N-glycoproteomic profiling of the hookworm Ancylostoma ceylanicum reveals heterogeneity and phosphorylcholine-modified glycans in larval and adult parasites.

Paraggio, C. L.; Chang, V.; Mahoney, K. E.; Chung, N.; Sunsunwal, S.; Archer-Hartmann, S.; Randi, S.; Bungiro, R.; Harrison, L. M.; Ledizet, M.; Azadi, P.; Cappello, M.; Malaker, S. A.

doi:10.64898/2026.10.26.762015

bioRxiv · 10.64898/2026.10.26.762015

N-glycoproteomic profiling of the hookworm Ancylostoma ceylanicum reveals heterogeneity and phosphorylcholine-modified glycans in larval and adult parasites.

Paraggio, C. L.·Chang, V.·Mahoney, K. E.·Chung, N.·Sunsunwal, S.·Archer-Hartmann, S.·Randi, S.·Bungiro, R.·Harrison, L. M.·Ledizet, M.·Azadi, P.·Cappello, M.·Malaker, S. A.

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Abstract

Glycans of parasitic worms (helminths) have been previously described with roles in immunomodulation and the recognition of helminth proteins by the host immune system. Despite their significance in other systems, little is known about the glycosylation patterns of the hookworm Ancylostoma ceylanicum, an intestinal parasite of animals and humans, due to challenges in sample collection and structural complexity of many nematode glycans. Herein, we describe results of a released N-glycan analysis of adult hookworms, which revealed oligomannose, hybrid, and complex structures, including those with non-mammalian features. Next, we report the results of glycoproteomic analyses to identify hookworm glycoproteins and their associated glycan structures. We compared three enrichment methods and found that strong anion-exchange electrostatic repulsion liquid chromatography (SAX-ERLIC) led to the identification of the most N-glycopeptides. Analyses of larval and adult extracts revealed 82 larval and 157 adult N-glycoproteins, the majority of which were stage-specific. Of particular interest, phosphorylcholine (PC), a known immunomodulatory glycan modification, was detected in both stages, and several unique PC-modified structures were identified in each. Collectively, these results provide the first site-specific characterization of intact N-glycopeptides in A. ceylanicum and, with an adult-stage released glycan analysis, establish a method for glycoproteomic characterizations of other helminths.

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Paraggio, C. L., Chang, V., Mahoney, K. E., Chung, N., Sunsunwal, S., Archer-Hartmann, S., Randi, S., Bungiro, R., Harrison, L. M., Ledizet, M., Azadi, P., Cappello, M., Malaker, S. A.. 2026-10-27. N-glycoproteomic profiling of the hookworm Ancylostoma ceylanicum reveals heterogeneity and phosphorylcholine-modified glycans in larval and adult parasites.. https://doi.org/10.64898/2026.10.26.762015

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