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 syndrome (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, and 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.

Keywords: Ehlers–Danlos syndrome; bikunin; glycopeptides; glycoproteomics; glycosaminoglycan linkage region; linkeropathies; liquid chromatography; tandem mass spectrometry; β3GalT6.

FIGURE 1

Schematic representation of proteoglycan linkage and GAG biosynthesis with the indication of the associated EDS types. The assembly of both CS/DS and HS GAGs starts with the biosynthesis of a common tetrasaccharide linkage region (GlcA‐Gal‐Gal‐Xyl‐O‐) at specific serine residues of the core proteins. The first step is initiated by adding a Xyl residue to a serine residue by the action of one of two xylosyltransferases XylT‐I or XylT‐II (encoded by XYLT1 and XYLT2, respectively). Subsequently, two Gal and one GlcA residue are added to the growing chain by sequential actions of the enzymes β4GalT7/GalT‐I (encoded by B4GALT7), β3GalT6/GalT‐II (encoded by B3GALT6), and GlcAT‐I (encoded by B3GAT3). Biosynthesis of the CS/DS and HS GAG chains continues by the enzymatic addition of GlcA and GalNAc (for CS/DS) or GlcA and GlcNAc (for HS GAGs). Finally, various monosaccharides undergo a series of enzymatic modifications including epimerization, N‐deacetylation, N‐, and O‐sulfations to become the mature GAGs. Abbreviations: mcEDS, musculocontractural Ehlers–Danlos syndrome; spEDS, spondylodysplastic Ehlers–Danlos syndrome

FIGURE 2

MS/MS spectra of bikunin/protein AMBP showing glycan fragmentations of two glycopeptides with either a canonical (A) or a non‐canonical (B) linkage region attached to the same peptide sequence. The urine sample analyzed was from individual PIV:1, one of three siblings diagnosed with spEDS with a β3GalT6 deficiency.