Mass spectrometric identification of aberrantly glycosylated human apolipoprotein C-III peptides in urine from Schistosoma mansoni-infected individuals

Abstract

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered.

Fig. 1.

MALDI-TOF MS analysis of urinary (glyco)peptides from control and S. mansoni-infected individuals. Urinary peptides were separated using strong cation exchange chromatography, and collected fractions were analyzed using MALDI-TOF MS. Numbers refer to MS numbers given to the samples. Open square, N-acetylhexosamine; open circle, hexose.

Fig. 2.

MS/MS fragmentation of a glycopeptide present in urine from a S. mansoni-infected individual. Urinary peptides from an S. mansoni-infected individual were separated using strong cation exchange chromatography. Fractions containing glycopeptides were analyzed using LC-ion trap MS, and glycopeptides were fragmented using collisional-induced dissociation. Shown is the MS/MS spectrum from a glycopeptide m/z 1068.2 [M + 3H]³⁺ present only in the infected individual with a glycan moiety composed of H₂N₄F₃. If not indicated differently, all ions containing the peptide moiety (pep) are doubly charged, and those lacking the peptide moiety are singly charged. No monosaccharide linkage information is obtained. Red triangle, fucose; yellow circle, galactose; blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine; open square, N-acetylhexosamine.

Fig. 3.

Preparative purification of glycopeptides from urine of S. mansoni-infected individual. Urinary peptides from an S. mansoni-infected individual were separated using strong cation exchange chromatography. Fractions containing highly fucosylated glycopeptides were further purified using HILIC HPLC and measured by MALDI-TOF MS (A). In addition to the peptide at 3201.6 [M + H]⁺ carrying H₂N₄F₃ (Fig. 2), truncated peptides (lacking Ala, Ala-Ala, or Val-Ala-Ala) with the same glycan moiety were detected. Moreover, heterogeneity in the glycan composition was observed. The heterogeneity in the peptide sequence and glycan moiety was confirmed by MALDI-TOF-TOF MS (B). pep, peptide; pep′, peptide lacking Ala; pep‴, peptide lacking Val-Ala-Ala. Red triangle, fucose; yellow circle, galactose; blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine; open square, N-acetylhexosamine.

Fig. 4.

Highly fucosylated glycopeptides from urine of S. mansoni-infected individuals consist of C-terminal peptide of human apolipoprotein C-III. A, MALDI-TOF-TOF analysis of the highly fucosylated glycopeptide at m/z 3201.6 ([M + H]⁺) after SCX and normal phase purification of urinary peptides from an S. mansoni-infected individual. y ions indicated with * have lost the complete glycan moiety. Ions indicated with # are similar to y₁₁ but have additionally lost one or more monosaccharides. B, normal human apolipoprotein C-III was digested with trypsin and analyzed with MALDI-TOF-TOF MS. Shown is a C-terminal tryptic glycopeptide containing one N-acetylgalactosamine-Gal element (corresponding to apoC-III₀). Note the similar series of y ions as observed in the MALDI-TOF-TOF spectrum in A. Red triangle, fucose; yellow circle, galactose; blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine; open square, N-acetylhexosamine. pep, peptide.

Fig. 5.

Aberrantly glycosylated apolipoprotein C-III-derived peptides from urine of S. mansoni-infected individuals contain multiple H₁N₁F₁ elements. Urinary peptides from an S. mansoni-infected individual (10413) were separated using strong cation exchange chromatography. Fractions containing highly fucosylated glycopeptides were further purified using HILIC HPLC and measured by MALDI-TOF MS. Shown are the C-terminal apoC-III peptides WDLDPEVRPTSAVA(A) carrying H₂N₄F₄ but also one or two extra H₁N₁F₁ units. Red triangle, fucose; yellow circle, galactose.

Fig. 6.

Characterization of fucose positions on aberrantly glycosylated apoC-III. Urinary peptides from an S. mansoni-infected individual (22824) were separated using strong cation exchange chromatography. Fractions containing glycopeptides were analyzed using LC-ion trap MS, and glycopeptides were fragmented using collisional-induced dissociation. From one specific aberrantly glycosylated apoC-III peptide (WDLDPEVRPTSA carrying H₂N₂F₃), CID MS/MS spectra were recorded from a fully protonated (A) and partially sodiated (B) species. Within the MS/MS spectrum from the fully protonated species, a [Fuc₂-Hex₁-HexNAc₁ + H]⁺ element was observed (m/z 658.1) potentially harboring a difucosyl (Fuc(α1–2)Fuc-) element. Subsequent fragmentation of the partially sodiated precursor indicated that no difucosyl elements were present. Red triangle, fucose; yellow circle, galactose; blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine. pep, peptide.

Fig. 7.

Profiling of apolipoprotein C-III glycoforms in serum of S. mansoni-infected and non-infected individuals. Proteins were extracted from human serum samples from non-infected and Schistosoma mansoni-infected individuals using C₁₈ ZipTips. Apolipoprotein C-III glycoforms in the eluates were measured by MALDI-TOF MS. A clear difference between the profile of the different apoC-III glycoforms in infected and non-infected individuals is apparent.