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 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 (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.

Mass spectrometric analysis of chondroitin sulfate-linked peptides

“…The bond between serine and chain initiating xylose is an O-glycosidic bond, which can undergo elimination in HCD (Riley et al 2020 ; Mao et al 2021 ) and peptides which contain two proximal serine residues, EtHCD can be beneficial for localizing the site unambiguously. However, in many cases, we did find evidence of site localization from HCD data, which is likely due to the fact that we employed a stepped collision energy strategy that has also proven useful previously (Noborn et al 2015 ; Nikpour et al 2021 ). When stepped collision energy is employed, every precursor is fragmented with three different specified collision energies and a composite MS/MS spectrum is reported.…”

Importance of Heparan Sulfate Proteoglycans in Pancreatic Islets and β-Cells

“…The expressions of the type XVIII collagen and SDC1 in T1DM patients, and a CD44 addition to the foregoing HSPGs in T2DM model mice, were reduced in pancreatic islets along with HS [ 15 , 16 ]. Recent findings using nano-liquid chromatography-tandem mass spectrometry method has reported that several proteins, including prohormones, not previously recognized as HSPGs, can become HSPGs in pancreatic β-cells [ 61 ]. However, there have only been a few studies on loss/gain in the functions of HSPG core proteins have provided the functional analyses of core proteins in pancreatic β-cells.…”

Expanding the Chondroitin Sulfate Glycoproteome — But How Far?

“…The identification of such "hybrid sites" is likely to be biologically important as HS and CS often display opposite effects on cellular physiology (Coles et al, 2011;Doody et al, 2015). Furthermore, similar glycoproteomic protocols have been used to identify novel HSPGs, and to investigate the functional role of HSPGs in neuronal synapse formation (Zhang et al, 2018;Montoliu-Gaya et al, 2021;Nikpour et al, 2021).…”

“…A list of all CSPGs known to date in humans was recently published by Toledo et al (2020). The number of human core proteins carrying HS are even less, and so far only a little more than 20 HSPGs have been established (Zhang et al, 2018;Nikpour et al, 2021). Moreover, while proteoglycans in vertebrates have been the focus of several structural studies, the information about proteoglycans in invertebrates is scarce and the reports are restricted to only a few species .…”

“…HS at the same C-terminal site, making this another "hybrid site" (Noborn et al, 2016;Nikpour et al, 2021). Although the glycosite is highly conserved between species (Noborn et al, 2015), this highlights the importance of cell-targeted mapping and that GAG modifications found in one cell type or tissue or species may not simply be inferred to another sample system.…”