Dataset Information

Proteoglycan profiling of human, rat and mouse insulin-secreting cells

ABSTRACT: Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, thus, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by ion exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). We identified CS modifications of chromogranin-A, islet amyloid polypeptide, secretogranin 1 and secretogranin 2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (chromogranin-A and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For chromogranin-A, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF, and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Type B Pancreatic Cell, Islet Of Langerhans, Cell Culture, Pancreatic Islet Cell

DISEASE(S): Type 1 Diabetes Mellitus

SUBMITTER: Jonas Nilsson

LAB HEAD: Göran Larson

PROVIDER: PXD024230 | Pride | 2022-02-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Byonic_results__Fig_3.xlsx	Xlsx
	Fusion_170416_56.raw	Raw
	Fusion_170416_56_MIN6_Media.xlsx	Xlsx
	Fusion_171109_35.raw	Raw
	Fusion_171109_35_human_beta_islet.xlsx	Xlsx

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Publications

Proteoglycan profiling of human, rat and mouse insulin-secreting cells.

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

Glycobiology 20210901 8

Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attac ...[more]

PMID: 33997891

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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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