Abstract
The inter-α-trypsin inhibitor 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 inter-α-trypsin inhibitor 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 (GlcAβ3Galβ3Galβ4Xylβ-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 nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(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. 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 GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence 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.
Highlights
From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Sweden; §The Proteomics Core Facility, Core Facilities, Sahlgrenska Academy at the University of Gothenburg, Sweden
Proteoglycans constitute a group of O-glycosylated proteins all carrying one or more complex glycan1 chains attached to Ser residues of the core proteins through a common linkage tetrasaccharide, called the GAG linkage region, which is composed of the innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) at the reducing end of the GAG chain (GlcA is glucuronic acid; Gal is galactose; and Xyl is xylose) [2]
Enrichment and MS/MS Fragmentation Analysis of Bikunin Linkage Region Glycopeptides—The urine, the plasma, and the cerebrospinal fluid samples were all selected for study as they represent accessible human biological fluids traditionally used for diagnostic purposes
Summary
GAG, glycosaminoglycan; CS, chondroitin sulfate; CSF, cerebrospinal fluid; CSPG, chondroitin sulfate proteoglycan; Fuc, fucose; ⌬GlcA, unsaturated glucuronic acid; Gal, galactose; GlcA, glucuronic acid; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; HCD, higher-energy collisional dissociation; H1, inter-alpha-trypsin inhibitor heavy chain 1 (UniProtKB P19827); H2, inter-alpha-trypsin inhibitor heavy chain 2 (UniProtKB P19823); HexNAc, N-acetylhexosamine; HSPG, heparan sulfate proteoglycan; I␣I, inter-alpha-trypsin inhibitor; NCE, normalized collision energy; Neu5Ac, N-acetylneuraminic acid; PG, proteoglycan; ppm, parts per million; SAX, strong anion exchange; UTI, urinary trypsin inhibitor, bikunin, inter-alpha-trypsin inhibitor light chain (UniProtKB P02760); Xyl, xylose. Chondroitin sulfate proteoglycans play a significant role in maintaining the structural integrity of most extracellular matrices In addition to their structural role as matrix components, eukaryote CSPGs are known to be involved in more specialized functions such as signal transduction, morphogenesis and regulation of stem cell behavior and differentiation [3,4,5,6,7,8,9]. In order to obtain integrated glycan-protein information, we recently developed a glycoproteomics approach allowing site-specific analysis of CSPG linkage region glycopeptides [20]. The fine-structure analysis of the CS glycopeptides and their substitution patterns was hampered by the relatively weak intensities of sulfated and/or phosphorylated fragment ions obtained under the HCD conditions used This problem became especially obvious for bikunin, the major component of the inter-␣-trypsin inhibitor (I␣I) complex and the most abundant and heterogeneous CSPG of those samples. Given that specific modifications of the linkage region may function as a molecular regulator of CS biosynthesis, these findings could assist in further elucidating the mechanisms of CS chain elongation and substitution
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