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, 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 exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, 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.

Keywords: chromogranin-A; glycosaminoglycan; insulinoma; islet cells; proteoglycan.

Fig. 1

Amino acid sequence, phosphorylation and O-glycosylation sites of human chromogranin-A pre-prohormone. Amino acids are shown as single letters, are numbered on top and indicated as bold at tryptic cleavage sites. Human, rat and mouse sequences were aligned using the multiple sequence alignment program Clustal Omega and the level of conservation among the three species is indicated below each residue. Sites of phosphorylation (P) and O-glycosylation (O-GalNAc; yellow squares and O-Xyl; orange star) of human CgA are marked above the corresponding amino acids. Established CgA-derived peptide hormones and fragments are annotated and their sequences underlined. The GAG site at Ser-424 (boxed sequence (Noborn et al. 2015) corresponds to Ser-433 of rat CgA and to Ser-430 of mouse CgA. Glycans are illustrated according to the Symbol Nomenclature for Glycans (Varki et al. 2015). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 2

MS² scan and spectra of GAG-glycopeptides of chromogranin-A from human islet cells. The spectral file was filtered for the MS² diagnostic ion at m/z 362.11, corresponding to the [ΔHexAHexNAc]⁺ ion (A). The MS² spectrum of the major glycopeptide is shown for NCE at 40%, to display the peptide sequence fragmentation (B) and at NCE 30% providing better glycan fragmentation (C). The positioning and distinction of sulfate and phosphate groups were made by manually evaluating the MS² spectra. The MS² spectrum of the precursor ion in (C) displays a mass shift of 79.958 u between the ions at m/z 362.110 and m/z 442.068, demonstrating the presence of a sulfate group on the GalNAc residue. A mass shift of 1153.1945 u between the precursor ion, m/z 1086.0616 (3+), and the peptide ions, m/z 1052.4841 (2+) and m/z 701.9922 (3+), demonstrates the presence of one sulfate group on the GalNAc residue and one phosphate group on the Xyl residue.

Fig. 3

Comparison of unmodified and modified peptides of chromogranin-A from different preparation protocols of human islets and rat INS-1 832/13 cells. Peptides were prepared from human islets (A-C) or rat INS-1 832/13 cells (D-F) either without (A, B, D and E) or with (C, F) digestion with trypsin and either not enriched (A, D) or enriched (B, C, E and F) on SAX-columns, and finally depolymerized with ChABC. The identified CgA peptides are aligned under the corresponding sequences and the type of peptide is illustrated with unique colors (unmodified; dark blue (without SAX), red (with SAX) and green (with trypsin and SAX), CS-glycopeptide; orange, O-glycopeptide; yellow and phosphopeptide; light blue). This figure is available in black and white in print and in color at Glycobiology online.

Fig. 4

Western blot analysis of chromogranin-A from INS-1 832/13 cell culture media after cultivation with different concentrations of glucose and different incubation times. Cells were incubated in 3, 11, 17 or 25 mM concentrations of glucose and the cell culture media collected after 6, 24 and 48 h (A, B). Western blot analysis was done with or without ChABC and heparinases II and III (Hep II/III) depolymerization on the fractions collected after 6 and 48 h (C, D). The numbers under the blots represent the relative distribution of glycosylated and nonglycosylated CgA isoforms (A-D).

Fig. 5

Identification of chromogranin-A as a hybrid PG with one single glycosite in rat INS-1 832/13 cells. Extracted ion chromatograms (XIC) of ChABC- and heparinases II and III-depolymerized INS-1 832/13 cell culture media were obtained by filtering for the presence of the MS² diagnostic ions at m/z 362.11 and at m/z 173.04 (A). XIC of the ions of m/z 881.3116 (3+) and m/z 728.2854 (3+) utilized for comparing the areas under the peaks corresponding to the relative distribution of CS- and HS-glycopeptides of the same peptide sequence (B). MS² spectra of the HS and CS CgA glycopeptides, taken at 11.02 and 11.77 min at NCE 20% (C, D) and at NCE 35% (E, F). The asterisk in panel (A) indicates an unknown HS-glycopeptide eluting at 34 min. The major glycopeptide eluting at 11.07 min corresponds to a monosulfated CS-hexasaccharide linkage region attached to the peptide 422-GDFEEKKEEEGSANR-436.

Fig. 6

MS² spectra showing the identification of novel CS- and HS-glycopeptides in rat INS-1 832/13, MIN6 and NIT-1 cells. The MS² spectra illustrate the identification of glycopeptides of islet amyloid polypeptide (A, B), neurexin-2/neurexin-2-beta (E, F) and secretogranin-1 (G, H) from rat INS-1 832/13 cells, and from a glycopeptide of immunoglobulin superfamily member 10 (C, D) from MIN6 and NIT-1 cells. The MS² spectra were acquired at NCE 20% for glycan fragmentation (left panels) and at NCE 35% for peptide fragmentation (right panels). The glycan and peptide fragmentation for secretogranin-1 are shown at NCE 30%, due to the lack of fragmentation at NCE 20% for this glycopeptides. Note the diagnostic ion at m/z 173.04 for HS-derived structures and the diagnostic ion at m/z 362.11 for CS-derived structures.

Fig. 7

Identification of two glycopeptides of human syndecan-4 modified with HS-tetrasaccharide linkage regions. MS² spectra of two different glycopeptides of SDC4 obtained from a human islet cell preparation, treated with both ChABC and heparinases II and III, and analyzed at NCE 20% for glycan characterization (A, C) and 30 or 35% (B, D) for peptide identification. The two precursor ions were extracted by filtering the MS² spectral file for the presence of the ion at m/z 173.04, diagnostic for HS structures. Only two major peaks appeared and the masses of the precursor ions, the glycan and peptide fragmentation patterns clearly identified HS tetrasaccharide linkage regions attached to these two glycopeptides.