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For parasitic nematodes, the host parasite interface can be defined as the cuticle or outer exoskeleton covering of the worm, excretory-secretory (ES) components, and extracellular vesicles. Early studies of filarial worm cuticle proteins identified two B. malayi glycoproteins. gp29 (Bm2151) is a major surface glycoprotein found in the cuticle of adult worms with homology to the glutathione peroxidases that are part of the oxidative stress response22. gp15/400 (Bm6083 or Bm6084, Bma-npa-1) is a nematode polyprotein allergen related glycoprotein found in the cuticle of the adult worms and is thought to play a role in acquiring the required fatty acids that the worms cannot synthesize de novo22,23. Recently, gp15/400 was described as an immunodominant antigen targeted by human IgE monoclonal antibodies produced from parasite infected human sera24. Later studies broadened the number of cuticle associated proteins but little is known about their glycosylation status25. ES-62 (Bm9816) is a major secreted glycoprotein in filarial nematodes26,27 and of special interest due to immunomodulatory phosphorylcholine substitution of its N-glycans8,9,26. Larger proteomic studies of excreted and secreted proteins28,29, extracellular vesicle proteins30 and membrane or surface enriched proteins31 highlighted more proteins that are exposed to the mammalian host. However, the possibility of glycan-mediated interactions with the host were not addressed in these strictly proteomic analyses. N-linked glycosite mapping in Caenorhabditis elegans, a soil living nematode, identified 829 N-linked glycoproteins in one study and 1010 N-linked glycoproteins in a second21,32. These studies that identified over a thousand N-glycoproteins included all stages (egg, L1, L2, L3, L4, and adult worms) of the nematode life cycle (2015)." href="/articles/s41598-023-34936-9#ref-CR33" id="ref-link-section-d12369696e568"33. A recent glycosite mapping of a Clade V34 parasitic nematode, Haemonchus contortus, identified 291 N-linked glycoproteins35 from only a mixed adult worm sample./p> pyro-Glu/ -17.026549, amidated @ D and E / -0.984016. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE40 partner repository with the dataset identifier PXD039002 and 10.6019/PXD039002./p> 200 and a peptide length > 4 for the Fbs1 enriched samples. For the Total samples, which had a higher complexity, we used a Byonic score of > 300 and a peptide length > 4. The Byonic score is a gauge of the accuracy of the peptide spectrum match41. Single unique peptides that were associated to only one protein, in one replicate of the sample, and were not present in the remaining five samples are marked by an asterisk in Supplemental Table S1 and not included in further data analysis. To compare the proteins identified by LC-MS/MS to previously published proteomic studies28,29,30,31,42,43 that used different protein IDs, we cross referenced the Wormbase38 and UniProt44 databases to correlate Wormbase ID from our data to the PUB_loci/pub_locus numbers or to UniProtKB ID for the B. malayi proteome and to the UniProt database to correlate NCBI ID with UniProtKB ID or wBm gene numbers for the Wolbachia proteome. The synonyms we found associated with each protein in our dataset used for cross referencing are listed in Supplemental Table S2./p>

We used NetNGlyc-1.046 to find the canonical N-X-S/T glycosites present in each N-glycoprotein. We used Weblogo47 to create a sequence logo for extracted -10 to + 10 amino acid sequences. We used BioVenn48 to generate a proportional Venn diagram. We used TMHMM-2.049 to predict transmembrane domains. We used UpsetR50 to generate UpsetR intersecting set plots. The gene ontology identification and functional enrichment analysis was performed using g:Profiler (version e105_eg52_p16_e84549f.) with g:SCS multiple testing correction method applying significance threshold of 0.0551. We used ggplot (2016)." href="/articles/s41598-023-34936-9#ref-CR52" id="ref-link-section-d12369696e755"52 to generate scatter plots from gene enrichment data analysis. We used DeepLoc to predict subcellular localization for each N-glycoprotein53. We used Parasite Biomart at Wormbase to search for both C. elegans and H. contortus orthologs to identified B. malayi N-glycoproteins54. We used Clustal Omega to generate a multiple sequence alignment55./p> (2015)./p>