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Identification of a chondroitin sulfate linkage region trisaccharide

ABSTRACT: Here, using LC-MS/MS, we describe for the first time a linkage region trisaccharide comprising [-GlcUA-Gal-Xyl-O-] as an alternative point of entry for glycosaminoglycan (GAG) biosynthesis. The trisaccharide was identified in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, [deltaHexUA-GalNAc(S)-GlcUA-Gal(S)-Xyl-O-] after chondroitinase ABC degradation. Furthermore, it was identified in chondroitin sulfate primed on xylosides isolated from human cell lines, present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation.

INSTRUMENT(S): Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Egor Vorontsov

LAB HEAD: Göran Larson

PROVIDER: PXD011855 | Pride | 2019-02-25

REPOSITORIES: Pride

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Identification of a non-canonical chondroitin sulfate linkage region trisaccharide.

Persson Andrea A Nilsson Jonas J Vorontsov Egor E Noborn Fredrik F Larson Göran G

Glycobiology 20190501 5

It is generally accepted that the biosynthesis of chondroitin sulfate and heparan sulfate is proceeding from a common linkage region tetrasaccharide comprising GlcA-Gal-Gal-Xyl-O-. The linkage region can undergo various modifications such as sulfation, phosphorylation and sialylation, and as the methods for studying glycosaminoglycan structure have been developed and refined, the number of discovered modifications has increased. Previous studies on the linkage region and the glycosyltransferases ...[more]

PMID: 30824935

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Project description:The inter-alpha-trypsin inhibitor (IαI) complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The IαI complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the non-sulfated non-reducing end, (GalNAc4GlcA3)n ,to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation (HCD). The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal two GalNAc residues. The fragmentation behavior of CS glycopeptides under variable HCD conditions displays an energy dependency that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

Project description:In order to study the function of the Campylobacter jejuni Cj0667 gene, a series of experiments were carried out. Two strains were constructed: a Cj0667 knockout strain and a strain with a second copy over-expressing Cj0667 from an fdxA promoter. The transcriptomes of these were all compared to the wild-type strain. The arrays are all from RNA isolated in mid-exponential growth. Batch cultures of Campylobacter jejuni NCTC 11168 were grown in 50 ml volumes of Brucella broth in 70cm tissue culture flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (5% Oxygen, 10% Carbon dioxide, 85% Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 37 M-BM-:C. RNA was extracted from C. jejuni cultures grown to an OD600 of approximately 0.4. Briefly, 0.1 volume of 5% phenol in ethanol was mixed with the broth culture, and after centrifugation RNA was isolated with Tri Reagent (Sigma) and chloroform. RNA was further purified using the RNeasy kit (Qiagen) according to the manufacturerM-bM-^@M-^Ys instructions. The RNA was treated with Turbo DNA-free (Ambion) to remove any residual DNA according to the manufacturerM-bM-^@M-^Ys instructions. RNA concentration was determined using the Nanodrop Spectrophotometer NS-1000 (Thermo Scientific). RNA preparations of each sample type were labelled and hybridized to custom-designed Agilent microarrays. Equal quantities of RNA from test and control cultures were labelled by using nucleotide homologues of dUTP containing either Cy3 or Cy5 fluorescent dye (Perkin Elmer). For each microarray slide, the test strain was labelled with Cy3-dUTP, while the wild-type sample was labelled with Cy5-dUTP. RNA (15 M-BM-5g) was primed with 5 M-BM-5g pd(N)6 random hexamers (Amersham Biosciences). The Affinity Script kit (GE Healthcare) was used to produce cDNA, and hybridized (Agilent Hi-RPM Gene Expression Hybridization Kit) to the microarray slide according to the manufacturerM-bM-^@M-^Ys instructions. Microarrays were scanned at 5 M-BM-5m using an Axon 4000A scanner, and images were acquired using GenePix Pro 3.0 software (Axon). Related analysis reference: Holmes K., Mulholland F., Pearson B. M., Pin C., McNicholl-Kennedy J., Ketley J. M., Wells J. M. (2005) Campylobacter jejuni gene expression in response to iron limitation and the role of Fur Microbiology-SGM 151 243-257 (PMID 15632442).

Project description:In order to study the function of the Campylobacter jejuni Cj1103 gene, a series of experiments were carried out. Three strains were constructed: a Cj1103 knockout strain, a strain where the Cj1103 knockout was complemented in trans, and a strain with a second copy over-expressing Cj1103 from an metK promoter. The transcriptomes of these were all compared to the wild-type strain. The arrays are all from RNA isolated in mid-exponential growth from independent biological replicates. Batch cultures of Campylobacter jejuni NCTC 11168 were grown in 50 ml volumes of Brucella broth in 70cm tissue culture flasks. Microaerophilic conditions were generated using a MACS-VA500 microaerophilic work station (5% Oxygen, 10% Carbon dioxide, 85% Nitrogen) from Don Whitley Scientific, Ltd which also maintained the growth temperature at 37 M-BM-:C. RNA was extracted from C. jejuni cultures grown to an OD600 of approximately 0.4. Briefly, 0.1 volume of 5% phenol in ethanol was mixed with the broth culture, and after centrifugation RNA was isolated with Tri Reagent (Sigma) and chloroform. RNA was further purified using the RNeasy kit (Qiagen) according to the manufacturerM-bM-^@M-^Ys instructions. The RNA was treated with Turbo DNA-free (Ambion) to remove any residual DNA according to the manufacturerM-bM-^@M-^Ys instructions. RNA concentration was determined using the Nanodrop Spectrophotometer NS-1000 (Thermo Scientific). Two or three independent RNA preparations (biological replicates) of each sample type were labelled and hybridized to custom-designed Agilent microarrays. Equal quantities of RNA from test and control cultures were labelled by using nucleotide homologues of dUTP containing either Cy3 or Cy5 fluorescent dye (Perkin Elmer). For each microarray slide, the test strain was labelled with Cy3-dUTP, while the wild-type sample was labelled with Cy5-dUTP. RNA (15 M-BM-5g) was primed with 5 M-BM-5g pd(N)6 random hexamers (Amersham Biosciences). The Affinity Script kit (GE Healthcare) was used to produce cDNA, and hybridized (Agilent Hi-RPM Gene Expression Hybridization Kit) to the microarray slide according to the manufacturerM-bM-^@M-^Ys instructions. Microarrays were scanned at 5 M-BM-5m using an Axon 4000A scanner, and images were acquired using GenePix Pro 3.0 software (Axon). Related analysis reference: Holmes K., Mulholland F., Pearson B. M., Pin C., McNicholl-Kennedy J., Ketley J. M., Wells J. M. (2005) Campylobacter jejuni gene expression in response to iron limitation and the role of Fur Microbiology-SGM 151 243-257 (PMID 15632442).

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