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Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain

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Abstract

Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50 %, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1.

As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.

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Abbreviations

CID:

Collision-induced dissociation

ETD:

Electron-transfer dissociation

FPLC / HPLC:

Fast protein / High pressure liquid chromatography

Gal:

Galactose

Gal1:

Galectin1

GlcNAc:

N-acetylglucosamin

HEK293 cells:

Human embryonic kidney cells

Hex:

Hexose

HexNAc:

N-acetylhexosamine

HUVEC:

Human umbilical vein endothelial cell

Neu5Ac:

N-acetylneuraminic acid

Nrp1:

Neuropilin-1

(O-)GalNAc:

(O-linked) N-acetylgalactosamine

(RP)-LC-ESI-MS:

(reversed phase)-liquid chromatography-electrospray ionization mass spectrometry

PGC:

Porous graphitic carbon

Q-TOF:

Quadrupole - Time of flight

SPE:

Solid phase extraction

VEGF:

Vascular endothelial growth factor

VEGFR2:

Vascular endothelial growth factor receptor 2

References

  1. Giger, R.J., et al.: Semaphorin III: role in neuronal development and structural plasticity. Prog. Brain Res. 117, 133–149 (1998)

    Article  CAS  PubMed  Google Scholar 

  2. Frankel, P., et al.: Chondroitin sulphate-modified neuropilin 1 is expressed in human tumour cells and modulates 3D invasion in the U87MG human glioblastoma cell line through a p130Cas-mediated pathway. EMBO Rep. 9(10), 983–989 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Shintani, Y., et al.: Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling. EMBO J. 25(13), 3045–3055 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Appleton, B.A., et al.: Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding. EMBO J. 26(23), 4902–4912 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chen, R., et al.: Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. J. Proteome Res. 8(2), 651–661 (2009)

    Article  CAS  PubMed  Google Scholar 

  6. Hsieh, S.H., et al.: Galectin-1, a novel ligand of neuropilin-1, activates VEGFR-2 signaling and modulates the migration of vascular endothelial cells. Oncogene 27(26), 3746–3753 (2008)

    Article  CAS  PubMed  Google Scholar 

  7. Meynier, C., Guerlesquin, F., Roche, P.: Computational Studies of Human Galectin-1: Role of Conserved Tryptophan Residue in Stacking Interaction with Carbohydrate Ligands. J. Biomol. Struct. Dyn. 27(1), 49–57 (2009)

    Article  CAS  PubMed  Google Scholar 

  8. Carlsson, M.C., et al.: Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients. PLoS One 6(10), e26560 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Quinta, H.R., et al.: Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury. Cell Death Differ. 21(6), 941–955 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Steentoft, C., et al.: Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J. 32(10), 1478–1488 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Windwarder, M., Altmann, F.: Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry. J. Proteomics 108, 258–268 (2014)

    Article  CAS  PubMed  Google Scholar 

  12. Pabst, M., et al.: Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140. Biol. Chem. 393(8), 719–730 (2012)

    Article  CAS  PubMed  Google Scholar 

  13. Edge, A.S., Spiro, R.G.: Presence of an O-glycosidically linked hexasaccharide in fetuin. J. Biol. Chem. 262(33), 16135–16141 (1987)

    CAS  PubMed  Google Scholar 

  14. Pabst, M., et al.: IL-1beta and TNF-alpha alter the glycophenotype of primary human chondrocytes in vitro. Carbohydr. Res. 345(10), 1389–1393 (2010)

    Article  CAS  PubMed  Google Scholar 

  15. Wuhrer, M., Deelder, A.M., van der Burgt, Y.E.M.: Mass Spectrometric Glycan Rearrangements. Mass Spectrom. Rev. 30(4), 664–680 (2011)

    Article  CAS  PubMed  Google Scholar 

  16. Everest-Dass, A.V., et al.: Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS. J. Am. Soc. Mass Spectrom. 24(6), 895–906 (2013)

    Article  CAS  PubMed  Google Scholar 

  17. Kolarich, D., et al.: The Minimum Information Required for a Glycomics Experiment (MIRAGE) Project: Improving the Standards for Reporting Mass-spectrometry-based Glycoanalytic Data. Mol. Cell. Proteomics 12(4), 991–995 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Taylor, C.F., et al.: The minimum information about a proteomics experiment (MIAPE). Nat. Biotechnol. 25(8), 887–893 (2007)

    Article  CAS  PubMed  Google Scholar 

  19. Stavenhagen, K., et al.: Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J. Mass Spectrom. 48(6), 627–639 (2013)

    Article  CAS  PubMed  Google Scholar 

  20. Marx, K., Kiehne, A., Meyer M.: amaZon speed ETD: Exploring glycopeptides in protein mixtures using Fragment Triggered ETD and CaptiveSpray nanoBooster, Bruker application note LC-MS-93, 04/2014, Bruker Daltonics GmbH, http://bdal.de (2014). Accessed 23 October 2014

  21. Liu, J., McLuckey, S.A.: Electron Transfer Dissociation: Effects of Cation Charge State on Product Partitioning in Ion/Ion Electron Transfer to Multiply Protonated Polypeptides. Int. J. Mass Spectrom. 330–332, 174–181 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  22. Good, D.M., et al.: Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6(11), 1942–1951 (2007)

    Article  CAS  PubMed  Google Scholar 

  23. Catalina, M.I., et al.: Electron transfer dissociation of N-glycopeptides: loss of the entire N-glycosylated asparagine side chain. Rapid Commun. Mass Spectrom. 21(6), 1053–1061 (2007)

    Article  CAS  PubMed  Google Scholar 

  24. Mormann, M., Paulsen, H., Peter-Katalinic, J.: Electron capture dissociation of O-glycosylated peptides: radical site-induced fragmentation of glycosidic bonds. Eur. J. Mass Spectrom. 11(5), 497–511 (2005)

    Article  CAS  Google Scholar 

  25. Thaysen-Andersen, M., et al.: Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS. Electrophoresis 32(24), 3536–3545 (2011)

    Article  CAS  PubMed  Google Scholar 

  26. Takahashi, K., et al.: Clustered O-glycans of IgA1: defining macro- and microheterogeneity by use of electron capture/transfer dissociation. Mol. Cell. Proteomics 9(11), 2545–2557 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Brockhausen, I.: Pathways of O-glycan biosynthesis in cancer cells. Biochim. Biophys. Acta 1473(1), 67–95 (1999)

    Article  CAS  PubMed  Google Scholar 

  28. Djordjevic, S., Driscoll, P.C.: Targeting VEGF signalling via the neuropilin co-receptor. Drug Discov. Today 18(9–10), 447–455 (2013)

    Article  CAS  PubMed  Google Scholar 

  29. Kolarich, D., et al.: Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms. Proteomics 6(11), 3369–3380 (2006)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors thank Daniel Kolarich for fruitful discussion. We wish to acknowledge the input of the OPPF (Oxford protein production facility, Didcott, UK) in supporting initial mammalian protein expression trials of the c/MAM domain.

Compliance with Ethical Standards

No ethical objections emanate from this work.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria

    Markus Windwarder & Friedrich Altmann

  2. Institute of Structural and Molecular Biology, University College London, Gower Street, Darwin Building, London, WC1E 6BT, UK

    Tamas Yelland & Snezana Djordjevic

Authors
  1. Markus Windwarder
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  2. Tamas Yelland
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  3. Snezana Djordjevic
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  4. Friedrich Altmann
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Corresponding author

Correspondence to Markus Windwarder.

Electronic supplementary material

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Supplementary Figure 1

Quality control of purified recombinant c/MAM domain. Size exclusion chromatography and SDS-PAGE demonstrate high protein purity and an apparent size of ~ 25 kDa. (GIF 58 kb)

High Resolution Image (TIFF 149 kb)

Supplementary Figure 2

Proof of novel α2-3,6,8 neuraminidase substrate specificity. Panel A shows the chromatograms of the two main O-glycan species, derived from reductive beta elimination of neuraminidase treated chymotryptic recombinant neuropilin-1. The structure of the core 1 glycan was verified by positive mode CID fragmentation, giving the feature ion 515.1 m/z (panel B). Panel C shows a CID fragmentation spectrum of the main fetuin O-glycan being sialylated on the galactose. In contrast to panel C, 515 m/z shows no signal whereas a specific feature ion at 454.1 m/z (Neu5Ac-Gal) appears. (GIF 76 kb)

High Resolution Image (TIFF 3405 kb)

Supplementary Figure 3

Positive mode CID-MS/MS fragmentation spectra of some c/MAM O-glycan species. Precursor mass and charge form as well as inferred glycan structure are given in the respective top right corners of Panels A-D. Peak * in Panel D results from a glycan rearrangement during CID, which did, however, not affect data interpretation. (GIF 135 kb)

High Resolution Image (TIFF 4326 kb)

Supplementary table 1

(DOCX 25 kb)

Supplementary table 2

(DOCX 30 kb)

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Windwarder, M., Yelland, T., Djordjevic, S. et al. Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain. Glycoconj J 33, 387–397 (2016). https://doi.org/10.1007/s10719-015-9602-x

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  • DOI: https://doi.org/10.1007/s10719-015-9602-x

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