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Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome

ABSTRACT: The spondylodysplastic type of Ehlers-Danlos syndromes (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, 59/41.We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

SUBMITTER: Proteomics Core Facility

LAB HEAD: Göran Larson

PROVIDER: PXD033230 | Pride | 2022-06-29

REPOSITORIES: Pride

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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:Chondroitin sulfate proteoglycans (CSPGs), an important class of carbohydrate-modified proteins, are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. CS polysaccharides interact with a multitude of protein ligands to control key events in homeostasis and development. Much effort has been devoted to CS structural characterization, as the specificities of the interactions are considered to be determined by the distribution of sulfate groups along the polysaccharide chains. Owing to their structural complexity however, CS chains are typically characterized after they have been released from their core proteins, which precludes site-specific structural information. Such information would likely assist in assigning CSPG-related functions and provide insights to potential differences in CS structures for different core proteins and their glycosites. Here we utilize a novel glycoproteomic approach for the site-specific sequencing of CS modifications of CSPGs from human urine using a combination of biochemical enrichments, enzymatic digestions, and positive mode nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. Trypsin-derived CS-glycopeptides were partially depolymerized with chondroitinase ABC, yielding peptide-attached CS chains, ranging from 6 - 18 monosaccharides in length. Software-assisted- and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that acidity of the attachment site motifs influenced the levels of CS sulfation and statistical analysis confirmed this relationship. These results demonstrate attachment site-specific CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.

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Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome

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