 and Supplementary data 1), similar to previous observations for S. mansoni9. To tackle this complexity and allow efficient comparison between schistosome species, we focused on glycans yielding ions with signal intensities > 1% of the total MALDI-TOF-MS spectrum intensity and presence in replicate measurements (Supplementary data 2, GSL glycans; Supplementary data 3, N-glycans and Supplementary data 4, O-glycans). Glycan species are referred to throughout the manuscript using their theoretical masses in negative-ion mode for GSL and N-glycans ([M-H]-) and in positive-ion mode for O-glycans ([M+Na]+).

A) S. haematobium GSL glycans are built on a trihexosyl core extended with oligomeric chains of \beta-linked N-acetylhexosamine (HexNAc) residues

MALDI-TOF-MS analysis of 2-aminobenzoic acid (AA)-labeled S. haematobium GSL glycans (Figure 1) showed striking differences to the previously described S. mansoni GSL glycan profiles9. For all three life stages examined, the S. haematobium GSL glycan backbone invariably contained a minimum of three hexose (Hex, H) residues (Supplementary Data 1-2). \beta-N-acetylhexosaminidase (\beta-HexNAcase, Figure 2) could cleave N-acetylhexosamine (HexNAc, N) residues from GSL glycans of composition H3N2-6-AA (m/z 1030.37; 1233.45; 1436.53; 1639.61; 1842.63) that were either present in the native samples or obtained through defucosylation of structures of composition H3N2-6F1-6 using hydrofluoric acid (HF) (Figure S2.A-C). Resulting structures with m/z < 700 could not be detected by MALDI-TOF-MS, implying that the three hexoses are located at the reducing end of the glycans. Y-type fragment-ions corresponding to Y2-H3-AA and Y3-H3-AA (m/z 462.16; 624.21) were visible in MALDI-TOF-MS/MS fragmentation spectra of GSL glycans indicating that the three Hex residues are present in a linear stretch at the reducing end of the GSL backbone (Figure S3.A-C). We used an ultra-high performance liquid chromatography (UHPLC) platform, optimized for the detection of short oligosaccharides29, to further analyze the core trisaccharide that was obtained from exoglycosidase digestion of HF-treated S. haematobium egg GSL glycans (Figure S4). While \beta1-3,4 galactosidase had no effect on the structure, digestion with \alpha-galactosidases - both with \alpha1-3,6 and \alpha1-3,4,6 galactosidases – resulted in a disaccharide product, that could be further digested using \beta1-4 and \beta1-3,4 galactosidase (Figure 2). These results indicated that S. haematobium GSL glycans are built on a trihexosyl core constituted of two galactoses most likely extending a ceramide (Cer)-linked glucose (Glc) and forming the isoglobo-series core Gal\alpha1-3Gal\beta1-4Glc\beta1-Cer that has also been reported in the GSL glycans of the liver fluke Fasciola hepatica30,31. The trihexosyl core could also be detected using porous graphitized carbon nano-liquid chromatography coupled to tandem mass spectrometry (PGC-nano-LC-MS/MS, Figure S5). Corroborating the UHPLC analysis, the S. haematobium GSL glycan core exhibited a different elution pattern in the PGC-nano-LC-MS analysis compared to the synthetic globotriose (Gb3) standard Gal\alpha1-4Gal\beta1-4Glc, but its elution profile was consistent with that of the isoglobotriose (iGb3) standard Gal\alpha1-3Gal\beta1-4Glc (Figure S5.A). The occurrence of this trihexosyl core structure in S. haematobium greatly contrasts with S. mansoni GSL glycans where the ceramide-linked glucose (Glc) is extended with \beta1-4-linked N-acetylgalactosamine (GalNAc)32,33.

The MALDI-TOF-MS/MS analysis (Figure S3.A-C) and \beta-HexNAcase digestions of HF-treated structures (Figure S2.A-C) mentioned previously showed that stretches of \beta-linked HexNAc residues extend the trihexosyl core in most S. haematobium GSL glycans. Resistance to treatment with Streptococcus pneumoniae \beta-N-acetylglucosaminidase (\beta-GlcNAcase) indicated that \beta1-4-linked GalNAc is capping the structures with H3N2-6F0-6 compositions (Figure S2.A), as \beta-GlcNAcase lacks activity towards \beta1-4 linked GalNAc (Figure 2). This is similar to previous observations in S. mansoni where HexNAc stretches of the GSL backbones were shown by monosaccharide analysis to consist predominantly of N-acetylglucosamine (GlcNAc) with the exception of the terminal and core-GalNAc residues32,34–36. Thus, unless determined otherwise, we assumed the backbone HexNAc to be primarily constituted of \beta-linked GlcNAc, by analogy (Figure 3).

B) Acidic GSL glycans in S. haematobium: identification of terminal glucuronic acid (GlcA) in adult worms and abundant subterminal GlcA-containing glycans in eggs

Several ions detected in the MALDI-TOF-MS spectra of S. haematobium worm and egg GSL glycans could not be assigned to glycan compositions containing solely monosaccharide constituents detected previously in S. mansoni GSL glycans9. PGC-nano-LC-MS/MS analysis of the ion with m/z 1087.37 detected in adult worms showed a clear loss of 176.03 Da directly from the parent ion resulting in an ion with m/z 911.34 (Y5-H3N2) (Figure S5.B). A mass increment of 176 Da matches the mass of either a methylated hexose or a hexuronic acid (HexA, A) two types of residues that have been observed in helminths and mollusk glycans, including in Biomphalaria snails37,38. Efficient ionization in negative-ion mode indicated that the ion species with m/z 1206.41 and m/z 1612.56 contained HexA, with compositions of H3N2A1 and H3N4A1-AA, respectively. In line with the fragmentation pattern, \beta-HexNAcase had no effect on these structures, while both could be digested using \beta-glucuronidase (Figure S2.B), showing that a terminal \beta-linked GlcA is capping these adult worm GSL glycans (Figure 1.B and Supplementary data 2). The \beta-glucuronidase digestion product (m/z 1030.37 (H3N2-AA)) was then sensitive to \beta-HexNAcase but was resistant to \beta-GlcNAcase (Figure S2.B) showing that GlcA was linked to GalNAc (Figure 2).

HexA-containing GSL glycans of composition of H3N2-7A1F0-3 constituted a major subset of S. haematobium egg GSL glycans (Figure 1.C and Supplementary data 2). Unlike in adult worms, MS/MS fragmentation patterns suggested a subterminal position of the HexA residue, with the visible loss of a HexNAc (203.08 Da) from parent ions immediately followed by the loss of HexA (176.03 Da) (Figure S3.C, MALDI-TOF-MS/MS and Figure S5.C, PGC-nano-LC-MS/MS). Further glycan sequencing work showed that \beta-HexNAcase could cleave a single HexNAc from these HexA-containing glycans (Figure S2.C). Upon removal of the capping HexNAc, these egg GSL glycans became sensitive to \beta-glucuronidase, confirming the presence of subterminal \beta-linked GlcA. The products of \beta-glucuronidase-treatment could then be further digested with \beta-HexNAcase to structures of composition H3N2-4F1-3. \beta-GlcNAcase was also effective on a substantial proportion of the \beta-glucuronidase digestion products (Figure S2.C, Supplementary data 2), suggesting that the GlcA can be \beta-linked to either GalNAc or GlcNAc in the egg GSL glycan backbone (Figure 2). For simplicity, we represented GalNAc-containing isomers in Figure 1, Figure 4 and Figures S2-5.

C) Lower abundance of acidic GSL glycans in S. mansoni eggs compared to S. haematobium

Acidic GSL glycans have not been reported for S. mansoni9, although preliminary data suggested that some S. mansoni egg GSL glycans contain a HexA residue39. To further investigate this and perform a direct comparison with S. haematobium, we conducted a parallel glycomic analysis of egg GSL glycans from both species (Figure 4). In line with previous data, all 10 major signals in the S. mansoni egg profile were assigned to neutral structures of composition H3N1-7F2-8, unlike in S. haematobium, where 7 out of the 10 major ions contain HexA (Figure 4.A, Supplementary data 2). When acidic and non-acidic glycans were separately eluted and subjected to MALDI-TOF-MS analysis (Figure 4.B-C), the HexA-containing glycans described above were recovered in the acidic GSL glycan fraction from S. haematobium eggs, while the m/z of the main ions detected in the neutral fraction corresponded to structures of compositions H3N2-7F0-3 (Supplementary data 2). For S. mansoni eggs, the m/z values observed in the neutral fraction accurately matched the GSL glycans already defined9, while the acidic profile revealed a substantial number of previously uncharacterized molecular ions with compositions including HexA, that constitute a minor subset of the total S. mansoni GSL glycan pool (Figure 4). Structural characterization indicated a similar subterminal context for the HexA residue in the backbone of S. mansoni egg GSL glycans (Figure 4 and Supplementary data 2). Digestion of a single HexNAc was obtained by applying \beta-HexNAcase to the HF-defucosylated glycans (Figure S2.E) and the invariable loss of one HexNAc followed by one HexA from parent ions was visible in MALDI-TOF-MS/MS (Figure S3.D) and PGC-nano-LC-MS/MS analysis (Figure S5.E-F). In both S. haematobium and S. mansoni eggs, the HexNAc residue capping acidic GSL glycans was never fucosylated (Figure S3.C-D and Figure S5.C, E-F) and comparison between \beta-HexNAcase and \beta-GlcNAcase activities (Figure 2) suggested the majority of these capping HexNAc residues to be a \beta1-4-linked GalNAc (Figure S2.C, E).

D) Differential fucosylation of S. haematobium and S. mansoni egg GSL glycans

Fucosylation levels varied substantially between S. haematobium and S. mansoni egg GSL glycans. While the main GSL glycans of S. haematobium have a maximum of three Fuc residues and several acidic GSL glycans are not fucosylated, the major structures identified in S. mansoni comprised three to eight fucoses (Figure 4 and Supplementary data 2). Both the acidic and neutral GSL glycans of S. haematobium eggs were sensitive to \beta-HexNAcase, that could cleave up to four HexNAc residues from the neutral ones (Figure S2.C). Resistance to treatment with \beta-GlcNAcase indicated that \beta1-4-linked GalNAc is capping neutral structures with H3N2-7F0-3 compositions, forming GalNAc\beta1-4GlcNAc (LacDiNAc, LDN) motifs, as in S. mansoni9,33,36. In S. mansoni egg GSL glycans, however, these terminal LDN motifs were found to be fucosylated, as previously reported9. Similarly, in the acidic fraction, HF-defucosylation was necessary for \beta-HexNAcase to act (Figure S2.E) due to the presence of multiple Fuc substituents near the non-reducing end of the GSL glycan backbone. Thus, Fuc residues are not only more abundant in S. mansoni egg GSL glycans, but their position on the GSL glycan backbone constitutes another major difference between species.

Resolving the structure of such (multi)fucosylated glycans by MALDI-TOF-MS/MS is notoriously challenging33,40, but is enabled by the high isomer separation power of PGC chromatography. Using PGC-nano-LC-MS/MS, we detected several isomeric structures resulting from variable Fuc distribution among the acidic GSL glycans of S. mansoni (Figure S5.F). We also found the HexNAc residue directly preceding HexA to be invariably fucosylated, and often with Fuc residues forming the di-fucosyl motifs (Fuc\alpha1-2Fuc\alpha1-3, DF). In contrast, DF elements were absent in S. haematobium egg GSL glycans for which HexNAc residues were found either unsubstituted or substituted only with a single Fuc (Figure S5.C-D). When present, Fuc was found preferentially attached to the HexNAc residue(s) close to the trihexosyl core. In the acidic GSL glycans, this left the HexA preceded by a chain of three or more unsubstituted HexNAc residues. Fewer isomeric structures were identified in the GSL glycans of S. haematobium than in S. mansoni egg GSL glycans, which is a direct consequence of the lower degree of fucosylation in S. haematobium (Figure 4). For S. mansoni, the abundance of highly fucosylated GSL structures was shown to increase with egg maturity9. To investigate a potential change in glycosylation during egg development in S. haematobium, we released and analyzed GSL glycans from separated immature and mature eggs. The resulting MALDI-TOF-MS spectra were virtually identical (Figure S2.D), suggesting that, during maturation, the S. haematobium eggs do not develop the highly fucosylated motifs ubiquitous in the mature eggs of S. mansoni.

E) Branched \beta-Gal in S. haematobium cercarial GSL glycans

Several non-fucosylated cercarial GSL glycans appeared to have H4-7N1-2 compositions, containing hexoses in addition to the trihexosyl core (Figure 1, Figure 3, Supplementary data 2). These glycans were terminated with \beta-1-4 linked Gal as shown by sensitivity to \beta-1-4 galactosidase (Figure S2.A). MALDI-TOF-MS/MS analysis of the \beta1-4 galactosidase digestion products at m/z 1354.48 (H5N2-AA) and 1516.53 (H6N2-AA) (Figure S3.A) each showed a loss of both one Hex (fragment ions with m/z 1192.43 and 1354.48) and one HexNAc residue (fragment ions with m/z 1151.40 and 1313.45), suggesting these structures to be branched, an unusual trait in the typically linear GSL glycans. Further digestion with \beta-GlcNAcase, indicated the branched HexNAc to be \beta-linked GlcNAc while sensitivity to \beta1-3,4 galactosidase but not with \beta1-4 galactosidase (Figure S2.A, Supplementary data 2) indicated the branched Hex to be a \beta1-3 Gal, in contrast to the terminal \beta1-4 Gal (Figure 2). These glycans did not resemble any of those previously described for S. mansoni.

F) Life stage-specific expression of (fucosylated) terminal LDN and LacNAc (LN) motifs in S. haematobium GSL glycans

Fucosylation, including \alpha1-2 linked Fuc residues forming DF or tri-fucosyl motifs (Fuc\alpha1-2Fuc\alpha1-2Fuc\alpha1-3, TF) is a unique and antigenic feature of Schistosoma glycans13,41,42. DF and TF motifs, which are particularly abundant in S. mansoni egg GSL glycans were not detected in the eggs of S. haematobium (Figure 4) but were found in the GSL glycans of S. haematobium cercariae and worms (Figure 1.A-B), where they form fucosylated terminal LDN motifs (Figure 3). In addition, terminal LacNAc (LN, Gal\beta1-4GlcNAc) and Lewis X (LeX, Gal\beta1-4(Fuc\alpha1-3)GlcNAc) motifs were observed in cercarial and adult worm GSL glycans (Figure 1, Figure 3, Supplementary data 2) as determined using MALDI-TOF-MS/MS analysis (Figure S3.A-B) and sensitivity to HF treatment and to a combination of \beta1-4 galactosidase and \beta-GlcNAcase (Figure S2.A-B). LN and LeX termination were found to be major features of S. haematobium adult worm GSL glycans, together with fucosylated LDN and the GlcA-containing motifs described above (Figure 1, Figure 3). This diversity of motifs contrasts with S. mansoni adult worms GSL glycans, largely terminated by (multi)fucosylated LDN motifs9. In addition, we did not find any evidence of pseudo-Lewis Y (Fuc\alpha1-3Gal\beta1-4(Fuc\alpha1-3)GlcNAc) in S. haematobium GSL glycans, while it is the most prominent terminal motif in S. mansoni cercarial GSL glycans9.

G) N- and O-linked glycans of S. haematobium show quantitative differences to those of S. mansoni

N-linked glycans were released by sequential use of PNGase F and PNGase A and structurally characterized using glycan sequencing approaches (Figure S6). Many of the features previously reported for S. mansoni9 were also detected in S. haematobium N-glycans, including presence of terminal LN, LeX and (fucosylated) LDN motifs as well as the non-mammalian core-modifications \beta1-2 linked xylose and \alpha1-3 linked core-Fuc (Figure 5 and Supplementary data 3). Stage-specific expression of many glycan motifs was observed. N-glycans carrying \alpha1-3 core-fucose, released with PNGase A, were exclusively detected in the mature eggs (Figure 5.D, Figure S6.D-E). In addition, these glycans were found to include high molecular weight structures (>2500 Da), most likely containing LN repeats based on their monosaccharide composition (Figure S6.G). Interestingly, the MALDI-TOF-MS profiles of glycans released from immature and mature S. haematobium eggs by PNGase F were virtually identical (Figure S6.F), which differs from the increase in complex, highly fucosylated N-glycans observed in S. mansoni during egg maturation9. Certain features of the evolution of N-glycan expression throughout the parasite life cycle appeared common to S. haematobium and S. mansoni such as the shift from cercarial terminal LN/LeX to LDN in adult worms and eggs9. However, many qualitative differences in the distribution of the glycan motifs were also observed between both species (Figure S7) particularly for cercarial N-glycans. Generally, we found that S. haematobium N-glycans were shorter, less complex, and less fucosylated than those of S. mansoni, following the trend shown for the GSL glycans.

Following reductive \beta-elimination, released O-glycans were permethylated and analyzed by positive-ion mode MALDI-TOF-MS (Figure 6). Glycan-derived signals were detected in each of the life stages, but with very low intensity for the adult worms, similar to S. mansoni9, preventing further interpretation. Structural characterization of S. haematobium cercarial and egg O-glycans was performed using MALDI-TOF-MS/MS (Figure S8) and indicated that this class of glycans is built either on the conventional O-glycan core-2 of composition H1N2 (Gal\beta1-3(GlcNAc\beta1-6)GalNAc), or on a H2N1 schistosome-specific core (Gal\beta1-3(Gal\beta1-6)GalNAc)43. The majority of the cercarial O-glycans were built on the latter, while core-2 was more abundant in the eggs (Figure 6, Supplementary data 4), as reported also for S. mansoni O-glycans9,44. In both life stages, HexNAc, Hex and Fuc residues extended these cores, most likely forming LN and LeX motifs, as well as (fucosylated) terminal GlcNAc (Supplementary data 4, Figure 6 and Figure S8). MALDI-TOF-MS/MS analysis indicated the presence of isomeric structures, for instance in the eggs, where the ion species at m/z 1780.92 (F2H3N3) corresponded to two structures, one presumably built on the branched Gal\beta1-3(Gal\beta1-6)GalNAc core and a linear version containing multiple LeX motifs (Figure S8.B). S. haematobium O-glycans also contain fucosylated LDN motifs (Figure 6 and Supplementary data 4), as S. mansoni9, although the presence of DF was exclusively observed in the cercariae for S. haematobium with the linear glycan at m/z 1720.90 (F4H1N3) and the structure with m/z 2907.42 (F8H1N5) (Figure 6, Figure S8.B). The latter corresponds to the highly fucosylated core-2 O-glycan of composition F8H1N5, also reported in S. mansoni eggs and cercarial glycocalyx45. The proportion of short, truncated O-glycans with m/z <1100 was scarcer in S. haematobium eggs than in S. mansoni.

II] Differential recognition of Schistosoma glycans by antibodies from infected children

Knowing that many S. mansoni glycans are antigenic, we constructed microarrays of native glycans isolated from S. mansoni and S. haematobium to comparatively assess the antigenicity of S. haematobium glycans. Glycan-containing fractions were obtained through UHPLC-purification (as detailed in Methods and in Figure S9) and selected to provide a broad coverage of the structural diversity of glycans from both species (data not shown), with a particular focus on GSL glycans among which major differences between S. haematobium and S. mansoni were observed. Details of glycan fraction compositions are provided in Supplementary data 5. A selection of monoclonal antibodies (mAbs) binding specific glycan motifs was used to ensure the printing quality of the generated microarrays (Figure S9, Supplementary data 6) prior to incubation with infected and control sera. In addition, binding patterns of the mAbs confirmed the structural assignment of terminal LeX, LDN and F-LDN(-F) motifs in S. haematobium GSL and N-linked glycans (Supplementary Data S2-3).

A) Immunogenic glycan motifs in S. haematobium and S. mansoni infections

Once validated, the glycan microarrays were screened for IgG and IgM binding with sera from children infected with either S. haematobium (n = 18) or S. mansoni (n = 21) and with sera from non-endemic individuals (n = 9). IgG (Figure 7) and IgM (Figure S10) responses yielded significantly higher median fluorescence intensity (MFI) values for both groups of infected children compared to the uninfected controls, with significantly higher binding to 117 (IgG) and 120 (IgM) array fractions out of 151 for the S. haematobium-infected group compared to the non-endemic control group (Supplementary data 7 and Supplementary data 8).

Infected individuals – with either of the two species – showed IgG and IgM responses higher than the control group to N- and O-linked glycans harboring LDN-F, fucosylated GlcNAc, (multifucosylated) LDN or high-mannosidic structures (Man8-9) (Figure S11). Higher IgM binding than the control group was measured to LeX-containing N-glycans but we noted the absence of anti-LeX IgG reactivity (fractions NG-7 and NG-8, Figure S11, Supplementary data 7 and Supplementary data 8), in line with previous reports in S. mansoni infections13. IgG and IgM binding to multifucosylated glycans yielded strikingly high MFI values in both infection groups compared to non-endemic sera (Figure 7 and Figure S10). Such structures harboring multifucosylated motifs were particularly abundant in S. mansoni GSL glycans and also present in some O-linked and GSL glycan-containing fractions isolated from S. haematobium cercariae and worms (Supplementary data 5), in line with our structural observations (Figure 1). The few non-reactive fractions mainly contained truncated and mammalian-like N-glycans or short GSL glycans (Figure 7 and Figure S10, Supplementary data 5).

B) Differential IgG binding to acidic egg GSL glycans

Contrasting the above similarities, clear differences between the two infection groups were observed for IgG responses to fractions containing GSL glycans derived from S. haematobium eggs (Figure 7). Significantly higher MFI values were measured to 24 fractions for IgG binding in S. haematobium-infection sera compared to S. mansoni-infection sera (Supplementary data 7). We further investigated IgG responses to egg GSL glycans by grouping fractions based on the species from which they originated with a distinction between intact glycans or glycans treated with HF for fucose removal (Figure 8). Reflecting the observations made at the individual fraction level, the average of the MFI values measured for S. haematobium egg GSL glycans was significantly higher in S. haematobium than in S. mansoni infection sera (Figure 8.A-1). When averaging MFI values recorded from IgG binding to S. mansoni egg GSL glycans (Figure 8.A-2), on the other hand, there were no significant differences between the two infection groups (p > 0.05). HF-defucosylated S. mansoni fractions yielded markedly lower MFI values than those containing intact GSL glycans, showing that, for both infection groups, a substantial part of the IgG reactivity to S. mansoni egg GSL glycans was fucose-dependent. This was also observed for the IgG response from S. mansoni-infected individuals to S. haematobium-egg GSL glycans (Figure 8.A-1). However, this decrease in MFI levels between intact and HF-treated S. haematobium-GSL glycans was not as marked for S. haematobium-infected individuals, which also contain IgG against non-fucosylated epitopes.

Strikingly, most of the fractions to which differential IgG binding was observed between S. haematobium- and S. mansoni-infection sera were derived from S. haematobium eggs and contained acidic GSL glycans, i.e. structures that comprise GlcA in their compositions (Supplementary data 5 and Supplementary data 7). Therefore, we separately evaluated IgG binding to fractions based on acidic or neutral content, keeping the distinction between HF-treated and native glycans to assess the role of fucosylation in IgG binding (Figure 8.B-C). This analysis highlighted a strong IgG response to acidic GSL glycans in S. haematobium-infected individuals which was not observed in S. mansoni-infected individuals (p < 0.001, Figure 8.B-1). Glycan defucosylation by HF treatment had a very limited impact on IgG binding from S. haematobium infection sera, showing that the HF-resistant, GlcA-containing glycan epitope is a major IgG target in S. haematobium infections, but this was not the case for S. mansoni infections. S. haematobium and S. mansoni infection sera both showed a strong IgG binding to the acidic GSL glycans isolated from S. mansoni eggs, which was fucose-driven as a sharp decrease of IgG responses was observed to the HF-defucosylated versions of these glycans (Figure 8.B-2). Finally, there were no significant differences between both infection groups for IgG responses to the neutral GSL glycans (p > 0.05), confirming the role of the acidic residue in the preferential IgG binding from S. haematobium infections (Figure 8.C). Comparison of IgG binding from infection and control sera to the neutral fractions emphasized again the major role for fucosylated epitopes in IgG responses during schistosomiasis.

To summarize, the present glycan microarray-assisted study revealed that sera from S. haematobium-infected individuals contain IgG and IgM targeting a wide range of Schistosoma-derived glycans. These antibodies are partly directed to the same antigenic glycan elements as anti-glycan antibodies in S. mansoni infection sera. However, whereas fucosylation appears to be the dominant element in S. mansoni, the newly identified Schistosoma egg GSL glycans containing GlcA are highly antigenic and targeted by IgG responses specifically in S. haematobium infections.

Methods
1. Structural characterization of glycans

Enzymatic release and AA labeling of N-glycans and GSL glycans from parasite proteins and lipids

The various life stages of S. haematobium (Egyptian NAMRU-3 strain)79 and S. mansoni (Puerto Rican strain)80 were obtained from the parasite life cycles maintained in male Golden Syrian hamsters (RjHan:AURA strain, Janvier labs, France) at Leiden University Medical Center in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Research and have received approval from the Dutch ‘Central Authority for Scientific Procedures on Animals’ (CCD, License number: AVD11600202215904). Worms and eggs were recovered from hamsters 6–7-week post-infection for S. mansoni and 16–20-week post-infection for S. haematobium. S. haematobium and S. mansoni cercariae were recovered from Biomphalaria glabrata and Bulinus truncatus snail shedding, respectively, at various time-points post-infection. About 1.2 million eggs, 125 000 cercariae and 100 worms from S. haematobium were used as starting material on two different occasions in order to generate biological replicates and about 0.8 million eggs, 150 000 cercariae and 240 worms were used from S. mansoni for direct comparison purposes. When unspecified, mixtures of eggs at different stages of maturity were used. To separate immature from mature eggs, a pool of 660,000 S. haematobium eggs was subjected to a Percoll gradient centrifugation protocol81. The eggs were first centrifuged in 40% Percoll in a saline solution of 1.7% sodium chloride (w/v). The immature egg layer was collected, and the pellet containing mature eggs was processed through a second centrifugation step in 50% Percoll prepared in saline to further eliminate residual debris. N-linked and GSL glycans were enzymatically released from ~100,000 of the mature and immature eggs thus isolated.

Glycoprotein/lipid preparation and glycan release were obtained through established protocols9. Pools of parasite material were ground in a Potter-Elvehjem homogenizer in 1 mL of ultrapure water (MQ, Milli-Q®, Millipore Sigma) and subjected to protein and lipid extraction by homogenization in methanol (MeOH) and chloroform in a final 4:7:13 ratio of MQ:MeOH:chloroform. Samples were sonicated and centrifuged at 1000 g for 5 minutes, the upper phase removed and replaced by 50% MeOH.

This step was repeated twice and (glyco)lipids from the upper phase of extraction were subsequently purified using octadecylsilane (C18) cartridges (#7020-03 BAKERBOND® spe™, JT Baker®). Cartridges were conditioned with MeOH and 10% MeOH and samples were applied to the cartridges that were then washed with MQ. Flow-through and wash were collected, combined and used for an additional C18 purification on new C18 cartridges following the same procedure. Glycolipids were eluted from both cartridges with chloroform:MeOH:MQ(10:10:1) and elutions were combined and dried under nitrogen (N2) flow. Dried (glyco)lipids were reconstituted in 500 µL 50 mM sodium acetate, pH5 containing 0.1% sodium taurodeoxycholate and lipid extracts were treated with 4 mU of recombinant rEGCase II (#4460, Takara) for 48h at 37℃. After 24h, another 4 mU of rEGCase II was added. Following enzymatic release, GSL glycans were purified sequentially on C18 and carbon cartridges (#57088 Millipore Sigma Supelclean™ ENVI-Carb SPE). Released glycans were loaded on C18 cartridges conditioned with MeOH and MQ. Flowthroughs from load and from washes with 10% acetonitrile (ACN) and MQ were combined for each sample. The collected flowthroughs were next loaded on carbon cartridges conditioned sequentially with ACN, 50% ACN containing 0.1% trifluoroacetic acid (TFA) and MQ. After washing with MQ, the glycans were eluted with subsequent addition of 25% ACN then 50% ACN containing 0.1% TFA. Both elutions were pooled and dried in a SpeedVac.

Simultaneously, (glyco)proteins in the lower phase from the protein/lipid separation procedure described above were pelleted using excess volumes of MeOH. Protein pellets were dried under N2 flow and then homogenized in PBS with 1.3% SDS and 0.1% \beta-mercaptoethanol. Denaturation was performed for 10 minutes at 95°C, samples were cooled to room temperature and Nonidet P-40 (1.3% final concentration) added to each sample. To generate tryptic peptides, 100 µL of trypsin-coated Sepharose beads prepared according to manufacturer’s instructions (GE Healthcare) were added to each sample and incubated overnight at 37℃. The beads were spun down, and peptide-containing supernatants collected. N-linked glycans were first released using 3500 units of PNGase F (#P0709, New England Biolabs (NEB)) and incubation at 37°C for 24h. Released glycans were cleaned up using C18 and carbon cartridges. Samples were loaded on C18 columns conditioned with ACN, 60% ACN containing 0.1% TFA and MQ and collected in the flowthrough of 10% ACN and MQ washes. Peptides were eluted from C18 columns using first 30% ACN with 0.1% TFA then 60% ACN with 0.1% TFA. Released glycans were further purified using carbon SPE as described for GSL glycans. Eluates from both C18 and carbon cartridges were dried in a SpeedVac. Peptide-containing C18 eluates were redissolved by sonication in 50 mM sodium acetate, pH 6 at 25°C (Glycobuffer 3, NEB). 25 units of PNGase A (#P0707, NEB) were added to each sample prior to incubation for 48h at 37℃ to release potential PNGase A specific N-glycans. Reactions were cleaned up using C18 and carbon cartridges purifications sequentially as described above. C18 and carbon column eluates were dried in a SpeedVac.

Released and dried glycans were resuspended in MQ and labeled with AA by reductive amination with sodium cyanoborohydride as described82. Labelling was performed for 2h at 65°C. To remove excess labelling reagent, ACN was added to a final concentration of 75% and the sample was loaded onto Bio-Gel P10 Gel resin (#1504144, Bio-Rad) previously conditioned with MQ and 80% ACN. Resin was washed with excess volume of 80% ACN, then glycans were eluted with MQ and dried using a SpeedVac.

Separation of acidic and neutral GSL glycans for in-depth glycomic studies

About 1.8 million S. haematobium eggs, 365 S. haematobium worms and 2.5 million S. mansoni eggs were homogenized using a Potter-Elvehjem. Lipid extraction and enzymatic glycan release using rEGCase II was performed as detailed earlier. Subsequent C18 and carbon purifications were also identical except that the two final elution steps from the carbon cartridges (25% ACN and 50% ACN containing 0.1% TFA) were kept separate, resulting in two fractions for each sample, containing “neutral” and “acidic” GSL glycans, respectively. These fractions were dried in a SpeedVac and reconstituted in MQ. A fifth of each sample was aliquoted for PGC-nano-LC-MS/MS analysis (see below) while the rest was labeled with AA, as described above, for MALDI-TOF-MS analysis.

Chemical release and permethylation of O-glycans

About 200 000 eggs, 20 000 cercariae and 100 worms from the S. haematobium life cycle were subjected to reductive \beta-elimination to release O-linked glycans. Biological material from each stage was ground in a Potter-Elvehjem homogenizer in 4 mL MQ and lyophilized by freeze-drying. The dried samples were redissolved by sonication in 200 µL MQ, then split into two aliquots of 100 µL to generate technical replicates. 100 µL of a solution containing sodium borohydride (2M) and sodium hydroxide (0.2M) was added to each replicate and incubated at 40°C for 24h. Samples were neutralized on ice by adding 4M acetic acid. MeOH with 1% acetic acid was added repeatedly with intermediate and final drying under a stream of N2 until a neutral pH was reached. Released glycans were dissolved in 1mL MQ and C18 purification identical to the N-glycan procedure was performed. C18 flowthroughs were subjected to sequential SPE using carbon cartridges previously conditioned in 80% ACN with 0.1% TFA and MQ After washing with MQ, the glycans were eluted with 25% ACN and 25% ACN with 0.05% TFA prior to concentration to dryness in a glass tube by N2 flow. To facilitate MALDI-TOF-MS analyses O-glycans were next permethylated according to83. For this, 100 µL of DMSO saturated with sodium hydroxide powder was added to each tube and left for 10 minutes at room temperature. Next, 100 µL of iodomethane was added followed by an additional 10 minute incubation at room temperature. Samples were then mixed by inversion in 400 µL of dichloromethane and 500 µL MQ. The aqueous layer was discarded and replaced with 500 µL MQ. This step was repeated at least five times or until neutral pH was reached. Finally, samples were dried under N2 flow and redissolved in 80% MeOH for MALDI-TOF-MS analysis.

Glycan sequencing using hydrofluoric acid (HF) and exoglycosidase treatments

In order to elucidate glycan structures, released N-linked and GSL glycans were subjected to selective digestion of glycans. HF was used to remove \alpha1-2/3/4-linked fucoses from the glycan backbone by mixing small aliquots of AA-labeled glycans suspended in MQ with cold 48% HF in a 1:100 ratio. Samples were incubated at 4°C for 48h and HF was then removed by evaporation under N2 flow. Multiple washes were performed by addition and subsequent N2 evaporation of MeOH. Finally, the HF-treated glycan samples were re-dissolved in MQ. A panel of exoglycosidases listed in Figure 2 was used to selectively remove monosaccharides from glycans. Digestions of a few µL of glycan samples were performed overnight in recommended conditions. All enzymes were from NEB and used at 37℃ except for the \beta-glucuronidase (Millipore Sigma) used at 50℃. Digestions with \beta-glucuronidase were performed in citrate buffer (100mM, pH4) from Teknova (#Q2444) while reactions with \alpha1-2,3,6 mannosidase and \beta1-3,4 galactosidase were performed in NEB glycobuffer 4 (50 mM sodium acetate, pH 4.5) supplemented with zinc for the \alpha1-2,3,6 mannosidase. All other enzyme reactions were performed in NEB glycobuffer 1 (5 mM CaCl2, 50 mM sodium acetate, pH 5.5), supplemented with BSA (1mg/mL) for the \alpha-galactosidases. Following HF and exoglycosidase treatment, clean-up and enzyme removal was performed using a previously detailed C18 clean-up84. Briefly, Millipore® Zip-Tips (Millipore Sigma # ZTC18S) were conditioned with 50% ACN containing 0.1% TFA and then with 0.1% TFA. AA-labelled glycans were bound to the C18 resin prior washing with 0.1% TFA. Finally, undigested controls and digestion products were eluted in 50% ACN containing 0.1% TFA.

MALDI-TOF-MS and MS/MS analysis

AA-labeled glycans released from different parasite life cycle stages and products of HF and exoglycosidase digestions were analysed by MALDI-TOF-MS using Bruker Daltonics mass spectrometers controlled by the software FlexControl 3.4 Build 119 (Bruker Daltonics). UltrafleXtreme® and Autoflex® instruments equipped with 1kHz Smartbeam lasers and a Rapiflex® equipped with a 10 kHz Smartbeam-3D laser technology were used based on availability. Samples were spotted on a 384 well steel polished target plate. AA-labeled glycans solubilized in MQ were mixed on the plate with 2,5-dihydroxybenzoic acid (DHB) matrix (#8201346, Bruker Daltonics, 20 mg/ml in 30% ACN) while products of exoglycosidase digestions were directly eluted onto the plate in 50% ACN, 0.1% TFA mixed with DHB (10 mg/ml) at the end of the enzyme removal with C18 Millipore® Zip-Tips. All spectra were obtained in the negative-ion reflectron mode using Bruker® peptide calibration mix (#8206195, Bruker Daltonics) for external calibration. Spectra were obtained over a mass window of m/z 700 – 3500 with ion suppression below m/z 700 for a minimum of 20,000 shots (2000 Hz) obtained by manual selection of “sweet spots”. The software FlexAnalysis (Version 3.4, Build 50, Bruker Daltonics) was used for data processing including smoothing of the spectra (Savitzky Golay algorithm, peak width: m/z 0.06, 1 cycle), baseline subtraction (Tophat algorithm) and manual peak picking. Peaks with a signal-to-noise ratio < 4 were excluded as well as known non-glycan peaks such as glucose polymers. Deprotonated masses of the selected peaks were assigned using the GlycoPeakfinder® tool of the free software GlycoWorkBench85 (Version 3, 29 June 2007). The AA label was taken into account as a fixed reducing-end modification and possible glycan composition was based on literature data on S. mansoni9 and general knowledge of helminth glycobiology (i.e. 0-15 deoxyhexoses, 0-5 hexuronic acids, 0-20 hexoses and N-acetylhexosamines, 0-1 pentose). A deviation of 300 ppm was allowed for initial assignment of compositions. MS/MS was performed for structural elucidation via fragmentation ion analysis by MALDI-TOF/TOF on selected ions using the UltrafleXtreme® or Rapiflex® mass spectrometers in negative-ion mode. Information obtained from MS/MS profiles and from the various glycan sequencing procedures (i.e. HF treatment and exoglycosidase digestions) were combined to determine the final glycan composition associated with the detected m/z values in the MALDI-TOF-MS spectra and to characterize the corresponding glycan structures.

Permethylated O-glycans

Reduced and permethylated O-glycans (1 µL) were spotted onto a 384 well steel polished target plate with 1 µL DHB (10 mg/ml in 80% MeOH). They were analysed by MALDI-TOF-MS, similarly to the N-linked and GSL glycans, but using the UltrafleXtreme® instrument in positive-ion reflectron mode. MALDI-TOF-MS/MS fragmentation of selected ions was also performed in positive-ion reflectron mode and results of this analysis were used to assign the O-glycan structures.

Structural characterization of GSL glycans using PGC-nano-LC-MS and MS/MS

GSL glycans isolated from eggs of S. haematobium and S. mansoni, and from S. haematobium worms with the acidic and neutral fractions processed separately (see above) were next reduced and desalted by PGC SPE based on previously established procedures86,87. Briefly, GSL-glycans were reduced to alditols in 20 μL of sodium borohydride (500 mM) in potassium hydroxide (50 mM) for 2 h at 50°C. Subsequently, 2 μL of 100% glacial acetic acid was added to neutralize the solution and quench the reaction prior to desalting by cation-exchange88. Glycans were eluted in MQ and dried by Speedvac. Dried samples were reconstituted in 0.1% TFA and loaded onto tips packed with 60 μL of bulk sorbent carbograph slurry (50 mg/mL in MeOH) previously conditioned three times with 100 μL of 80% ACN containing 0.1% TFA and three times with 100 μL of 0.1% TFA. Samples were washed three times with 50 μL of 0.1% TFA and eluted from tips twice with 30 μL of 60% ACN containing 0.1% TFA. Collected GSL glycan samples were dried by SpeedVac and reconstituted in MQ. Aliquots of released and reduced GSL glycans were treated with HF as described above. The defucosylated neutral GSL glycans from S. haematobium eggs were additionally subjected to digestion with \beta-HexNAcase using the reaction conditions detailed above. The GSL glycan samples resulting from HF and exoglycosidase treatments were cleaned again using C18 cartridges followed by PGC SPE as described above. After clean-up, reduced GSL glycans – native or HF treated/digested – were dried in a SpeedVac and re-suspended in 10 µL MQ and 2 µL were injected for PGC-nano-LC-MS/MS analysis. GSL glycan standards Gb3 and iGb3 were obtained by releasing the glycan moiety of synthetic trihexosylceramides from Avanti Research (Gb3‑Ceramide #860699 and iGb3‑Ceramide # 860722) prior to reduction and clean-up as described above. A Dionex Ultimate 3000 nano-LC system (Thermo Fisher Scientific) equipped with a home-packed PGC trap column (5 μm Hypercarb, 320 μm x 30 mm) and a home-packed PGC nano-column (3 μm Hypercarb 100 μm x 150 mm) connected to an amaZon speed ETD ion trap (Bruker Daltonics) were used for the detection of glycan alditols. Mobile phase B contained 60% ACN and 10 mM Ammonium Bicarbonate Buffer (ABC), while mobile phase A was 10 mM ABC. To analyze GSL glycans, a gradient of B (1-51% at 0.5%/min) at a flow rate of 0.6 μL/min was employed. The temperature of the column was maintained constant at 45°C. Ionization was achieved using the CaptiveSpray nanoBooster source (Bruker Daltonics) with a capillary voltage of 1000 V, dry gas temperature of 280 °C at 5 L min−1 and nebulizer at 3 pound per square inch (psi). Isopropanol-enriched dopant N2 was used. MS spectra were acquired within an m/z range of 340–1850, smart parameter setting was set to m/z 800. MS/MS spectra were generated using collision-induced dissociation over an m/z range from 100 to 2500 on the top three most abundant precursors. Data were analyzed in Compass DataAnalysis, Version 5.0, Build 203 (Bruker Daltonics). Glycan structures were assigned on the basis of the known MS/MS fragmentation patterns in negative-ion mode, elution order, and general glycobiological knowledge, with the help of GlycoWorkBench85 (Version 3, 29 June 2007) as detailed above.

Raw MS and MS/MS data generated during this study have been deposited in Glycopost89 (https://glycopost.glycosmos.org/, project ID: GPST000602).

Ultra-performance liquid chromatography (UHPLC) and mass spectrometry analysis of GSL glycans

AA-labeled GSL glycans isolated from S. haematobium eggs, HF-treated and digested with various exoglycosidases as described above were analyzed using UHPLC. UPHLC profiling was performed as described previously29 with slight adjustments. A Waters Acquity H-class instrument (Waters Corp., Milford, MA, USA) composed of a binary solvent manager, a sample manager, a fluorescence detector (excitation wavelength 250 nm; detection wavelength 425 nm) and a QDa mass detector (settings: positive mode; target sampling rate: 10 point/sec; gain: 1; capillary voltage: 1.5 kV; probe temperature: 600 °C) was used. Glycans were separated using an Acquity BEH Amide Column (130 Å, 1.7 µm, 2.1 mm x 150 mm; Waters Corp.) with 50 mM ammonium formate, pH 4.4 as solvent A and ACN as solvent B. A linear gradient of 12 to 47% of solvent A was applied from 0 to 35 min; 47 to 70% solvent A from 35 to 35.5 min; 70% solvent A from 35.5 to 36.0 min; 70 to 12% solvent A from 36 to 36.5 min and 12% solvent A from 36.5 to 40 min. Samples were kept at 5°C prior to injection, and the separation temperature was 40°C. Data from both the fluorescence and the mass detectors was acquired, processed and analyzed using Empower 3 software (Waters Corp.).

2. Comparative glycan microarray

Glycan microarray construction

A microarray was prepared using native AA-labeled GSL and N-linked glycans isolated from S. mansoni and S. haematobium that were fractionated following established UHPLC-based procedures13,15,63,90 detailed in Figure S9. To provide a broad coverage of the structural diversity of the GSL glycans from both species, 12 fractions derived from S. haematobium cercariae, 10 fractions from S. mansoni cercariae, 25 fractions from S. haematobium eggs and 36 fractions from S. mansoni eggs were included in the array together with subsets of GSL glycans from both species that were treated with HF to remove \alpha1-2/3-linked fucoses to create unsubstituted GSL glycan backbones (Supplementary data 5). In addition, 16 N-glycan fractions from cercariae, adult worms and eggs from S. haematobium and S. mansoni were included to represent the various Schistosoma N-glycans. As a reference, crude antigens from eggs (soluble egg antigen, SEA) and from cercariae (cercarial antigen, CA) were included for each species, and five fractions containing O-glycans derived from S. mansoni cercariae and eggs were generated using material obtained during a previous microarray study15 (more information is provided in Figure S9). Aliquots of 20 pmol of the samples listed above were dispensed into a 384‑well V‑bottom polypropylene plate (#784201, Greiner Bio‑One). When sufficient material was available, larger aliquots of 60, 100, 200, and 600 pmol were included. The plate was evaporated in a vacuum centrifuge, and glycans were redissolved in 10 µL of 1× spotting buffer (Nexterion Spot, #1066029, Schott AG) containing 10% DMSO, creating a glycan library for microarray printing at a minimum concentration of 2 µM, with higher concentrations of 6, 10, 20, and 60 µM used when available. The crude antigens (CA and SEA) were loaded to the plate at a concentration of 10 \mu g/mL, while the O-glycan pools had variable concentration comprised between 0.08 and 1.04 µM as specified in Supplementary data 5. Nine negative controls lacking biological material and consisting solely of buffer were included on the plate. Thus, the generated glycan library was constituted of 288 samples that were printed in triplicate to epoxysilane-coated glass slides (Slide E, Nexterion #1066643, Schott AG), using the Microgrid 600 microarrayer (Genomic Solutions) as further detailed in Figure S9.

Glycan microarray screening

Glycan microarray validation

Upon printing, the glycan microarray was incubated with four mAbs reactive with known glycan motifs for validation purposes (Figure S9).

Infection and control sera

S. haematobium infected sera (n = 18) were obtained from the highly endemic area of Lambaréné, Gabon. Children were enrolled in the previously described study91 during which consent for anonymized use of sera for further serological studies was given. This study was approved by the ethics committees of the University of Tübingen and the International Foundation of the Albert Schweitzer Hospital, Lambaréné, Gabon. Infection was assessed using microscopic egg counting following urine filtration. Sera from children infected with S. mansoni (n = 21) were collected during a different previous study conducted in the fishing community of Piida, located on the shore of Lake Albert, Uganda92. Details on informed consent given by the participants and sample collection have been detailed in the original study, which was approved by the Uganda National Council for Science and Technology and cleared by the Office of the President. The study also received support from the Cambridge Local Research Ethics Committee. Diagnosis of infection was performed using the Kato-Katz technique on stool samples. Finally, anonymized sera from 9 individuals from non-endemic area were obtained from Sanquin (Dutch blood bank) and were used as uninfected controls.

Binding assay

The comparative glycan microarray was screened with infected and control sera for IgG and IgM binding. The protocols for slide incubation, scanning and data handling have been reported previously13,72. Briefly, residual epoxides on the glycan array slides were blocked using a solution of 2% BSA and 50 mM ethanolamine in PBS. Slides were then incubated with serum diluted 1:100 in PBS with 1% BSA and 0.01% Tween 20. Anti-glycan IgG and IgM binding was detected using goat anti-human IgG (Fc-specific) Cy3-conjugate (Millipore Sigma #C-2571) and goat anti-human IgM (heavy chain) Alexa Fluor™ 647 (Invitrogen #A-21249) as secondary antibodies. Fluorescence was measured using a G2565BA scanner (Agilent Technologies) operated and controlled using Agilent’s Feature Extraction Software (version 7.0) at 10 µm resolution using two laser channels at 532 nm and 633 nm and with the sensitivity level of the photomultiplier tube set at 10%. Image analysis was processed with GenePix Pro 7.0 software (Molecular Devices) according to published methods93. Raw output files were deposited to Github (https://github.com/lmcpetralia/Major-human-schistosome-species-express-different-glycans) and the acquired data were exported to Excel. For each glycan-containing fraction, fluorescence intensities of triplicates were averaged and corrected for background using average fluorescence intensity of “blank” spots - i.e. spots printed from wells containing spotting buffer only - as a baseline. Finally, GLycan Array Dashboard (GLAD)94, version 3.1, from the glycotoolkit website (glycotoolkit.com/Tools/GLAD/) hosted by the National Center for Functional Glycomics, was used for data visualization, data mining, generation of heatmaps and boxplot graphs.

Statistical analysis

Background corrected fluorescence intensities were normalized using log2 transformation and all the subsequent statistical data analyses were conducted using the publicly available statistical programming language R (http://CRAN.R-project.org/, version 3.5.0). Significant differences between log2 normalized datasets were evaluated using the R/Bioconductor software package limma. This package, optimized for statistical analysis of microarray data uses a linear model approach to analyze microarray experiments and empirical Bayesian methods to assess significant differences between groups95,96.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript described the characterization of glycans in N- and O-glycans in glycoproteins and in glycosphingolipids from parasites in Schistosoma haematobium and S. mansoni. The results indicate differences in glycosylation, also during the stages of development, that may relate to pathology and immunogenicity. The differences in glycosphingolipids between the two species were substantial, with differences apparent in fucosylation, glucuronic acid, and sequences. The work is interesting at many levels and represents one of the most advanced studies on glycans in these parasites conducted thus far. The studies, however, are highly descriptive, and yet not all linkages are defined, and the sequences are largely predicted. A key aspect of the study is the potential biological relevance of the glycans, which is provided through glycan microarrays, in which selected glycans were printed and examined for recognition by antibodies in sera from young patients with parasitic infections. The results indicate selective recognition of glycans from each species. The descriptive nature of the work overall is excellent and provides new information about developmental and species-specific difference in glycosylation. However, there are aspects to the study in terms of biological impact that are incremental in several ways and somewhat lacking in terms of novel insights beyond the glycan structural differences. Several of the major points of concern are listed below.\n• The core structure of the GSLs from S. haematobium were not fully characterized and an unknown hexose and linkage was identified.\n• The precise linkages of monosaccharides in the glycans were not identified, so this raises questions about whether there are many isomers of such structures in regard to linkage, and the enzymes that might be important in generating them.\n• While the studies on antigenic nature of glycans is interesting, it is already well known from many prior studies that glycans from these parasites are antigenic and recognized by antibodies (IgG/IgM) in sera from infected individuals. It is not clear from the result whether each of the glycans is recognized by unique antibodies or that different epitopes shared between glycans are being recognized. These results in this manuscript are certainly well done and provide additional information, but much of the conclusions are consistent with prior efforts in the field.\n• The overall results do not provide specific diagnostic or immunogenicity information relative to the glycan structures, but rather is a broad survey of the glycome. This is not a criticism, per se, since such detailed information is certainly important in directing future work perhaps in this direction. Already, a lot of glycans in these parasites are known to be antigenic in infected individuals, as the parasite synthesized glycans largely difference in most respects from their mammalian hosts. The present study adds to that diversity.\n• The relationship between the glycome and the genome in terms of enzymatic machinery to synthesize these glycans is somewhat speculative as no detailed genetic information is provided in this regard.\n• The parasites are derived from hamsters, and not from people, obviously, but that does raise the question as to whether the host might influence the production of glycans by the parasite. No information in this regard is provided or discussed.\n• The antigenic nature of the glycans in hamsters is not discussed either, and that might be very interesting in terms of differences in mammalian responses to infection and glycan antigenicity.\n• The lack of information about glycoproteins and glycoproteomics is striking in this regard, as one might expect that from such a huge diverse set of glycans they might be distributed somewhat uniquely among glycan species, as the authors indicate from prior studies on Omega-1.\n• There are multiple monoclonal antibodies to the glycan determinants identified in table 2 for example. Did the authors examine the recognition of the relevant glycans under study for their binding to such antibodies, which would be an additional verification or potential validation of structure?\n• Although the authors raise great interest in the presence of glucuronic acid in glycans from S. haematobium, such residues are found also in S. mansoni, and thus perhaps not unique to either. In addition, the finding of glucuronic acid in glycans has been known for some time. Prior studies on the glucuronic acid residues in glycans from S. mansoni and studies on chemical synthesis and immunogenicity of such glycans are not discussed [see for example, Halkes et al PMID: 9741076].\n• The expression of glycans within the parasites are not known, as the total homogenates were used, and questions as to whether glycans are potentially expressed on the worm surface, secretions, or within other organs in some specific manner are not discussed.\n• The results are discussed as being quantitative but it is unclear what is meant by that, as no specific quantitation is provided. Perhaps differences in abundance is what is meant in this context, and even then it might be ‘relative abundance’. This reviewer is not certain of what is meant exactly. Given the glycans are fluorescently labeled, it might be possible to actually define the quantity of glycans. MALDI-TOF information is certainly not quantitative in its own right.\n• There appear to be some significant differences in responses of individual sera from different donors, but that is not clear or well described in the study. The potential cause of that is unclear. Are the individuals infected with more than one parasite or type of parasite? The donor information in that regard is not clear.", "reviewer_2": "Nature Commun Petralia\nOverall, the manuscript is of interest due to the variations in glycan structures between the two Schistosoma species as well as due to the glycan array data. The data is new, the insights into S. haematobium are novel. The methodology is typical of that from the authors' laboratory. With such a large dataset, it is not possible to assess all files, but I have a number of questions.\nPage 6/8 - does the removal by SpHex suggest a chondroitin-type and/or CAA-type terminus, or should the terminal HexNAc be undefined \"white\"? In the Excel files, then there are 'mixed' blue/yellow squares for GlcNAc or GalNAc, but \"white\" undefined hexose residues. It could be mentioned on page 8 that a HexA has more-or-less the same delta m/z (176) as methylhexose, a modification not unknown in helminths or snail hosts.\nFigure 3D - none of the higher Mr glycans above m/z 2300 are annotated, are they triantennary - although a composition is given in one of the Excel files. Inspection of the corresponding mzxml files on glycopost suggests the m/z window was not wide enough to detect the entire range as an obvious fucose series suddenly is cut of at m/z 3500. (In the Excel file cut off at m/z 3321.)\nFigure 4 - it is a bit strange that there is a gap of some 1200 amu between the largest and second largest glycan (although there are traces apparently of intermediate peaks shown in the Excel file) - this assumes an almost 'quantitative' complete biosynthetic modification once the second 'core 2-like' arm is generated.\nPage 15 - the authors say \"once validated\" without briefly stating at this point what they mean (except when the reader digs into the supplement).\nMethods - replace MQ by water - other manufacturers also have machines capable of generating pure water.\nRef 84 - check author names, perhaps for McShaneb and Doddb, b is a superscripted indication of affiliation. Ref 90 is the same as Ref 29.\nFigure S2 - what is meant by \"purified glycans\", \"purified fraction\" ... if it corresponds to a certain HPLC fraction, then this should be stated in terms of fraction number or time. Three decimal places for standard MALDI is over-exact - also in the Excel files. Are all the annotated m/z in the supplementary figures actually glycans or glycan fragments or just what the software annotates without human interpretation? What is the blank spectrum for the glucuronidase? The enzyme seems particularly full of contaminants.\nFigure S5 - not all the glycosidic bonds are easily seen.\nFigure S8 - refers to a previous study for the O-glycans - however, the basic characteristics of these samples should be stated here (e.g., also AA labelled?). Which entire O-glycan pools are meant in the methods? Were the concentrations defined on the basis of comparison to a standard?\nExcel file \"631032_0_data_set_11107602_sz8r9r\" - the \"accuracy\" varies between the N-and O-glycans - was the MALDI calibrated with an external standard on a daily basis? Are the \"null\" masses 'junk'? Perhaps there are limitations to what the rather old Glycoworkbench program can find. For the O-glycans, there is a mix of H and Na forms - some have only an H form, which is suspicious - considering the Na-rich release method, finding only Na or having both H and Na seems logical, rather than having some glycans solely as protonated forms.\nExcel file \"631032_0_data_set_11107609_sz8r9r\" - although the glycan fractions were printed in triplicate, I guess only mean values are shown. No individual data points are shown in the relevant supplementary figures S8-S10. What are the corresponding wet or dry weights of the numbers of eggs, worms, etc (methods and legend to Figure S8)?\nThe Excel files should be headed in the file with the same Table number as in the text, considering the renumbering by the editorial management system."}, "author_response": "We sincerely thank the reviewer for their constructive comments. The feedback has contributed to strengthening the manuscript by tightening loose ends of our structural study and improving the data presentation of our glycan microarray dataset. We also appreciated the insights highlighting important directions for future research. Below we provide a point-by-point response to the comments and questions raised.\n1. The core structure of the GSLs from S. haematobium were not fully characterized and an unknown hexose and linkage was identified\nWe acknowledge that the undefined trihexosyl core structure of S. haematobium GSL glycans represented a notable gap in our study. We are grateful to the reviewer for giving us the opportunity to further characterize this glycan class. To do so, we used a UHPLC-based method optimized for the detection of short, labeled oligosaccharides to address previous limitations of our workflow. With this experimental platform in combination with exoglycosidase digestions, we were able to determine that the ceramide-linked glycan portion of S. haematobium GSL glycans is built on a sequence of Galα-Galβ-Glcβ1-Cer. This new data has been included in the manuscript as Figure S4. These additional results are now described L.90*, discussed L.341(342), L.356(359)-362(365) and the UHPLC method is detailed L.634(648)-647(661) (M&M section).\n*NB: Please note that line numbers refer to the version of the manuscript without track changes, while numbers in brackets correspond to the version with track changes —except when they are identical in both versions.\n2. The precise linkages of monosaccharides in the glycans were not identified, so this raises questions about whether there are many isomers of such structures in regard to linkage, and the enzymes that might be important in generating them.\nWhile it is true that the precise linkages were not elucidated for every single glycan structure, our workflow provides substantial information in that regard. With the exception of the O-glycans for which we primarily relied on informed assumptions and analogies with S. mansoni, exoglycosidase digestions provided linkage information (at least in terms of α or β anomers) for monosaccharides in most GSL and N-linked glycans. Special attention was given to elucidating the linkages in glycan structures that diverge from those already characterized in S. mansoni, in particular the acidic GSL glycans detected in both S. haematobium and S. mansoni eggs, the GSL glycans of S. haematobium cercariae containing branched galactose residues, or the trihexosyl core of S. haematobium GSL glycans. PGC-nano-LC-MS/MS was employed to resolve some of the isomeric complexity of the GSL glycans. Our analysis clearly shows that isomeric variants are frequent in schistosomes, and we do not claim to have completed the daunting and perhaps impossible task of elucidating each glycan structure at the isomeric level. We hope to have now clarified this for the reader in the expanded discussion section (L.342(343)-348(351)).\nThe reviewer raises an interesting point by linking the observed structural diversity to the enzymatic machinery responsible for glycan synthesis. As further discussed in question 5, our current understanding of the enzymatic pathways involved in glycan synthesis in Schistosoma species remains extremely limited, and the present observations suggest that the underlying biosynthetic machinery must indeed be highly complex and diverge significantly from known mammalian pathways.\n3. While the studies on antigenic nature of glycans is interesting, it is already well known from many prior studies that glycans from these parasites are antigenic and recognized by antibodies (IgG/IgM) in sera from infected individuals. It is not clear from the result whether each of the glycans is recognized by unique antibodies or that different epitopes shared between glycans are being recognized. These results in this manuscript are certainly well done and provide additional information, but much of the conclusions are consistent with prior efforts in the field.\nWe do agree with the reviewer that many of our findings regarding anti-glycan antibody responses corroborate observations already made in the context of S. mansoni infections. We hope to have acknowledged this at L.334-335 by stating “These antibodies are partly directed to the same antigenic glycan elements as anti-glycan antibodies in S. mansoni infection sera”. Although the antigenicity of several schistosome glycans was previously known, this study is the first to investigate the antigenicity of S. haematobium glycans, and importantly, the first to address differences in term of anti-glycan antibody responses between individuals infected with different Schistosoma species.\nAlthough certain antibodies might be specific for entire glycan structures, it is clear that many serum antibodies from Schistosoma-infected individuals rather bind to glycan motifs or “glycotopes”. This was shown in previous studies with binding to synthetic glycan elements (neoglycoconjugates) printed on glycan microarrays (ref#13-15, 19 in manuscript). In the present work, antibodies in serum from S. haematobium-infected individuals bind to S. mansoni GSL glycans of compositions H N F while S. 1 4-7 2-8 haematobium does not synthesize such structures, constituting a clear example of cross-reactive glycotopes. In this example, the antibodies raised against the multifucosylated glycans of S. haematobium cercariae likely bind the multifucosylated S. mansoni GSL glycans due to the shared DF/TF glycotopes - see discussion L.380(388)-385(393).\n4. The overall results do not provide specific diagnostic or immunogenicity information relative to the glycan structures, but rather is a broad survey of the glycome. This is not a criticism, per se, since such detailed information is certainly important in directing future work perhaps in this direction. Already, a lot of glycans in these parasites are known to be antigenic in infected individuals, as the parasite synthesized glycans largely difference in most respects from their mammalian hosts. The present study adds to that diversity.\nWe agree with the reviewer’s analysis of the present work and the fact that such studies are necessary steps towards exploiting the antigenicity of parasite glycans for diagnostics or vaccines.\n5. The relationship between the glycome and the genome in terms of enzymatic machinery to synthesize these glycans is somewhat speculative as no detailed genetic information is provided in this regard.\nWhile high-quality nuclear genome assemblies are now available for S. mansoni and S. haematobium, at this stage, the relationship between the glycome and the genome is indeed highly speculative, with the exception of highly conserved N-glycan structures. The majority of the enzymes involved in glycan synthesis and their complex regulation remain uncharacterized (mentioned L.362(365)-364(367) with ref #27), particularly those responsible for the synthesis of antigenic, non-mammalian motifs (mentioned L. 389(397)-390(398) for DF/TF). We have chosen to limit ourselves to brief discussion points as providing any further information in that direction would require functional characterization of predicted glycogenes via challenging studies beyond our current scope.\n6. The parasites are derived from hamsters, and not from people, obviously, but that does raise the question as to whether the host might influence the production of glycans by the parasite. No information in this regard is provided or discussed.\nIt is fair to question the influence of the host on the parasite glycan synthesis, as the parasite material in this study was obtained from hamsters. Hamsters are not known to be a host of S. mansoni or S. haematobium in nature, but they are fully permissive to infections with these human schistosomes.\nIt is not known or expected that the enzymatic machinery of the parasite rapidly adapts to a different host, as host-specificity is observed for most other definitive and intermediate (snail) hosts, making drastic variations in glycan expression in the hamster host unlikely. Moreover, the glycan array results clearly demonstrate that infected (human) individuals have antibody responses to the glycans isolated from schistosomes obtained from hamsters, confirming that the glycans expressed by schistosomes obtained from hamsters accurately reflect those expressed by schistosomes infecting a human host.\n7. The antigenic nature of the glycans in hamsters is not discussed either, and that might be very interesting in terms of differences in mammalian responses to infection and glycan antigenicity.\nComparing glycan antigenicity across different mammalian species is indeed of major interest. While studying antigenicity in hamsters may yield interesting insights, it is even more relevant in species resistant to schistosome infections for exploiting glycan antigenicity in therapeutic applications for human schistosomiasis. Previous studies have notably identified specific anti-glycan antibody responses associated with infection clearance in monkey species resistant to Schistosoma infection (refs#14, 51, 52 in manuscript). We, however, believe that this constitutes a distinct research question that falls outside the scope of the present study.\n8. The lack of information about glycoproteins and glycoproteomics is striking in this regard, as one might expect that from such a huge diverse set of glycans they might be distributed somewhat uniquely among glycan species, as the authors indicate from prior studies on Omega-1.\nThe reviewer raises a valid point and highlights an important direction for future research. Since glycoproteomic analyses rely on robust glycan databases, we hope that the present comprehensive glycomic study will contribute to such efforts. We have edited the discussion section (L.435(448)-449(462)) to share this perspective with the reader.\n9. There are multiple monoclonal antibodies to the glycan determinants identified in table 2 for example. Did the authors examine the recognition of the relevant glycans under study for their binding to such antibodies, which would be an additional verification or potential validation of structure?\nWe did incubate the constructed glycan microarrays with several mAbs binding to specific glycan motifs. We show the results for four of these mAbs in Figure S9 (previously Figure S8) and Table S6 of the manuscript namely 273-3F2-A (anti-LDN); 291-2G3-A (anti-LeX); 291-5D5-A (anti-F-LDN(-F)) and 100-4G11 (that binds to the trimannosyl branching of N-glycans). We conducted this experiment primarily for validation of the array print quality, but it also provided confirmation of the structural assignment. These results are presented in Figure S9, where we briefly mentioned that mAb binding was consistent with predicted glycan structures in the array fractions, confirming the expression of putative LeX, LDN and fucosylated LDN motifs. In the revised manuscript, we added the sentence L.276-279 in the main text for the reader’s reference and we incorporated mAb binding results side by side with the structural assignment in Table S2 (GSL glycans) and S3 (N-glycans). We thank the reviewer for the suggestion, allowing a more effective presentation of the data.\n10. Although the authors raise great interest in the presence of glucuronic acid in glycans from S.haematobium, such residues are found also in S. mansoni, and thus perhaps not unique to either. In addition, the finding of glucuronic acid in glycans has been known for some time. Prior studies on the glucuronic acid residues in glycans from S. mansoni and studies on chemical synthesis and immunogenicity of such glycans are not discussed [see for example, Halkes et al PMID: 9741076].\nThe fact that glucuronic acid (GlcA) is clearly not unique to schistosome glycans is indeed worth noting, and we hope to have done so in the discussion L.398(407)-411(414). In summary, GlcA in helminth glycans has been observed so far:\n- As part of the repeated [-6(GlcAβ1-3)GalNAcβ1-] units of schistosomal CAA (ref #56 in manuscript)\n- As a terminal residue capping LDN in S. haematobium adult worm GSL glycans (this study) & in the N-linked and GSL glycans of other helminths (refs #62-64 in manuscript)\n- As a subterminal residue capped with HexNAc and associated with different fucosylation patterns in S. haematobium and S. mansoni egg GSL glycans (this study)\nThus, the present work adds to our knowledge of acidic glycans in parasitic helminths by describing novel GlcA-containing structures in schistosomes. Importantly, these different contexts of GlcA-containing motifs greatly matter for their immunogenicity. In the case of CAA, the glycosaminoglycan-like molecule is bound specifically by antibodies in serum from Schistosoma-infected individuals making it a relevant pan-schistosome diagnostic target (ref #19 in manuscript). We believe that the quoted study together with the added sentence L.413(422)-415(425) should effectively address previous work on immunogenicity of CAA as suggested here by the reviewer.\nWe would like to emphasize, however, that the serum antibody responses targeting the CAA molecule differ from those binding to GlcA-containing GSL glycans, in line with their vastly different structures. Based on previous work, it is known that several repeated units, and thus several GlcA residues are needed for antibody binding to synthetic CAA, while clearly, a single GlcA residue is sufficient to create an immunodominant epitope targeted by serum antibodies in the context of the GSL glycans. The differential recognition of this epitope by IgG from S. haematobium and S. mansoni-individuals discussed L. 415(425)-423(433) adds to the importance of the context of GlcA within the glycans for their specific antigenicity.\n11. The expression of glycans within the parasites are not known, as the total homogenates were used, and questions as to whether glycans are potentially expressed on the worm surface, secretions, or within other organs in some specific manner are not discussed.\nWe agree with the reviewer’s rightful comment that points out one of the limitations of our study. The present work is indeed not informative in terms of localization of the identified glycans, although the glycan microarray results indicate that most structures must be sufficiently exposed to trigger antibody responses in infected individuals. While interesting, the question of the spatial distribution of glycan falls outside the scope of this study, which aims to characterize the glycan repertoire of S. haematobium and provide insights into their antigenicity. This point echoes question #8: as for glycoproteomics, we believe that it is a critical area for future investigation, with the potential to significantly advance our understanding of glycan function. Thus, we hope to have tackled this in the paragraph we added L.435(448)-449(462) discussing future research directions and proposing potential analytical approaches and tools that could be employed to investigate these aspects in greater depth.\n12. The results are discussed as being quantitative but it is unclear what is meant by that, as no specific quantitation is provided. Perhaps differences in abundance is what is meant in this context, and even then it might be ‘relative abundance’. This reviewer is not certain of what is meant exactly. Given the glycans are fluorescently labeled, it might be possible to actually define the quantity of glycans. MALDITOF information is certainly not quantitative in its own right.\nWe fully agree with the reviewer’s comment and understand their concern. Different approaches were taken with respect to (relative) quantitation, depending on glycan classes.\n- For the O-glycans, all data are MALDI-based, and S. haematobium and S. mansoni samples were not processed side-by-side. Instead, the comparison relies on previously published S. mansoni data (ref #9 in manuscript). The same analytical workflow as in this previous study was applied to S. haematobium in the present work, and the identified O-glycans harbor similarities with those of S. mansoni. Although MALDI-TOF-MS is not quantitative, as rightfully pointed out by the reviewer, it still provided a sense of relative abundance based on signal intensity, which we have used cautiously to complement our qualitative observations.\n- For the N-linked and GSL glycans, on the other hand, S. haematobium and S. mansoni samples were processed side-by-side in our analytical workflow, allowing for a more robust comparison.\nImportantly, in addition to MALDI-TOF-MS, these samples were also analyzed using LC platforms, providing us with quantitative data:\nDuring the glycan microarray construction, N-linked and GSL glycans underwent UHPLC fractionation, and the amount of glycan collected was estimated by comparison with a RNAse B standard (as described in Figure S9). The resulting quantitation was used to build the glycan library for the microarray construction and confirmed our structural observations. For instance, although similar numbers of eggs were used as starting material for both species, over 300pM of acidic GSL glycans were isolated from S. haematobium eggs distributed across 16 fractions (Table S5). In contrast, the 12 acidic fractions obtained from S. mansoni eggs total approximately 150pM of glycans, a direct consequence of the smaller amount of acidic glycans in S. mansoni.\nWe presented the marked quantitative differences observed in the egg GSL glycans in Figure 2.\no We used PGC-nano-LC-MS to assess the differences regarding the expression of fucosylated motifs (right panel “Fucosylation of major egg GSL glycans). To estimate the relative proportion of fucosylated and acidic GSL glycans in the total GSL pool (left panels: “Fucosylated” and “Acidic”), we used intensity signals in MALDI-TOF spectra, as specified in the figure legend. These results were confirmed beforehand using UHPLC (data made available to the reviewers in Figure R1) as we acknowledge the inherently non-quantitative nature of MALDI-TOF-MS.\nWe hope that we have clarified the approaches to quantitation used in this MS-based study, and that the reviewer finds our rationale justified.\n13. There appear to be some significant differences in responses of individual sera from different donors, but that is not clear or well described in the study. The potential cause of that is unclear. Are the individuals infected with more than one parasite or type of parasite? The donor information in that regard is not clear.\nIt is indeed possible that coinfections with other pathogens harboring cross-reactive glycans account for some of the observed individual-to-individual variations, and we cannot rule out infections with parasites other than schistosomes in the cohorts used in this study. However, the magnitude of individual-to-individual variations observed in the present work is consistent with previous glycan-microarray assisted studies. Specifically, controlled human schistosome infections (ref #19 in manuscript) comprise the most homogenous groups of human subjects for which antibody responses have been studied using glycan microarrays. Even in these healthy Dutch adult volunteers from non-endemic areas, similar variations in anti-glycan antibody responses were observed. Importantly, the impact of S. haematobium and S. mansoni infections eliciting strong anti-glycan antibody responses, is clearly discernible despite inter-individual variations.\nReviewer #2\nOverall, the manuscript is of interest due to the variations in glycan structures between the two Schistosoma species as well as due to the glycan array data. The data is new, the insights into S. haematobium are novel. The methodology is typical of that from the authors' laboratory. With such a large dataset, it is not possible to assess all files, but I have a number of questions.\nWe would like to sincerely thank the reviewer for their careful and insightful review. We greatly appreciate their knowledgeable comments, keen eye for detail, and interest in our work. We have done our best to clarify all points raised and we hope to have made all necessary and appropriate changes.\n1. Page 6/8 - does the removal by SpHex suggest a chondroitin-type and/or CAA-type terminus, or should the terminal HexNAc be undefined \"white\"? In the Excel files, then there are 'mixed' blue/yellow squares for GlcNAc or GalNAc, but \"white\" undefined hexose residues. It could be mentioned on page 8 that a HexA has more-or-less the same delta m/z (176) as methylhexose, a modification not unknown in helminths or snail hosts.\nBy comparing the activity of β-HexNAcase with the activity of β-GlcNAcase, we determined the capping HexNAc in acidic egg GSL glycans to be mainly β1-4-linked GalNAc as these structures were mainly resistant to β-GlcNAcase, while β-HexNAcase could cleave off the terminal HexNAc residue, both for S. haematobium (Figure S2.C, 3rd panel vs 5th panel) and for S. mansoni, (Figure S2.E, 4th panel vs 5th panel). Unlike β-GlcNAcase, β-HexNAcase can digest β1-4 linked GalNAc (Table 1). We specified this in the text, L.135 & L.166-168.\nUsing the same enzymatic investigation, we determined that GlcA could be linked to either GlcNAc or GalNAc in the backbone chain of S. haematobium egg GSL glycans (Figure S2.C, 9th panel vs 10th panel, L.139-142). A “white” HexNAc suggests that the nature of the HexNAc is unknown or has not been investigated, while in this case, we actually know that both β-linked GlcNAc and GalNAc occur. Thus, mixed blue/yellow square seemed more appropriate to convey this message. For convenience, we decided to represent the GalNAc-containing isomer in the manuscript main figures and to show both possibilities in the supplementary table S2 as well as in Table 2, that has been updated to address this comment.\nWe believe that it may not be fully accurate to qualify the terminal acidic epitope characterized in schistosome GSL glycans of chondroitin-type and/or CAA-like. In the context of CAA, GlcA is branched in repeated [-6(GlcAβ1-3)GalNAcβ1-] units, differing from the linear GalNAcβ1-4GlcAβ-HexNAcβ-R sequence characterized in the GSL glycans. We hope to have clarified this by adding a sentence in the discussion L393(400)-398(407)*. The absence of repeat or polymerization of the motif in the egg GSL glycans constitutes a major difference with CAA but also with glycosaminoglycans. These structural variations have major implications regarding the antigenicity of these acidic motifs as anti-CAA antibodies do not bind to the acidic GSL glycans (data not shown) and vice versa.\nWe have put substantial effort into the characterization of the HexNAcs constituting terminal motifs due to their importance as antigenic epitopes. We have, however, not confirmed the exact nature of all single HexNAcs within the GSL backbones. We assumed the backbone HexNAc to be GlcNAc, based on analogy with S. mansoni for which HexNAc stretches of the GSL backbones have been shown by monosaccharide analysis to be constituted predominantly of GlcNAc during previous studies (refs #30, 32-34). This is specified in the figure captions, and we have now added this information in the main text L.106-114. In addition, we have resolved the previously uncharacterized (white) hexose part of the S. haematobium GSL glycan core structure. Using UHPLC, we determined this trisaccharide sequence is Galα-Galβ-Glc-Cer. This new data has been incorporated into the manuscript in Figure S4, L.90 (result description), L.341(342), L.356(359)-362(365) (discussion) and L.634(648)-647(661) (M&M section). Thus, the white hexoses were converted to Gal.\nLastly, we agree with the reviewer that the possibility of a methylated hexose and the rationale for its exclusion as a potential monosaccharide in the composition of the glycans characterized here are worth mentioning. We have revised the text L.121-125 accordingly.\n*NB: Please note that line numbers refer to the version of the manuscript without track changes, while numbers in brackets correspond to the version with track changes —except when they are identical in both versions.\n2. Figure 3D - none of the higher Mr glycans above m/z 2300 are annotated, are they triantennary – although a composition is given in one of the Excel files. Inspection of the corresponding mzxml files on glycopost suggests the m/z window was not wide enough to detect the entire range as an obvious fucose series suddenly is cut of at m/z 3500. (In the Excel file cut off at m/z 3321.)\nTo tackle the complexity of the data presented in this study, we arbitrarily decided to solely annotate the “ions with signal-to-noise ratios > 4 and intensities above 1% of the total spectrum intensity” (specified in figure legends) which indeed resulted in ions with m/z > 2300 not being annotated in Figure 3.D, as they do not fulfill these criteria.\nBased on composition and digestion with β-HexNAcase and β-GlcNAcase (Figure S6.E) these structures are expected to be multiantennary and to harbor a variety of terminal motifs including LN, LeX and (fucosylated) LDN (Figure R2-A). Many of these masses are also observed in S. mansoni eggs, although a poly-LacNAc series (m/z 3029, 3175, 3321…) present in S. haematobium was not detected in S. mansoni (Figure R2-B). As highlighted by the reviewer, it is very likely that this series encompasses larger structures with m/z > 3500 not measurable using this workflow. The amount of glycans released from S. haematobium eggs using PNGase A was limited, preventing further structural characterization of minor glycan subsets such as this one. For the same reason, these structures could not be included in the generated glycan array. However, we would not expect N-glycans containing terminal LN, which are widespread also in mammalian systems, to be antigenic. We focused this study on the most divergent glycan structures between S. mansoni and S. haematobium, which centered the manuscript on the GSL glycans and the corresponding antibody responses from infected individuals. Thus, although interesting, we opted not to elaborate on minor N-glycan subsets.\n3. Figure 4 - it is a bit strange that there is a gap of some 1200 amu between the largest and second largest glycan (although there are traces apparently of intermediate peaks shown in the Excel file) – this assumes an almost 'quantitative' complete biosynthetic modification once the second 'core 2-like' arm is generated.\nWe thank the reviewer for their interesting observation that may give some insights regarding the O-glycan synthesis in schistosomes. Although less visible than in S. haematobium, a similar pattern can indeed be observed in the O-glycans of S. mansoni mature eggs (ref #9 in manuscript) where ions with m/z 1157.5 and 1780.8 also appear relatively more abundant than the intermediate structures.\n4. Page 15 - the authors say \"once validated\" without briefly stating at this point what they mean (except when the reader digs into the supplement).\nTo clarify this, we have added a brief statement in the main text (L.276-278) and a short paragraph in the Materials and Methods section (L.673(691)-675(693)), both pointing to Figure S9 (formerly Figure S8), which provides the detailed version of the experimental validation procedure. We hope that these additions sufficiently clarify how the microarray validation was conducted while keeping the corresponding results and data in the supplementary materials for the sake of restricting the main text length.\n5. Methods - replace MQ by water - other manufacturers also have machines capable of generating pure water.\nWe appreciate the reviewer’s point, however, for the sake of methodological reproducibility, we prefer to specify that ultrapure water was used. Therefore, we retained the abbreviation 'MQ' but changed its meaning to ultrapure water (see abbreviation list).\n6. Ref 84 - check author names, perhaps for McShaneb and Doddb, b is a superscripted indication of affiliation. Ref 90 is the same as Ref 29\nWe apologize for the oversight which has now been rectified, and we thank the reviewer for their attention to detail.\n7. Figure S2 - what is meant by \"purified glycans\", \"purified fraction\" ... if it corresponds to a certain HPLC fraction, then this should be stated in terms of fraction number or time. Three decimal places for standard MALDI is over-exact - also in the Excel files. Are all the annotated m/z in the supplementary figures actually glycans or glycan fragments or just what the software annotates without human interpretation? What is the blank spectrum for the glucuronidase? The enzyme seems particularly full of contaminants.\n“purified glycans” and “purified fractions” in Figure S2, indeed referred to UHPLC fractions generated during the glycan array construction. They have been replaced by their respective fraction ID numbers as listed in Table S5.\nWe have standardized the decimal number to two decimal places, for uniformity between the various MS-based techniques employed in this study. This also aligns with the resolution and mass accuracy typically achievable in reflector mode on Bruker MALDI-TOF instruments (Autoflex, Ultraflex, Rapiflex) and is commonly seen in publications presenting MALDI-TOF-MS spectra of glycans.\nAll the glycan and glycan fragment annotations both in main and supplementary figures were interpreted and assigned manually by the authors with the help of GlycoWorkBench. Currently available tools for annotation of MALDI-TOF-MS data are designed for mammalian N-glycans and are not able to handle non-mammalian structures such as those found in schistosomes. Therefore, manual annotation remains necessary.\nThe β-glucuronidase appears to indeed contain a few contaminant peaks (Figure R3). The ion with m/z 1418 for instance can be distinguished in β-glucuronidase digestion spectra in Figure S2.B. The MS spectrum shown in Figure S2.B (panel 2) appears particularly noisy due to the low amount of glycan material available from the pool of acidic worm GSL glycans used as starting material. Unfortunately, this sample was exhausted, preventing us from generating a cleaner, more concentrated replicate. However, when more abundant material was available, such as in the case of GSL glycans derived from eggs (Figure S2.C), a life stage yielding significantly higher quantities of acidic GSL glycans, the contaminant peaks became negligible and were no longer detectable.\n8. Figure S5 - not all the glycosidic bonds are easily seen. Figure S8 - refers to a previous study for the O-glycans - however, the basic characteristics of these samples should be stated here (e.g., also AA labelled?). Which entire O-glycan pools are meant in the methods? Were the concentrations defined on the basis of comparison to a standard?\nThe glycosidic bonds represented in Figure S6 (former Figure S5) appear clearly visible to us, and we are uncertain about the issue the reviewer is experiencing.\nGlycan amounts were indeed estimated by comparing UHPLC peak heights to those of RNase B standards. Height values were converted to pmol amount based on calibration curves generated using glycans released from known quantities of RNase B. This information has been added to the caption of now Figure S9 (previously Figure S8) for the reader’s reference. We have also edited the M&M section “Glycan array construction and validation” (L.659(673)-671(689)) to clarify the points brought to our attention by the reviewer.\n9. Excel file \"631032_0_data_set_11107602_sz8r9r\" - the \"accuracy\" varies between the N-and O-glycans - was the MALDI calibrated with an external standard on a daily basis? Are the \"null\" masses 'junk'? Perhaps there are limitations to what the rather old Glycoworkbench program can find. For the O-glycans, there is a mix of H and Na forms - some have only an H form, which is suspicious - considering the Na-rich release method, finding only Na or having both H and Na seems logical, rather than having some glycans solely as protonated forms.\nExternal MALDI calibration was performed using the Bruker Peptide Calibration Standard II (#8206195, Bruker Daltonics) as specified in the M&M (L.586(601)) prior to each measurement. Measurements were recorded over several months and years using different MALDI instruments, which likely account for some of the observed variation. This had been omitted in the M&M section and has now been added L.579(594)-581(596). Additionally, while GSL and N-glycans were measured in negative-ion mode, O-glycans were measured in positive-ion mode.\nThe null masses fall into two categories: known non-glycan peaks (DHB matrix or hexose polymer contaminants) and masses to which a plausible glycan composition could not be assigned. GlycoWorkbench, while reliable and widely used, has inherent limitations, in particular the fact that its annotation capabilities are constrained by various user-defined settings including the predefined pool of allowed monosaccharides and ppm threshold. It is unclear whether “null” masses are not annotated because they truly are non-glycan peaks or because the proper monosaccharide were not provided as an input for the software. Of note, peaks with “null” masses largely correspond to low-intensity ions and capturing all relevant glycan signals while avoiding the inclusion of noise is inherently challenging. This is part of our rationale for excluding masses with signal-to-noise ratio below 4 from our MALDI-TOF-MS analysis.\nWe thank the reviewer for pointing out the inconsistencies in the O-glycan assignments in Table S1. As correctly noted, ions with protonated ([M+H]+) adducts should not have been included in the analysis, since our workflow exclusively yielded sodiated ions ([M+Na]+). We have carefully reexamined Table S1 and revised the annotations of the raw MALDI data accordingly.\n10. Excel file \"631032_0_data_set_11107609_sz8r9r\" - although the glycan fractions were printed in triplicate, I guess only mean values are shown. No individual data points are shown in the relevant supplementary figures S8-S10. What are the corresponding wet or dry weights of the numbers of eggs, worms, etc (methods and legend to Figure S8)?\nThe reviewer is correct, only mean values of triplicates are shown in Figures S9-S11 (previously Figures S8-S10), as averaging fluorescence intensities is one of the initial data processing steps alongside background correction using the average fluorescence intensity of buffer spots (see M&M L.697(715)). Raw data for individual triplicates are available in the GitHub repository (see Data Availability section).\nIn Figures S9 (previously S8) and S11 (previously S10), we chose to display averaged MFI (median fluorescence intensity) values to simplify visualization and facilitate interpretation. These figures are intended to provide a clear overview of mAb binding (Figure S9) and Ig binding to specific array fractions (Figure S11). In Figure S10, however, the heatmap allows visualization of the variation in IgM responses to schistosome glycan antigens from the different individuals.\nWe did not record the weight of the starting biological material used for glycan microarray construction. However, we believe that the information provided in the manuscript should support experimental reproducibility, as quantification by counting eggs, worms, or cercariae is a standard and widely accepted method in the field for estimating and handling Schistosoma material.\n11. The Excel files should be headed in the file with the same Table number as in the text, considering the renumbering by the editorial management system.\nWe regret the confusion caused by the editorial management system’s handling of the file names. We appreciate the reviewer’s suggestion and have added headers indicating the corresponding table numbers as referenced in the main text in the resubmitted manuscript, in hopes of making the review process more straightforward."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has addressed several key concerns, namely characterization of the core structure of the GSLs, clarified the limitations of their studies in regard to linkages of sugars, expanded on their insights into the differential antibody responses to glycans previously observed versus their current observations, addressed the limited nature of the study’s insights regarding linkages to the genome, clarified the nature of glycosylation in worms isolated from hamsters compared to human hosts, addressed the limitation of the study in regard to lack of glycoproteomic information, provided additional information in Figure S9 regarding structure of glycans recognized by antibodies, revised their discussion regarding the presence of glucuronic acid in glycans, the lack of quantitation of glycan expression, which is a common limitation of current technologies, and the individualistic nature of immune responses to glycan antigens.\nOverall, the revised manuscript provides excellent information about glycan structures in these parasites, as well as developmental and species-specific difference in glycosylation.", "reviewer_2": "Petralia revised\nOverall, the manuscript is of interest due to the variations in glycan structures between the two Schistosoma species as well as due to the glycan array data. The data is new, the insights into S. haematobium are novel. The methodology is typical of that from the authors' laboratory. With such a large dataset, it is not possible to assess all files, but spot checks still result in some questions. The authors have revised their manuscript, adding further data and explanations as well as making corrections. My line numbers refer to the 'tracked changes' version which follows the revised manuscript in the single \"manuscript\" pdf.\nHeading - line 77 - \"chains\" or \"oligomers\" rather than \"stretches\"\nLine 93 - if both a general and a specific b-galactosidase worked, then the linkage is Galb1,4Glc and if the a-Gal is not a1,4, then it can be hypothesised that it is Gala1,3Galb1,4Glc (Gala1,6 in an animal would probably be unusual). Possibly in vitro a1,3-galactosylation of lactose with a commercially available enzyme to generate an alternative standard would resolve this in the absence of enough material for linkage analysis - alternatively Elictyl actually sell Isoglobotriose.\nLine 108 - specify S. pneumoniae in the text, not just the table.\nLine 370 - certainly one alpha and one beta galactosyltransferase\nComment 2: The S/N ratio of 4 and a 1% intensity as a cut-off for defining glycan structures seems arbitrary. Can these larger glycan structures be annotated by careful manual interpretation or are the MS2 spectra uninterpretable? Also, it should be possible to detect and fragment glycans above m/z 3500 using at least the Rapiflex mentioned in the methods. The result of these omissions is an underestimation of the glycomic potential of the organism. Testing interactions of the long LN repeats with either mammalian lectins or antibodies would be of interest; again, it is the authors' assumption that they will be immunologically invisible, which if true is also of interest in terms of 'glycan mimickry'.\nComment 8: Importing from Glycoworkbench does have the tendency to make slightly blurry pictures and one has to really zoom in to distinguish the rather small alphas and betas, which could be added rather with a drawing program for legibility.\nComment 9: It is indeed good that the authors found the mistakes in the table due to misassignments as protonated ions. The m/z 738 glycan shown in Tab S4 is not found in both replicates, but m/z 779, 1402, 1432, 1475 found in one replicate are not in Tab S4, while m/z 1636 is in both replicates, but is not in Tab S4. Is Tab S4 meant to be a consensus set - if so, then m/z 738 should not feature if it was only found in one experiment, while m/z 1636 should be included. Without me checking: has the Table S1 - Raw MALDI-TOF-MS data been cross-checked with the Tabs S2-S4?"}, "author_response": "POINT-BY-POINT REPLY TO REVIEWER COMMENTS\nRevision of NCOMMS-25-53181A (Major human schistosome species express different glycans with immunological and diagnostic implications.)\nReviewer #1 (Remarks to the Author):\nThe revised manuscript has addressed several key concerns, namely characterization of the core structure of the GSLs, clarified the limitations of their studies in regard to linkages of sugars, expanded on their insights into the differential antibody responses to glycans previously observed versus their current observations, addressed the limited nature of the study’s insights regarding linkages to the genome, clarified the nature of glycosylation in worms isolated from hamsters compared to human hosts, addressed the limitation of the study in regard to lack of glycoproteomic information, provided additional information in Figure S9 regarding structure of glycans recognized by antibodies, revised their discussion regarding the presence of glucuronic acid in glycans, the lack of quantitation of glycan expression, which is a common limitation of current technologies, and the individualistic nature of immune responses to glycan antigens.\nOverall, the revised manuscript provides excellent information about glycan structures in these parasites, as well as developmental and species-specific difference in glycosylation.\nWe are pleased to have been able to provide satisfactory responses to the reviewer’s comments, and we would like to thank the reviewer once again for the careful evaluation of our work and for the many constructive suggestions that have substantially improved the manuscript.\nReviewer #2 (Remarks to the Author):\nOverall, the manuscript is of interest due to the variations in glycan structures between the two Schistosoma species as well as due to the glycan array data. The data is new, the insights into S. haematobium are novel. The methodology is typical of that from the authors' laboratory. With such a large dataset, it is not possible to assess all files, but spot checks still result in some questions. The authors have revised their manuscript, adding further data and explanations as well as making corrections. My line numbers refer to the 'tracked changes' version which follows the revised manuscript in the single \"manuscript\" pdf.\nWe thank the reviewer for confirming the relevance of our work and the continued detailed evaluation of our manuscript in order to achieve further improvements and clarity\nHeading - line 77 - \"chains\" or \"oligomers\" rather than \"stretches\"\nThe section title has been changed from “S. haematobium GSL glycans are built on a trihexosyl core extended with β-linked N-acetylhexosamine (HexNAc) stretches” to “S. haematobium GSL glycans are built on a trihexosyl core extended with oligomeric chains of β-linked N-acetylhexosamine (HexNAc) residues” (see L.76-77).\nLine 93 - if both a general and a specific b-galactosidase worked, then the linkage is Galb1,4Glc and if the a-Gal is not a1,4, then it can be hypothesised that it is Gala1,3Galb1,4Glc (Gala1,6 in an animal would probably be unusual). Possibly in vitro a1,3-galactosylation of lactose with a commercially available enzyme to generate an alternative standard would resolve this in the absence of enough material for linkage analysis - alternatively Elictyl actually sell Isoglobotriose.\nWe fully concur with the reviewer that the isoglobo series Galα1-3Galβ1-4Glcβ1-Cer is the most likely composition of the core of S. haematobium GSL glycans. Thus, we have modified the text accordingly in the results (L.97-102) and discussion (L.361-371).\nLine 108 - specify S. pneumoniae in the text, not just the table.\nThe organism has been specified (L.110-111).\nLine 370 - certainly one alpha and one beta galactosyltransferase\n“While S. mansoni uses a putative β1-4-N-acetylgalactosaminyltransferase to extend the glucosylceramide with GalNAc, one or more galactosyltransferase(s) must be involved to synthesize the trihexosyl core in S. haematobium” was changed to “While S. mansoni uses a putative β1-4-N-acetylgalactosaminyltransferase to extend the glucosylceramide with GalNAc, S. haematobium employs both an α- and a β-galactosyltransferase to synthesize the trihexosyl core.” (L. 376-378).\nComment 2: The S/N ratio of 4 and a 1% intensity as a cut-off for defining glycan structures seems arbitrary. Can these larger glycan structures be annotated by careful manual interpretation or are the MS2 spectra uninterpretable? Also, it should be possible to detect and fragment glycans above m/z 3500 using at least the Rapiflex mentioned in the methods. The result of these omissions is an underestimation of the glycomic potential of the organism. Testing interactions of the long LN repeats with either mammalian lectins or antibodies would be of interest; again, it is the authors' assumption that they will be immunologically invisible, which if true is also of interest in terms of 'glycan mimickry'.\nIndeed, we applied a criteria of a S/N ratio of 4 and a minimum intensity of 1% to define the cut-off in our MALDI MS analysis. However, these thresholds are not arbitrary but rather were selected because they provide a conservative and robust cut-off for reliably distinguishing true signal from noise in our MALDI MS workflow. We aimed to apply consistent and fair criteria across all samples, and, in our opinion altering this cut-off for a specific sample or glycan class would be difficult to justify without introducing bias and fundamentally affecting the rationale of our study.\nWe however fully agree with the reviewer that the upper limit of the mass range (normally set at m/z 3500) should be extended when the presence of structures with higher molecular weight meeting the aforementioned criteria may be suspected, such as in the case highlighted by the reviewer, in order not to overlook any important glycosylation feature.\nTo address this, we acquired new mass spectrum with a mass range extended to 5000 Da.\nUnfortunately, the original sample containing the N-glycans released using PNGase A from the S. haematobium mature eggs was exhausted. A fifth of this sample was concentrated using C18 ziptip (as detailed in the M&M section) and subjected to MALDI analysis to obtain the spectrum presented in Figure 3 while the remaining amount was used for exoglycosidase digestions presented in Figure S6-D. Applying a newly generated sample containing S. haematobium eggs at different stages of maturity, we detected the structures of composition H N F X (m/z 2591.1, 2737.1, 2794.2, 5-7 6-8 2-3 1 2940.2, 3321.1, 3467.0,) previously pointed out by the reviewer, originating from the mature eggs. The resulting spectrum is presented in Figure R. This N-glycan series is continued by ions with m/z 3623.9, 3670.8, 3920.5, 4082.3 (H N F X ) and several high mass structures of composition 7-9 8 2-3 1 H N F (m/z 3512.8, 3658.7, 3804.6, 3950.5) were also visible. However, the intensities of these ions 9 8 2-5 only decrease further with increasing molecular weight, indicating that no significant glycans with m/z values beyond the mass range applied in this study were overlooked.\nWe were unable to obtain interpretable MS2 spectra for ions detected in the 2500–5000 m/z range of the S. haematobium egg N-glycans released using PNGase A, due to their low intensities. Indeed, these ions exhibit low intensities in the range of 600-2000 arbitrary intensity units (a.u.). For comparison, the MS/MS data shown in Figure S6-E were obtained by fragmenting parent ions with intensities between 4x104 and 5x104 a.u.. Rather than being instrumentation-related, this limitation is due to the scarce amount of material available. Mature S. haematobium eggs are obtained through a specific separation procedure (detailed in M&M) from mixed-age eggs isolated from infected hamsters. Their quantity is therefore very limited. For this reason, it is also not possible to perform functional experiments to investigate the potential interactions of these particular structures with lectins or (infected) sera, although we fully agree with the reviewer that such studies could be valuable to confirm our suggestion that LN repeats are not relevant from an antigenic point of view.\nThe minimum amount needed of these native isolated glycans of interest for microarray printing (low pmol) is far higher than the obtainable amount.\nAlthough we are unable to further address the structural and antigenic features of this low abundance mature egg-specific glycan subset, we agree with the reviewer that its uniqueness, being exclusively found in mature eggs and released with PNGase A, deserves acknowledgment as it constitutes an additional feature of the parasite glycosylation potential. We hope that the newly added Figure S6‑G and the reference in the main text (L.241-243) address this point by showing the specific expression of these structures of higher molecular mass, even though they do not meet our criteria nor the minimally required amount for further quantitative or qualitative study.\nComment 8: Importing from Glycoworkbench does have the tendency to make slightly blurry pictures and one has to really zoom in to distinguish the rather small alphas and betas, which could be added rather with a drawing program for legibility.\nWe appreciate the reviewer’s suggestion to improve the manuscript quality and have added the linkage information using a different program to Glycoworkbench in the following tables and figures:\nTable 2, Figure S3, Figure S5, Figure S8.\nComment 9: It is indeed good that the authors found the mistakes in the table due to misassignments as protonated ions. The m/z 738 glycan shown in Tab S4 is not found in both replicates, but m/z 779, 1402, 1432, 1475 found in one replicate are not in Tab S4, while m/z 1636 is in both replicates, but is not in Tab S4. Is Tab S4 meant to be a consensus set - if so, then m/z 738 should not feature if it was only found in one experiment, while m/z 1636 should be included. Without me checking: has the Table S1 - Raw MALDI-TOF-MS data been cross-checked with the Tabs S2-S4?\nIndeed, data included in Table S4 (and Figure 4) were selected based on the following criteria: ions with S/N > 4, intensities above 1% of the total spectrum intensity, and presence in both technical replicates. The reviewer is correct in noting that the ion at m/z 738 does not meet these criteria and should therefore not have been included in Table S4 or Figure 4. Both have now been corrected. Ion with m/z 1636, however, although present in both duplicates is less than 1% of total intensity, and thus does not fulfill the cut-off criteria.\nTables S1 and S2–S4 have been thoroughly cross-checked. Table S1 contains the complete dataset, which serves as the basis for the subsets presented in Tables S2–S4 (ions with S/N > 4, intensities above 1% of the total spectrum intensity, and presence in both technical replicates)."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have again revised their manuscript, adding some 'zoomed' spectra for the higher molecular weight range. Unfortunately, their attempt at another preparation did not work well as the 'only for referees' supplement shows a spectrum full of polyhexose contaminants. Nevertheless a statement could be included in the legend to Supp 6 that: \"In experiments with total eggs, there were indications of higher molecular weight N-glycans of approximately 4000 Da, whereby no interpretable MS2 could be obtained to verify whether these had either a fourth antenna or extra LacNAc repeats.\" As previous indicated, a perfect \"corroboration\" for the GSL core would be to do PGC-nano-LC-MS with isoglobotriose from Elictyl. Line 585 - does the Rapiflex® have a 1 kHz Smartbeam II laser or a 10 kHz one?"}, "author_response": "POINT-BY-POINT REPLY TO REVIEWER COMMENTS\nRevision of NCOMMS-25-53181B (Major human schistosome species express different glycans with immunological and diagnostic implications.)\nReviewer #2:\nThe authors have again revised their manuscript, adding some 'zoomed' spectra for the higher molecular weight range. Unfortunately, their attempt at another preparation did not work well as the 'only for referees' supplement shows a spectrum full of polyhexose contaminants. Nevertheless a statement could be included in the legend to Supp 6 that: \"In experiments with total eggs, there were indications of higher molecular weight N-glycans of approximately 4000 Da, whereby no interpretable MS2 could be obtained to verify whether these had either a fourth antenna or extra LacNAc repeats”.\nWe typically observe polyhexose contaminants when pushing towards the detection limits in MALDI-TOF MS. Despite the presence of these unwanted peaks in the spectrum, it can be still concluded that high molecular weight N-glycans are not present in a significant amount that would fall within the study criteria set. To convey the point raised by the reviewer that [minor amounts of] N-glycans with m/z up to 4000 Da may still be present we included the sentence below in the caption of Figure S6.\n“Of note, in experiments in which PNGase A was applied to a mixture of eggs at various stages of maturity, minor ions corresponding to N-glycan structures up to ~4000 Da were observed (data not shown). The lack of interpretable MS2 spectra prevented verification of whether these carried four antennae or polyLacNAc repeats.”\nAs previously indicated, a perfect \"corroboration\" for the GSL core would be to do PGC-nano-LC-MS with isoglobotriose from Elictyl.\nWe proceeded with the recommended experiment consisting of running an isoglobotriose standard alongside our biological sample, indeed yielding additional confirmation of the core structure of the S. haematobium glycosphingolipid glycans. We have edited Figure S5-A to incorporate the newly acquired data and referd to the GSL glycan standards in the Methods section (lines 637-639). The main text was also slightly modified accordingly in the Results (lines 102-105) and Discussion (lines 354-360) sections.\nLine 585 - does the Rapiflex® have a 1 kHz Smartbeam II laser or a 10 kHz one?\nWe appreciate the reviewer’s attention to this discrepancy. Unlike the UltrafleXtreme® and Autoflex® instruments, the Rapiflex® is indeed equipped with a 10 kHz Smartbeam laser. We have updated the Methods section to provide the correct value (lines 590-592).\nWe thank the reviewer for their valuable suggestions and hope that our revisions satisfactorily address them."}]

Decision letter
[Editorial Note: See attachment]