title: Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
abstract: Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.
authors.affiliation: From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden;
authors.forename: Fredrik
authors.initials: F
authors.lastname: Noborn
authors.affiliation: From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden;
authors.forename: Alejandro
authors.initials: A
authors.lastname: Gomez Toledo
authors.affiliation: §Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Box 413, SE-405 30, Sweden;
authors.forename: Carina
authors.initials: C
authors.lastname: Sihlbom
authors.affiliation: §Proteomics Core Facility, Sahlgrenska Academy, University of Gothenburg, Box 413, SE-405 30, Sweden;
authors.forename: Johan
authors.initials: J
authors.lastname: Lengqvist
authors.affiliation: ¶Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Fries
authors.affiliation: ¶Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Kjellén
authors.affiliation: From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden;
authors.forename: Jonas
authors.initials: J
authors.lastname: Nilsson
authors.affiliation: From the ‡Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; goran.larson@clinchem.gu.se.
authors.forename: Göran
authors.initials: G
authors.lastname: Larson
mesh_terms: D000510:Alpha-Globulins / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D002766:Cholecystokinin / Q000032:analysis; D011508:Chondroitin Sulfate Proteoglycans / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D053379:Chromogranin A / Q000032:analysis; D053378:Chromogranin B / Q000032:analysis; D002864:Chromogranins / Q000032:analysis; D006020:Glycopeptides / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000378:metabolism / Q000652:urine; D006801:Humans; D008297:Male; D009479:Neuropeptides / Q000032:analysis
chemical_list: D000510:Alpha-Globulins; C506134:CHGB protein, human; D011508:Chondroitin Sulfate Proteoglycans; D053379:Chromogranin A; D053378:Chromogranin B; D002864:Chromogranins; D006020:Glycopeptides; C467731:NPW protein, human; D009479:Neuropeptides; C506136:SCG3 protein, human; C016001:alpha-1-microglobulin; D002766:Cholecystokinin