Development of a method to study glycoproteins modified with ribitol-5-phosphate

Alpha-dystroglycan (αDG) is a heavily glycosylated protein that is part of the dystrophin-glycoprotein complex present on the membrane of mammalian cells. The function of this complex is to give rigidity to a tissue through binding laminin in the extracellular matrix. αDG has a unique O-mannosyl glycan, essential for its function, consisting of a phosphorylated trisaccharide core M3 elongated with two ribitol-5-phosphate (Rbo5P) residues. The terminal Rbo5P acts as a linker to which a long chain of alternating xylose and a glucuronic acid residue, named matriglycan, is attached. Rbo5P is currently found in mammalian cells uniquely on αDG. Enzymes responsible for the synthesis of the alditol donor CDP-Rbo and for the Rbo5P transfer onto the O-mannosyl glycans have been identified.
The driving force of the research presented here is the hypothesis that other mammalian proteins might also have glycans with Rbo5P and share a similar function to αDG. This work aimed to identify novel Rbo5P glycoconjugates through chemical tagging of Rbo5P residues and subsequent enrichment of tagged glycoproteins from cultured cells. A method for tagging, enriching and analysing by mass spectrometry glycoproteins modified with Rbo5P has been developed and tested. The method consists of oxidising glycoproteins bearing Rbo5P with sodium periodate to generate aldehydes, which can then be conjugated with aminooxy-biotin. The biotin tag was exploited for the enrichment of Rbo5P glycoconjugates from mammalian cell lysates using streptavidin pulldown. Proteins recovered this way were then digested into peptides and analysed by liquid chromatography coupled to mass spectrometry. Two putative Rbo5P-containing glycoproteins were identified using this method. Moreover, the chemical tagging to recombinant αDG revealed a new glycosylation site for the Rbo5P modification.