1 to 3 HexNAc units (Fig. 4, A and B). Manual assignment of EThcD spectra for glycopeptides with several HexNAc units revealed that every HexNAc is connected to a distinct residue (Fig. S3 and Information S1). Given preceding reports of mucin-type glycosylation in C. parvum, these O-glycans are probably to become Tn structures (GalNAc1-Ser/ Thr) (41, 42). For the CpTSP family members of proteins, numerous glycopeptides had been identified (Table 1) with EThcD evaluation confirming that4 J. Biol. Chem. (2023) 299(3)Characterizing the TSP protein household in C. parvumFigure three. C-mannosylated proteins in Cryptosporidium parvum sporozoites. A, Western blot analysis of SDS-extracted proteins from C. parvum sporozoites utilizing the 5G12 mAb as main antibody. B, volcano plot demonstrating the enrichment of C-mannosylated peptides from C. parvum sporozoite lysate utilizing the 5G12 mAb, as compared with an isotype handle. C, tandem mass spectra for the singly modified CpTSP11 peptide 531TCETTEWSR539 and (D) for the doubly modified CpTSP2 peptide 912SDCVLGEWTEWGVCSK927. A red “W” indicates a Trp(Man) residue, whereas “c” represents acetamidylcysteine.these delta masses corresponded to N-glycosylation events with both Hex5HexNAc2 and Hex6HexNAc2 localized for the peptide 212 YYFDDKNLYYVNSTGIDEK231 of CpTSP8 (Figs. 4C) and 89 ELKFNGLNITSYENR103 of CpTSP11 (Fig. 4D), respectively.SKI II These minimally processed N-linked glycans are unlike those created by mammalian, insect, and yeast cells, suggesting thatglycoengineered cell lines will likely be required to create C.Naloxone (hydrochloride) parvum antigens with genuine glycosylation profiles.PMID:24065671 Localization of CpTSP proteins in C. parvum sporozoites Together with the exception of CpTSP8 (CpMIC1) (43), no published localization information exist for the CpTSP household of proteins. WhileJ. Biol. Chem. (2023) 299(3)Characterizing the TSP protein family members in C. parvumTable 1 N-, O-, and C-glycosylation web sites identified on CpTSP proteins in Cryptosporidium parvum sporozoites following 5G12 or ZIC-HILIC enrichmentNo. of glycosylation websites Protein CpTSP1 (TRAP-C1) CpTSP2 CpTSP3 CpTSP4 CpTSP7 CpTSP8 (CpMIC1) CpTSP11 UniProt ID Q5CSA5 Q5CRC0 Q5CSA4 Q5CQ00 Q5CQ18 Q5CXK1 Q5CXC2 Peptide YTEWSAWSSCDCSGTQTR DADCDTGTCIHNEWSSWTTCKDPCSNTETMSR ESCNKDVECPHVQCELGEWSSWSPCSVTCGCGTTTR SDCVLGEWTEWGVCSK NGGETCGALKAEETGCNSHIPCPLSCTVSEWGNWSR VGEWSSWSECDAK YGECDINCVLGDWTQWSGCDSALCSDGK CFVGEWSNWSK VEDCQISQWTDWSTCSKTCSTGSK ELTHSAPGCDSLLKETSSCNSSPCPVDCVLSFWSPW TGCSK YYFDDKNLYYVNSTGIDEK TCETTEWSR LSSIKNETEQNSSIQTGDLLTK ELKFNGLNITSYENR W-Hex two 2 2 2 2 two 2 2 two 2 — 1 — — S/T-dHexHex — — 1 — — — — — 1 — — — — — N-Hex5 HexNAc2 — — — — — — — — — — 1 — 1Bold and underlined residues are confirmed glycosylation web-sites, whereas bold-only residues are most likely but unconfirmed web-sites of O-glycosylation.these proteins are all targeted for secretion (Fig. 1A) and assumed to be around the cell surface and/or within secretory organelles, confirming their presence around the surface of sporozoites is important if they are to be considered as potential vaccine antigens. Towards the very best of our information, no welldefined antibody tools are readily available to probe the localization of CpTSP12 individually. Nevertheless, our immunoprecipitation and proteomics experiment (Fig. 3) demonstrated that these proteins are exclusively recognized by 5G12: the C-mannosyl tryptophan pecific mAb (31, 32). As a result, we utilized this antibody to probe the collective localization of the CpTSP protein family in C. parvum sporozoites by immunofluorescence micr.