Analysis of Folate Binding Protein N-linked Glycans by Mass Spectrometry

Abstract

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.

Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol‐anchored proteins through selective glycan enrichment

“…In addition to its GPI anchor, most characterized GPI-APs also possess additional carbohydrate modifications such as N-and/or O-linked glycans (Fig. 1A) [1][2][3][4].…”

The complex interplay between ligand binding and conformational structure of the folate binding protein (folate receptor): Biological perspectives

“…This was verified by mass spectrometry of glycopeptides from trypsin digested bovine FBP, which showed partial N-glycosylation at positions 49 (94%) and 141 (74%) [27]. A detailed structural characterization of N-linked glycans from bovine and human FBPs was performed by mass spectrometry [28]. Both FBPs contained abundance of high mannose hybrid/complex type N-glycans.…”

“…N-glycans originating from bovine milk mostly do not contain fucose and sialic acid but many Nglycans of this type are present in human milk FBP. The MALDI-TOF mass spectrum of deglycosylated bovine FBP (26.4 kDa) contained dimeric and trimeric peaks [28] just as the spectra of glycosylated bovine and human FBP [29]. Furthermore, SDS-PAGE and Western blotting studies of bovine and human FBPs [28] revealed heterogeneity with double bands due to different glycosylation, and high molecular weight bands of aggregated/self-associated FBPs.…”

“…The MALDI-TOF mass spectrum of deglycosylated bovine FBP (26.4 kDa) contained dimeric and trimeric peaks [28] just as the spectra of glycosylated bovine and human FBP [29]. Furthermore, SDS-PAGE and Western blotting studies of bovine and human FBPs [28] revealed heterogeneity with double bands due to different glycosylation, and high molecular weight bands of aggregated/self-associated FBPs. The latter findings are similar to those reported in another study [29], where the identities of the heterogenous SDS-PAGE bands of purified bovine and human apo-FBP were verified by ingel-digestion with trypsin followed by MALDI-TOF mass spectrometric peptide mass finger print analyses.…”

“…Self-association of bovine and human FBP has been analyzed through a variety of techniques, e.g., size exclusion chromatography, analytical ultracentrifugation, surface plasmon resonance, MALDI-TOF, SDS-PAGE-Western blot and dynamic light scattering [13,39,26,41,43,29,28] over a concentration range from 10 nM (less than one tenth of the FBP concentration in cow's milk) to 200 μM. They revealed a concentration-dependent reversible self-association of apo-FBP into dimers at low concentrations and octamers at the highest concentrations.…”